I AN INTRODUCTION TO THE CHEMISTRY OF PLANT PRODUCTS LONGMANS, GREEN AND CO. Ltd. 39 PATERNOSTER ROW, LONDON, E.C. 4 6 OLD COURT HOUSE STREET, CALCUTTA 53 NICOL.ROAD, BOMBAY 167 MOUNT ROAD, MADRAS LONGMANS, GREEN AND CO. 55 FIFTH AVENUE, NEW YORK 221 EAST 20TH STREET, CHICAGO TREMONT TEMPLE, BOSTON 210 VICTORIA STREET, TORONTO H // ^/ AN INTRODUCTION TO THE CHEMISTRY OF PLANT PRODUCTS Vol. I. ON THE NATURE AND SIGNIFICANCE OF THE COMMONER ORGANIC COMPOUNDS OF PLANTS BY PAUL HAAS, D.Sc, Ph.D. READER IN PLANT CHEMISTRY IN THE UNIVERSITYrOF LONDON, UNIVERSITY COLLEGE AND T. G. HILL, D.Sc, A.R.C.S. READER IN VEGETABLE PHYSIOLOGY IN THE UNIVERSITY OF LONDON, UNIVERSITY COLLEGE FOURTH EDITION WITH DIAGRAMS LONGMANS, GREEN AND CO. LONDON ♦ NEW YORK ♦ TORONTO 1928 Made in Great Britain PREFACE TO THE FOURTH EDITION. The original intention of this work was to provide students with an account of the chemistry and physio- logical significance of some of the more important substances occurring in the plant. The founding of Chairs of Biochemistry during recent years, with the consequent dissemination of biochemical knowledge, would appear to give just cause for the discontinuance of the present work, but it is not possible for all students to avail themselves of the facilities offered, and it is primarily for such students that the work is intended. The enormous output of papers and the recent advances in knowledge have necessitated much revision, and, in the main, the present edition has been rewritten. In so doing we have borne in mind the requirements of those approaching the subject from different angles and have included a certain amount of somewhat elementary information, both botanical and chemical, and also have admitted certain rather more advanced aspects of the subject even though they be matters of controversy. We fully recognize that this involves some dispro- portion, some lack of balance, but this is inevitable. R H. T. G. H. June, ig28. PREFACE TO THE FIRST EDITION. The importance to the botanist of a working knowledge of chemistry can hardly be overestimated, since vege- table physiology is replete with problems awaiting solution by the combined application of botanical and chemical methods. Teachers of vegetable physiology, however, not in- frequently find that their students' knowledge is deficient in just those branches of chemistry which are of parti- cular importance to the botanist, which is, no doubt, largely due to the fact that those compounds which are of interest to the botanist do not necessarily fit into the scheme of instruction of the chemist. The present work is an attempt to provide such students, who are assumed to have some acquaintance with chemistry, with an introductory account of the chemistry and biological significance of some of the more important substances occurring in plants. In compiling this book various sources of informa- tion have been laid under contribution, and although the point of view is, in the main, purely chemical and botanical, the economic aspect has not been lost sight of, and, where possible, mention has been made of the uses of plant products and of the manufacturing processes employed in their preparation. P. H. T. G. H. December^ igi2. VI PREFACE TO THE THIRD EDITION. The necessity for a third edition has afforded an oppor- tunity for making certain changes in the arrangement of the subject-matter. In order to give the more purely physiological aspect of the subject fuller treatment, with- out at the same time unduly increasing the size of the volume, the work now appears in two parts. Volume I. is essentially the same in scope as the earlier editions and deals primarily with the more chemical side of the subject : a sufficiency of plant physiology has, however, been retained to make the account reasonably complete and to preserve the character of the work. Volume II., which is in preparation, will be devoted to more purely physiological problems, and will contain some of the matter previously found in the original volume. The present volume has been brought up to date as far as is possible ; some portions have been rewritten. Section VIII. for example, and in other sections a certain amount of rearrangement has been deemed advisable. P. H. T. G. H. October, ig20. vn CONTENTS. PREFACE SECTION I.— FATS, OILS, AND WAXES Fats .... Occurrence Constitution Chemical properties Saponification Extraction Characterization Quantitative estimation Quantitative methods for characterization Acid number . Saponification value. Unsaponifiable residue Iodine value . Reichert Meissl value Acetyl value . Spontaneous changes Rancidity Drying and resinification Industrial uses Hardening of oils Physiological significance Microchemical reactions Waxes Properties Sterols Cholesterol Reactions Phytosterols Distinction between cholesterol and phytosterol Estimation of sterol content of an unsaponifiable residue Lipins .... Phospholipins Lecithin . Kephalin Cerebrosides or galactolipins Occurrence Physiological significance PAGE V I I 4 9 lo 12 14 i6 20 21 21 22 23 26 27 28 28 29 31 34 34 44 44 45 46 46 47 48 49 50 51 53 53 55 56 56 57 SECTION II.— ALDEHYDES AND ALCOHOLS Formaldehyde Alcohols Occurrence 60 65 69 69 IX 37638 CONTENTS Inositol Preparation Identification . Manufacture of ethyl alcohol PAGE 71 72 72 73 SECTION III.— THE CARBOHYDRATES Classification .... Solubilities ..... General tests .... Constitution and isomerism of sugars Oxidation products of sugars Characterization of sugars Monosaccharides .... Pentoses .... General properties . Properties of individual pentoses Arabinose Xylose Ribose Apiose Methyl pentoses Hexoses .... Distinction between aldoses and ketoses Glucose or dextrose . Occurrence Preparation Properties Reactions Microchemical tests Fructose or levulose Occurrence Preparation Properties Reactions Constitution Sorbose Galactose Occurrence Preparation Properties Detection Mannose Occurrence Preparation Properties Detection Heptoses Disaccharides Action of enzymes on disaccharides Cane sugar, sucrose or saccharose Occurrence Preparation Constitution Properties Reactions Turanose Maltose Properties and reactions Isomaltose Cellobiose Iso-cellobiose 76 77 78 79 80 84 86 88 88 90 92 92 92 93 93 93 94 96 96 96 97 98 98 99 lOI loi lOI 102 102 102 103 104 104 104 105 105 106 106 106 106 107 107 107 108 109 109 III "3 "3 114 "5 "5 116 117 119 119 CONTENTS XI Gentiobiose . Trehalose . . - Lactose or milk sugar Melibiose ... Disaccharides produced by the union of a hexose with Primeverose . Vicianose Strophanthobiose Trisaccharides Raffinose Detection Melecitose Stachyose Gentianose . . Sugars of unknown molecular weights or sugar saccharides . . . . • Abnormal or ill-delined sugars Estimation of sugars . . . . • Volumetric methods .... Estimation by means of Fehling's solution Estimation of pentoses Estimation of reducing sugars Estimation of galactose and mannose Estimation of cane sugar . Estimation of maltose Estimation of mixtures of sugars Estimation by means of Pavy's solution Estimation by means of Benedict's solution Estimation by Bertrand's method Gravimetric methods Estimation of pentoses Reducing sugars other than pentose Estimation of glucose as osazone Estimation of natural mixtures of sugars Polarimetric methods Polysaccharides Hexosans Glucosans Starch or amylum Preparation Purification Properties Composition of the starch grain Action of acids on starch . Action of malt diastase on starch Action of bacteria on starch Reactions Estimation of starch Dextrins Occurrence Formation from starch General properties . Commercial dextrin Glycogen Preparation Properties Identification Estimation Lichenin and isolichenin Paradextrane and paraisodextrane Fructosans . Inulin . a pentose like poly- PAGB 119 120 120 120 121 121 121 122 122 122 124 124 125 126 126 126 126 126 128 128 130 130 132 134 135 137 137 139 140 140 141 146 146 146 149 149 154 161 163 163 164 165 167 167 169 170 170 171 171 173 173 173 xn CONTENTS Preparation Characters Identification . Physiological significance . Inulin-like substances Hemicelluloses .... Properties .... Constitution .... Mannan .... Paramannan . Carubin or secalane . Xylan .... Araban .... Wood gum Galactan Mixed galactans Amyloid Gums ...... Microchemical reactions . Gum-arabic .... Reactions Gum-tragacanth Wound gum .... Mucilage .... Function Pectic bodies Isolation of pectins from the tissues Properties Microchemical reactions . Estimation Action of enzymes on pectins Origin and constitutional relationship Changes taking place on ripening Cellulose ...... Classification .... Properties ..... Solubility .... Action of various chemicals on cellulose Oxycellulose .... Properties Constitution ..... Microchemical reactions Lignified membranes .... Chemistry of lignin Isolation and constitution of lignin . Estimation ..... Estimation of cellulose in lignified tissues Nature of the union between lignin and cellulose Microchemical reactions . Cutinized membranes Suberized membranes Microchemical reactions of suberized and membranes . Industrial uses of cellulose and cellulose products Commercially valuable derivatives of cellulose cutinized 174 174 175 176 178 179 180 180 181 182 182 182 183 183 184 185 185 186 187 187 188 189 189 190 191 192 193 198 199 201 201 203 203 205 206 208 208 209 212 213 214 216 216 219 222 226 227 228 229 230 232 234 235 236 SECTION IV.— GLUCOSIDES Constitution Physiological significance Sinigrin Coniferin 239 240 243 245 246 CONTENTS Xlll Salicin ..... Monotropitin Aucubin .... Orobanchin .... Asperulin .... Gein ..... Melilotosin .... Indican .... Identification . Cyanogenetic glucosides Reactions .... Amygdalin .... Prunasin, prulaurasin, and sambunigrin Dhurrin .... Phaseolunatin or linamarin Lotusin .... Saponins ..... Physical and Chemical Properties Isolation .... Constitution .... Reactions .... Physiological action General properties and uses 246 248 248 249 249 250 250 250 251 253 255 257 259 259 260 261 261 261 262 263 263 264 265 SECTION v.— TANNINS . Occurrence . Microchemical reactions Physiological significance Phenolic constituents Catechol Reactions Resorcinol . Reactions Hydroquinone Reactions Protocatechuic acid Reactions Pyrogallol or pyrogallic acid Reactions Phloroglucinol Reactions Gallic acid . Reactions Galloyl-gallic acid or digallic EUagic acid Properties and reactions Classification of tannins Properties and description of Pyrogallol tannins . Gallotannic acid Extraction Reactions Constitution and synthesis acid EUagitannic acid Tannins as glucosides Catechol tannins Cutch or catechu Constitution of the catechu tannins Oak-bark tannin or quercitannic acid Phlobaphenes . . . • • acid individual tannins of natural gallotannic 266 267 270 272 278 278 278 279 279 279 280 280 281 281 281 282 282 283 283 284 285 286 286 288 288 288 289 290 291 292 293 294 294 296 297 297 XIV CONTENTS Relationship between catechol tannins and flavonols, etc. Fxonomic uses of tannins ....... Composition of certain dye-woods and barks and their extracts Old fustic Jack wood Quercitron bark Depsides Lecanoric acid Evernic acid . Chlorogenic acid Properties 298 301 302 302 302 303 304 304 305 305 306 SECTION VI.— PIGMENTS Chlorophyll . Constitution . Action of acid and alkali on chlorophyll Action of alkalis Action of acids Crystalline and amorphous chlorophyll . Relationship between chlorophyll and haemoglobin Extraction of chlorophyll .... Carotinoids or yellow pigments accompanying chlorophyll Carotin Lycopin Xanthophyll . Rhodoxanthin Fucoxanthin . Anthoxanthins Flavones and xanthones Yellow colouring matters derived from flavone Yellow colouring matters derived from xanthone Properties of anthoxanthins Anthocyanins .... Occurrence, conditions of formation and physiological significance Preparation and properties Reactions and properties Chemical constitution The colour of petals Connection between anthocyanins and anthoxanthins Phycoerythrin Preparation Reactions Phycophaein Phycocyamn Preparation SECTION VII.— NITROGEN BASES Alkaloids Occurrence Classification General properties General reactions Isolation Origin of alkaloids in the Ptomaines . Purine bases Physiological significance Nucleic acids Hydrolj'tic products plant of nitrogen bases 307 307 316 317 317 319 319 322 324 328 328 330 330 330 331 331 331 332 335 335 336 336 344 345 345 349 351 353 354 354 356 356 356 358 361 361 361 365 366 367 368 371 374 378 381 381 CONTENTS XV SECTION VIII.— THE COLLOIDAL STATE Suspensoids .... General properties . Optical properties Electrical properties Protective action of colloids . Emulsoids .... Genera] properties . Swelling of colloids or imbibition Gel formation General properties of gels Nature of gels Adsorption Colloidal electrolytes Enzyme action of colloids Colloidal nature of protoplasm SECTION IX.— PROTEINS animal proteins d their ph ysiological signifi Extraction .... Classification Comparison between vegetable and Physical and chemical properties Physical properties Solubilities of proteins an cance . Iso-electric points Chemical properties Microchemical reactions Decomposition products Amino acids obtained as cleavage products of proteins Occurrence of amino acids in plants Synthesis of amino acids in the plant A note on the chemical composition of protoplasm SECTION X.— ENZYMES . Classification Isolation and purification Chemical constitution . Mode of action Conditioning factors Temperature . Reaction of medium Concentration of enzyme and of substrate Influence of end products Paralysers ..... Radiation ..... Reversibility of enzyme action Antienzymes ..... A consideration of selected enzymes Lipase ...... Preparation .... Properties .... Diastase (Amylase) Isolation Quantitative determination of activity Takadiastase . Maltase Preparation Proteolytic enzymes Occurrence 385 389 389 389 390 393 394 395 400 402 403 404 405 409 410 413 419 421 424 430 433 433 434 436 438 440 441 443 449 450 453 455 459 460 463 464 466 467 468 469 471 472 472 473 474 474 474 476 476 477 478 478 479 480 481 481 481 jcvi CONTENTS - Isolation ....... :, General considerations ..... .,; Zymase and alcoholic fermentation .... Activity of different species of yeast Mechanism of fermentation .... Co-enzyme of zymase ..... Isolation of zymase ...... Role of phosphate in yeast juice fermentation Oxidases ......•■ General considerations ..... Isolation ....... Peroxidase ...... Preparation ..... Reagents used for detection of oxidases and peroxidases ..... Tyrosinase ...... C>atalase •.••••• APPENDIX.— HYDROGEN ION CONCENTRATION INDEX 483 485 486 487 489 492 493 494 498 499 502 503 503 504 505 506 507 517 SECTION I. FATS, OILS, AND WAXES. In ordinary parlance, no clear distinction is made in the use of the terms fat and wax, which are applied more or less in- discriminately to any solid substances which have a greasy feeline to the touch and do not mix with water. Chemically, however, there is a marked difference between the two classes ; the fats are compounds of the trihydric alcohol glycerol, whereas the waxes are compounds of the higher monohydric alcohols, such as cetyl alcohol CigHggOH, myristic alcohol CgoHfiiOH, and cholesterol C27H45OH. The tendency to rely on physical properties only, and to regard waxes as having generally a harder consistency than fats has given rise to several cases of incorrect nomenclature. For example, wool fat and sperm^aceti being compounds of cholesterol and cetyl alcohol are in reality waxes, though they are usually regarded as fats, whereas the substance ordinarily known as Japan wax is actually a fat, since it is a compound of glycerol. The term oil, as used in the ordinary sense to imply a liquid which is immiscible with water, must not be taken to have any chemical significance, since substances having this physical property are found in almost every class of chemical compound. Used in connection with fats, the term oil simply implies a fat that is liquid at ordinary temperatures ; any solid fat on melting becomes an oil, and, on the other hand, any fatty oil on solidifying becomes a fat. OCCURRENCE. Fats are very widely distributed in the vegetable kingdom, and occur in both vegetative and reproductive structures ; m fact, it is highly probable that all living cells contain a certain I 2 FATS, OILS, AND WAXES amount of fat. Amongst the Protophyta, fat is the charac- teristic food reserve of the Heterokontae, Chrysophycese, Bacillariales, and Chloromonadales. In the Phaeophyceae, the amount of fat, or fat-Hke substances, would appear to vary with the conditions of Hfc. Thus Pelvetia canaliculata, var. libera, which is submerged only during the spring tides, may contain 8 per cent of ether-soluble material, whilst Laminaria digitata, which is exposed only at low water of spring tides, contains but 0-5 per cent. The fucoids of the intermediate zones contain amounts of ether-soluble substance intermediate between these extremes. The Rhodophycese which are charac- teristic of the submerged zone would appear to contain less fat, thus Chondrus crispus yields 0*2 per cent of ether-soluble material.* The fats of the Fungi, which are rich in fatty acids associated with lecithins and ergosterols, vary much in amount; thus the sclerotia of Claviceps purpurea (ergot) may contain as much as 60 per cent, whilst the mycelium of Laclarius deliciosus contains about 6 per cent. In Angiosperms fats are widely distributed, especially in seeds where they may replace the carbohydrates as a reserve food-material and are not uncommonly associated with protein reserves ; to mention a few examples, colza oil is obtained from the seeds of Brassica Napus, palm oil from the pericarp of the fruits of Elceis guineensis , cotton-seed oil from Gossypium herbaceum, linseed oil from Linuni usitatissimum, olive oil from the sarcocarp of Olea europcea, castor oil from the seeds of Ricinus, and cacao butter from the fruits of Theobrotna. Oils of lesser economic importance occur in the fruits or seeds of the sunflower, almond, hemp, willow, and many other plants. The amount of oil present in sueh structures may be quite considerable, thus in the kernel of the Brazil nut nearly 70 per cent may obtain, and in the almond about 54 per cent. Oils also occur in the vegetative organs to a greater or lesser extent ; substances of an oily nature are found in association with the chloroplasts and, in some cases, to a relatively large extent, e.g. in Strelitzia ; sometimes it is present as a definite * Authors' observations hitherto unpublished. OCCURRENCE 3 reserve food-material as in the tubers of Cyperus esculenhis, where it is associated with starch, and in the roots of some orchids. This particular form of food reserve is doubly of value since its presence may lessen the danger arising from drought, and also more energy can be stored up in the form of oil than in an equal bulk of carbohydrate ; in this connection may be mentioned the fact that in some cases the appearance of oil may be transient, thus in some trees the starch stored up in the parenchyma of the stem may be converted into fat during the winter's cold ; the starch, however, reappears on a rise in temperature. Also fat or fat-like substances may appear in the leaves of evergreen plants during the winter months. The fat-like substances, according to Meyer,* who studied Vinca, Taxus, and Ilex, do not show a seasonal variation in amount, but continually increase with the age of the leaf. A low tem- perature would appear to be a significant factor in this con- nection. Thus Tuttle f found that plants of Linncsa borealis exposed to a low temperature contained fat but no starch ; on raising the temperature to 20° C, starch appeared in the course of a day or two in a few plants, and in all cases after the lapse of a week, during which period the plants were kept in the dark. The controls, on the other hand, kept in the dark at the low outside temperature gave no reaction for starch. Plants containing much starch, on exposure to a moderately low temperature, — 2° C, were found to lose their starch and, concurrently, fat appeared. But if such starch-containing plants were immediately exposed to very low temperatures, — 15° to — 28° C, no reconversion ensued and death took place. Lipase is present in the leaves of plants showing these changes, and this, presumably, is part of the mechanism of the change. Tuttle also found that all evergreens growing in Northern Alberta contained little or no starch but much fat by the end of October. All of the many plants examined, Populus, Salix, Betula, Pyrola, Picea, etc., with the exception of Lonicera glaucescens and Craiosgus, * Meyer : " Ber. deut. bot. Gesells.," 1918, 36, 5. t Tuttle : " Ann. Bot.," 1919. 33, 201 ; " Bot. Gaz.," 1921. 71, 146. 4 FATS, OILS, AND WAXES contained fat as a food reserve during the winter months. Even the leaves of deciduous plants at the time of leaf-fall were devoid of starch but contained fat. Whilst the power of form- ing fat from starch is not uncommon in plants naturally ex- posed to extreme winter cold, the ability to form starch on the advent of warmer weather does not necessarily follow. Thus many alpine Ericaceae and Salicaceae possess both starch and fat during the vegetative season, and Gaultheria ovalifolia, a lowland plant, has only fat. Wherefore the ability to form starch is not entirely to be associated with the climatic con- ditions resulting from high altitudes. These phenomena are similar to those which will be mentioned in connection with the conversion of starch into sugar under the influence of low temperature (p. 176). The majority of vegetable fats are fluid at ordinary tem- peratures ; a few, however, are solid, for instance, cacao butter and the fat in the seeds of Myristica. CONSTITUTION OF FATS. The naturally occurring fats are mixtures of esters of glycerol with fatty acids such as palmitic C15H31COOH or stearic C17H35COOH acids, or with the unsaturated acid oleic acid C17H33COOH. A wax, on the other hand, is an ester of a monohydric alcohol as illustrated by the equation : — C15H31COOH + CaoHeiOH = C16H31COOC30K,, + H^O Palmitic acid Myricyl Myricyl palmitate alcohol myricyl palmitate being the chief constituent of beeswax. Lapworth and Pearson * have shown that the glycerol in fats may be directly replaced by a higher polyhydric alcohol such as mannitol. This replacement may be brought about by distilling olein or stearin with mannitol under reduced pressure in the presence of sodium ethoxide. By this treat- ment much of the glycerol of the fat is expelled, the maximum yield being reached when the proportion of fat to the mannitol corresponds with two molecules of the former to three mole- * Lapworth and Pearson : " Biochem. Journ.." 1919. I3. 296. See also Irvine and Gilchrist : " J. Chem. Soc," 1924, 125, 10. CONSTITUTION cules of the latter. The composition of the mannitol olein, or mannitol stearin, corresponds with that of a mixture of dioleates or distearates of mannitan and isomannide. It has been shown by feeding experiments that mannitol olein is utilized by animals to the same extent as olive oil, but there is no evidence that mannitol fats occur in nature.* The classification and identification of fats is based upon the acids which they contain. Thus it is found that whereas beef suet and mutton fat consist chiefly of esters of the higher fatty acids, such as palmitic and stearic acids, butter contains a considerable quantity of the lower members of this same fatty series such, for example, as butyric, caproic, caprylic, and capric acids ; these acids, which are low boiling liquids readily volatile with steam, are known as volatile fatty acids, and their presence or absence in a given sample of fat may be used for characterizing the fat. Thus, for example, the estimation of the amount of volatile fatty acid serves to distinguish genuine butter from its substitute margarine, which is relatively poor in volatile acids and contains chiefly higher fatty acids. The more important members of the fatty acid series are given in the following list : — HCOOH or CH^Oj Formic acid t CH3COOH .. C2H4O2 Acetic acid C2H5COOH 1. CgHgO^ Propionic acid t CsH-COOH „ QHsO^ Butyric acid ^2'>CH CH2CH2COOH .. QHijOa Isobutyl acetic or caproic acid CH3(CH2),COOH „ QHjoO, Caprylic acid CHglCH^jgCOOH ,. ^10^20^2 Capric acid CH3(CH2)ioCOOH .. Ci2f^2<02 Laurie acid CH3(CH.,)iXOOH .. Ci4H2802 Myristic acid CHalCHali^COOH ., ^16^32^2 Palmitic acid CHjICHjIioCOOH ,, C^lflH.3802 Stearic acid CHjICH^^isCOOH ,, C20H40O2 Arachidic acid CH,(CH2)2oCOOH .. ^22"44^'2 Behenic acid It should be noted that these acids all conform to the general formula for the fatty acids, CnH2n02, in which " n " may have any value, odd or even, but only those in which " n " is an even number are found to occur naturally in fats ; ♦Halliburton, Drummond, and Cannan : " Biochem. Journ.," 1919. 13, 301. t These acids do not occur in fats. 6 FATS, OILS, AND WAXES the alleged occurrence in natural fats of acids with an uneven number of carbon atoms has in every case, so far recorded, been refuted on careful re-examination. It appears probable, moreover, that all naturally occurring fatty acids have a straight and not a branched carbon chain, so that it is open to question whether the iso-huty\ acetic acid which is said to have been found in fats was not, in reality, normal caproic acid of the formula CH3(CH2)4COOH. Besides acids of the fatty series whose general formula is CnHgnOg, acids belonging to several other series, poorer in hydrogen than the above, are found in fats. The simplest example of such a series of acids is furnished by the acids of the Oleic series, the members of which differ from the corre- sponding members. of the fatty acid series in having two atoms of hydrogen less. Some of the more important acids of this group are given below. I. Acids of the Oleic or Acrylic series. General formula CnHgn _ o^a- CjHgOa Tiglic acid C18H34O2 Oleic acid CigHg^Oa Elaidic acid C18H34O2 Iso-oleic acid C22H42O2 Erucic acid C22H42OJ Brassidic acid The most widely distributed of these acids is undoubtedly oleic acid, which, in the form of its glyceride triolein, Ci7H33COOCri5, C„H33COO in C„H33COOCH2 forms an important constituent of most vegetable and animal oils. 2. Acids of the Linolic series. General formula CnHgn _ 4O2. (a) Open chain compounds, CigHgaOg Linolic acid and its various isomers. (b) Cyclic compounds, CigHggOa Hydnocarpic acid. CjgHggOg Chaulmoogric acid. CONSTITUTION . 7 3. Acids of the Linolenic series. General formula CnHan.eOa- CigHgoOa Linolenic acid and its isomers. 4. Acids of the Clupanodonic series. General formula CnHan-gOg. C^gH-jgOa Clupanodonic acid. 5. Acids of the Ricinoleic series. General formula CnHgn _ 203- CjgHjjOj Ricinoleic acid and its isomers. The relationship between the five series of acids, which differ from each other successively by two atoms of hydrogen, as shown by the formulae, CnHgijOg, CnHgn _ 2^)2, Cnrljn _ 402> ^"-211 — 6^2' ^■iid CnH^n — f\^2' is similar to that subsisting between the three series of hydro- carbons having the general formulae, ^-Ti"^2n + 2) Curi2ni ^n"^2ii — ?• The hydrocarbons of the first or Paraffin series arc said to be saturated, by which is meant that each of the four valencies of their carbon atoms are fully satisfied, as shown by the follow- ing graphic formulae : — H H H H H H— C— C— H H— C— C— C»-H Ethane C2HJ Propane CgHg When, however, the graphic formulae of the corresponding members of the second or define series are written, it is found that if the tetravalency of carbon is maintained, there are not enough hydrogen atoms to satisfy all these valencies, and, in order not to leave any unsatisfied, the remaining valencies must be united to each other, thereby joining two carbon atoms to each other by more than one bond : — H H H H H I I I u—o-c=c—u u H H H Ethylene CjH^ Propylene C^Ug 8 FATS, OILS. AND WAXES In the next series of hydrocarbons, the acetylenes, by the loss of two more hydrogen atoms, the process has been carried a step farther, with the result that two carbon atoms are united by a triple bond : — H HC=CH H— C— C^CH 1 H Acetylene C^H^ Allylene C3H4 All such substances containing two carbon atoms united together by more than one bond are said to be unsaturated, and are able to form additive compounds with many sub- stances, notably the halogens. Thus, while the saturated hydrocarbon will only react with chlorine or bromine by the replacement of one atom of hydro- gen for each atom of halogen introduced into the molecule, C^Hg + Br^ = CjHsBr + HBr Ethyl bromide an unsaturated compound, such as ethylene, will add on the halogen directly, C2H4 + Bra = CjH^Br, Ethylene dibromide the resulting additive compound being saturated. It will thus be seen that it requires two atoms of bromine to saturate an unsaturated compound containing one double bond, and similarly it requires four atoms of halogen to saturate a compound containing two double bonds. In this way it is shown that since the acids of the oleic, linolic, and linolenic series require two, four, and six atoms of halogen respectively for saturation, they must contain respectively one, two, or three double bonds. A measure of the degree of unsaturation of a given acid may accordingly be obtained by determining how much bromine it will absorb ; as, however, the interaction with bromine is liable to be violent, it is found more convenient to employ iodine, which, in addition to being less violent in its action than bromine, is also easier to handle. A description of the method employed in determining what is known as the " iodine value " of fats is given below (p. 23). PROPERTIES 9 PHYSICAL PROPERTIES OF FATS. The naturally occurring fats vary in consistency from oils to wax-like solids ; the solid fats have mostly a low melting- point which is, however, rarely a sharp one, as natural fats are not simple substances, but are, as a rule, mixtures of several different chemical individuals ; such mixtures never have sharp melting-points. All fats and fatty oils are lighter than water, their specific gravity varying from about O-poo to 0-970 at 15°. They are insoluble in water and at ordinary temperatures are sparingly soluble in cold alcohol, excepting castor oil which dissolves readily in this solvent ; they, however, dissolve readily in ether, chloroform, petroleum ether, benzene, carbon tetra- chloride or carbon disulphide. CHEMICAL PROPERTIES OF FATS. One of the most important chemical properties of fats is their decomposition by hydrolysis. The term hydrolysis, which literally means loosening by water, is applied to any reaction in which a substance is broken up into two or more simpler ones with the fixation of water. The following examples taken from a variety of different classes of compounds all illustrate this reaction : — CH3COOC2H5 + H2O = CH3COOH + C2H5OH . . (i) Ethyl acetate Acetic acid Ethyl alcohol CH3CN + 2H2O = CH3COOH + NH3 . . . (2) Methyl cyanide CaHjCONHCHXOOH + H,0 = CgHjCOOH + NH^CHjCOOH . (3) Hippuric acid Benzoic acid Glycine Ci2H,,0,i + HP = 2CeHi,Oe . . . . (4) Malt sugar Glucose C,oH„NO„ + 2HaO = CjHjCHO + 2CeHi208 + HCN . (5) Amygdalin Benzalde- Glucose Hydro- hyde cyanic acid It will be seen from reaction (l) that the conversion of an ester into an acid and an alcohol is an example of hydrolysis, and since fats are esters it follows that they also must be capable of hydrolysis. The reaction — Ci,H35COOCH2 CH2OH C17H35COOCH H- 3H2O = sCi.HjsCOOH + CHOK C^Hg^COOCH^ CH2OH Stearic acid Glycerol lo FATS, OILS, AND WAXES is, however, not readily brought about by water alone at ordinary temperatures ; in the presence of enzymes, however, the hydrolysis may be effected at a moderate temperature with comparative ease. The hydrolysis of fats for the purpose of preparing the free fatty acids may be effected in either of the following ways : — 1. By acting on the fat with superheated steam in the presence of a little lime or magnesia, which acts as a catalytic agent. This method is the one most commonly adopted by candle-makers for the preparation of fatty acids required in the manufacture of candles. The fat is subjected to the action of steam under pressure at 170° in large copper vessels in the presence of a small quantity of lime. The resulting mixture is then treated with sulphuric acid sufficient in amount to combine with the lime, after which the free fatty acids rise to the surface in a molten condition. 2. By the action of concentrated sulphuric acid. The molten fats are stirred up in leaden vessels with 9 per cent of concentrated sulphuric acid, the mixture being heated to about 120° C. The mixture is then warmed with water to remove the acid, and the acids arc further purified by distillation with steam. SAPONIFICATION OF FATS. Closely related to hydrolysis is the reaction known as saponification ; this reaction, which literally means " soap- making," is that which takes place when a fat is boiled with caustic alkali. The alkali acts in much the same way as water, breaking up the ester into glycerol and the fatty acid which, however, in this case, combines with the alkali to form a salt : — C„H„COOCH, CH,OH C^HsbCOOCH + 3KOH = 3C„H35COOK + CHOH C^HajCOOCHg Potassium stearate, CHgOH Tristearin, a fat a soap Glycerol It SO happens that the sodium and potassium salts of pal- mitic, stearic, and oleic acids dissolve in water, forming opales- SAPONIFICATION ii cent alkaline solutions which readily give a lather, and can, therefore, be used as soaps,* and hence the process by which they are made from fats is called saponification. Although alkali metal salts of other organic acids do not exhibit the characteristics of soap, the term saponification is commonly extended to include all cases of the decomposition of an ester into the corresponding alcohol and the salt of the acid, even though that salt may have none of the characteristic proper- ties of a soap. The saponification of a fat on a small scale t in the labora- tory may be effected as follows : Boil the fat under a reflux * The sodium and potassium salts of oleic acid and of the higher fatty acids, such as palmitic and stearic acids, when dissolved in water, are, to a large extent, hydrolysed into free fatty acid and caustic soda, according to the equation — CijHjjCOONa + H2O = Ci,H35COOH + NaOH Sodium stearate Stearic acid The stearic acid combines with some of the unhydrolysed soap to form an insoluble acid salt, giving rise to an opalescent or turbid solution. It is this insoluble acid salt which is responsible for the formation of a lather on shaking such a solution. The detergent or cleansing action of soap is dependent on the above reaction, since the caustic soda detaches the greasy dirt which then becomes enveloped in a layer of soap solution from the lather, and is so carried away. In this connection it is interesting to note the similar effect of soap on the formation of emulsions. An emulsion may be defined as a mixture, under special conditions, of two otherwise immiscible liquids. Thus, for example, if olive oil is shaken up with water, the two liquids rapidly separate as soon as the shaking ceases. If, however, a little soap solution or some other substance such as gum acacia, tragacanth, s?.ponin (see p. 261), or white of egg be added and the shaking repeated, an emulsion results owing to the oil particles being en- veloped in a layer of soap or other substance which prevents their coalescing. Milk is an example of a naturally occurring emulsion ; so also is latex, contained in plants. If pure olive oil, free from oleic or other acid, is shaken up with caustic soda no emulsion is produced ; on the other hand, olive oil which has been kept some time and contains free oleic acid, when shaken up with caustic soda does produce an emulsion, thus showing that the emulsifying agent is not the free alkali but the soap produced in the second case from the soda and the oleic acid. This may be also illustrated by Biitschli's experiment, which consists in placing a drop of old olive oil containing 9 per cent of oleic acid on a little o-o6 per cent aqueous solution of sodium carbonate. If examined under the microscope it will be seen to consist of a fine honeycomb structure, consisting of particles of oil, the whole apparently exhibiting amoeboid move- ments ; these latter are due to difference in surface tension. + For commercial soap manufacture, see p. 33. 12 FATS, OILS, AND WAXES condenser with alcoholic potash in the proportion of about 5 gms. of fat to 50 c.c. of alcohol containing from 2-3 gms. of caustic potash. The heating should be continued until on pouring a little of the solution into a large volume of water an opalescent solution free from undecomposed fat results. The time required for this may vary from a few minutes to half an hour or more. When the saponification is complete, the contents of the flask should be heated in an evaporating basin over a water bath, and thoroughly stirred to get rid of the alcohol. If the free fatty acids are required, the residual soap is dissolved in water and suflficient sulphuric acid is then added to make the solution strongly acid, whereupon the fatty acids separate out and rise to the surface. The aqueous layer contains the glycerol together with the excess of sulphuric acid and potassium sulphate. In addition to the trihydric alcohol glycerol, all fats contain a sniall quantity of the monohydric alcohols, cholesterol and phytosterol * which constitute what is known as the unsaponi- fiable residue of fats (cf. p. 22). These substances may be isolated from fats according to the following method devised by Kossel and Obermuller.f An ethereal solution of the fat is mixed with a solution of sodium in alcohol ; saponification takes place in the cold and the soap which is precipitated from solution can be filtered off ; the filtrate, which is a mixture of alcohol and ether, contains the glycerol together with the so-called unsaponi- fiable residue consisting of phytosterol or cholesterol, which may be obtained by evaporating the solvent. EXTRACTION OF FATS. The isolation of fats from admixture with other substances may be effected by extraction by means of fat solvents. * The term phytosterol, though employed by many authors to indicate a single definite substance, is beginning to be used as a generic term for a whole group of closely allied substances, the number of which is rapidly increasing as the investigation of vegetable fats proceeds. t Kossel and Obermiiller : " Zeit. physiol. Chem.," 1890, 14, 599 ; 1891, 15. 321. EXTRACTION 13 The principle of the extraction is to treat the dried mixture with a solvent which will dissolve only the fat and leave the other substances unchanged. The solvents most commonly used for this purpose are ether, light petroleum, carbon tetra- chloride, and carbon disulphide, the two latter being used chiefly on a commercial scale. It must be borne in mind that besides extracting fats, ether will also dissolve essential oils, chlorophyll, cholesterol, lecithin, and allied substances variously known as lipoids, lipins, etc. Moreover, other substances which are of themselves in- soluble in ether may become soluble in the presence of fats. Whatever solvent is employed must be tested before use to see that it leaves no residue on evaporation and is free from moisture. A rough and ready method of extracting fat from a given sample is to place the finely divided and dried material on a filter paper folded into a funnel and to pour the fat-solvent on to it. The filtrate will contain most of the fat, which may be recovered by evaporating off the solvent. When it is desired to extract the fat quantitatively, the operation is most conveniently carried out in a Soxhlet apparatus (see below). Previous to extraction, the substance must be thoroughly dried. For this purpose it must either be gently heated in a current of dry air or else desiccated by means of alcohol or anhydrous salts. The first method, which is the most convenient, should, however, be used with caution, as many fats may undergo chemical change during the process, as a result of which the material extracted by ether after drying may be very different from the substance originally present in the moist sample. The second method, which consists in treating the sample to be dried with absolute alcohol for some hours and then filtering and pressing, depends on the fact that the alcohol withdraws the water without dissolving away any appreciable quantity of the fat ; if treated two or three times in this way the substance will be practically free from moisture and can then be extracted under a Soxhlet with ether. The wet 14 FATS, OILS, AND WAXES alcoholic filtrates on careful evaporation yield a residue which may be separately treated with ether to extract any fat con- tained in them. It is obvious that the method cannot be employed if the fat to be extracted is soluble in alcohol. The third method of drying, which involves the use of anhydrous salts such as sodium sulphate, depends on the fact that the anhydrous salt when ground up with the moist tissue withdraws the water from it, forming the hydrated crystals. In a few hours the substance is sufficiently dry to be powdered. The chief objection to this process is the fact that a considerable bulk of salt has to be employed and consequently the volume of the material to be extracted is much increased. Whilst ether is one of the most commonly used solvents for the extraction of fats, Leathes recommends a preliminary extraction with alcohol, since this helps to dry the material and frequently renders easier the subsequent extraction by ether (see under Lipins, p. 51). In some cases a preliminary mild hydrolysis by boiling with dilute hydrochloric acid is necessary to set free the fat in a condition in which it can be readily extracted by the appropriate solvent. CHARACTERIZATION OF FATS. The unequivocal establishment of the true fatty nature of a given substance is not always easy, especially if only a small amount of material is available. 1. In the first instance, the solubilities of the substance should be determined by placing it on a watch-glass and adding a drop or two of the appropriate solvent. All fats dissolve readily in the so-called fat solvents, namely, ether, petrol, chloroform, benzene, acetone, and carbon disulphide ; they are sparingly soluble in cold alcohol, but more soluble in hot alcohol ; all are insoluble in water. These solvents will, how- ever, also dissolve waxes, lipins, hydrocarbons, essential oils, terpenes, resins, and chlorophyll, wherefore some further method of characterization is essential. 2. Fats leave a translucent mark on paper, and many of the aforementioned substances will do the same ; but in the CHARACTERIZATION 15 case of substances which are volatile, the mark will sooner or later disappear, whereas in the case of a true fat, the mark is permanent, since fats are not volatile. 3. Fats, waxes, and lipins are all saponified by boiling with alcoholic potash. In the case of most fats 2 grams can be completely saponified by boiling for a quarter of an hour with 25 c.c. of 3 per cent alcoholic potash. The resulting mixture of potassium soap and glycerol should be completely soluble in water, and, after boiling off the alcohol and acidifying the solution, the free fatty acids should be precipitated. Waxes being, on the whole, less easy to hydrolyse, may not have been completely decomposed under these conditions, but lipins would behave like fats. To distinguish between fats and lipins, special tests have to be applied {vide under Lipins, p. 51). 4. The only certain way of distinguishing between a fat and a wax is to establish the presence or absence of glycerol. This may be done either by heating the substance with a crystal or two of potassium hydrogen sulphate, or, better, if sufficient material is available, by preparing a concentrated solution of glycerol free from fatty acids as follows : Saponify the material as above, boil off the alcohol, take up with a little water and acidify ; filter off the precipitated fatty acids and evaporate the filtrate over a water bath ; extract the residue with a small quantity of alcohol, which dissolves out the glycerol, leaving the salts in solution. Evaporate off the alcohol ; if sufficient material remains divide it into two portions a and h \ a \s heated with a crystal of potassium hydrogen sulphate ; the presence of glycerol is confirmed by the production of acrid vapours of acrolein — CH2OH CHOH CHjOH = 3H2O + CH2 = CH . CHO, which blackens a filter paper moistened with ammoniacal silver nitrate solution. The second portion h is dissolved in a little water and warmed in a water bath with 10 c.c. of freshly prepared bromine water for 20 minutes ; any excess of bromine is then evaporated off and the resulting solution is tested for the presence of dihydroxy acetone, CHgOH . CO . CHjOH, as follows : — i6 FATS, OILS, AND WAXES To half a cubic centimetre add 2 c.c. of sulphuric acid to which have been added o-i c.c. of a 5 per cent solution of either j8-naphthol or resorcinol ; the former should give a green colour with a marked fluorescence, while the latter should give a bright red coloration. QUANTITATIVE ESTIMATION OF FATS. I . By Means of Soxhlefs Extraction Apparatus. — The fact that oils and fats are readily dissolved by ether, chloroform, and light petroleum is made use of in their estimation ; but it must be borne in mind that the method only yields correct results provided other substances, which would be extracted by the solvent em- ployed, are absent from the material under examination. The general arrangement of the ap- paratus required is given in Fig. i. The flask F, which is half-filled with the sol- vent to be employed, is connected to the extractor by a closely fitting cork. The material to be extracted is put into a thimble made of special quality filter paper and placed in the extractor, which is connected to a reflux condenser (C). The method may be conveniently em- ployed for determining the proportion of oil in the reserve food of the castor-oil seed, for example. A number of seeds, freed from their testas, are weighed in the thimble, which is then placed inside the extractor ; a few small chips of porcelain are placed in the flask F, which is then weighed and after being half-filled with freshly distilled ether it is attached to the Soxhlet. The apparatus is then connected up. The ether in the flask F volatilizes and passes up the tube T into the extractor and condenser, and gradually fills the Soxhlet ; on reaching a certain level it siphons over into the flask, carrying with it the fat in I ^ U H. ^ I \ f Fig. I. ESTIMATION 17 solution ; once in the flask the ether is again vaporized and goes through the same process as before, the oil, how- ever, remains behind. The ether is allowed to siphon over at least a dozen times,* and then, when most of the ether is in the extractor, the flask is disconnected. The ether in the flask is evaporated off and the flask is placed in a steam oven for half an hour, it is then allowed to cool in a desiccator and finally weighed. Weight of seeds . ,, flask, chips and oil ,, ,, and chips oil . Per cent fat = 100 (y — z) X y z y-z If the ether has extracted substances other than fats, the result obtained will, of course, be too high. In such cases the ether extract may be saponified and the amount of fatty acid determined, from which the amount of fat originally present can be estimated. Appended are a few figures giving approximately the fat content of some of the commercially exploited seeds : — Hemp 30-35 per cent. Rape • 39-42 „ „ Arachis . . 46-50 .. ,. Ricinus . • 45-55 .. .. Sesame . . 50-54 .. .. Cocos (copra) . . 64-70 „ „ 2. By Saponification. — Apart from the fact that in some cases it is not possible to extract the fat quantitatively by Soxhlet's method with less than forty-eight hours' continuous extraction, the method is open to the objection that the sub- stance must be dried previous to extraction, and this may involve loss or alteration of the fat ; furthermore, the residue which is weighed as fat may not consist entirely of fat, but may contain other substances which are extracted by the same solvents as the fats. The following method, which is due to Liebermann and * The number of times the liquid should be allowed to siphon off varies in every case. In order to ensure complete extraction, the only- safe method to adopt is to weigh the fat extracted after a certain time, then to attach the flask again and continue the extraction for some time longer and again weigh. i8 FATS, OILS, AND WAXES kl I *3-6 Cm/.-' Szekely * has the advantage of giving in a short time a re- liable value for the percentage of fat in almost any substance, and is specially convenient for the estimation of fat in fodder, meat, faeces, and physiological work in general. Five grams of the sample are placed in a flask, of the dimensions given in Fig. 2, with 30 c.c. of 50 per cent caustic potash (sp. gr. 1*54). The mixture is boiled over a wire gauze for half an hour and frequently shaken. After cooling, 30 c.c. of 90-94 per cent alcohol are added and the heating is continued for another ten minutes ; the mixture is then cooled again and carefully mixed with 100 c.c. of 20 per cent sulphuric acid (sp. gr. 1-145) ^^'^ thoroughly shaken after each addition ; the temperature must be kept low so as to avoid any loss of volatile fatty acids. When quite cold 50 c.c. of light petroleum (sp. gr. 0-6-0-7 ; b.p. about 60° C.) are added, and the flask is then closed with a tightly fitting rubber stopper, and is thoroughly shaken for about ten seconds ; the shaking is repeated about thirty times at intervals of one or two minutes without removing the stopper. Saturated salt solu- tion is then added until the lower aqueous layer reaches up to the 240 c.c. graduation which is marked on the neck. After shaking again a few times the flask is set aside in a vessel of cold water. When the petroleum containing the fatty acids in solution has separated, 20 c.c. are withdrawn by means of a pipette and are placed in a wide-mouthed 150 c.c. flask; 40 c.c. of 96 per cent alcohol, free from acid, are now added, together with I c.c. of a solution of phenolphthalein (made by dissolving I gram of accurately weighed phenolphthalein in 100 c.c. of 96 per cent alcohol), and the solution is titrated with N/io alcoholic potash. The titrated liquid is then carefully transferred in small portions at a time to a tared weighing bottle of about 80 c.c. * Liebermann and Szdkely : " Pfliiger's Archiv," 189S, 72, 360. 2,^0 ■15 c<*»/.- FlG. 2. ESTIMATION 19 capacity, which is warmed over a gently boiling water bath ; when the whole liquid has been evaporated to dryness, the residue is heated in an air oven for an hour at 100°, and, after cooling in a desiccator, is weighed with the glass stopper in- serted to prevent the hygroscopic soap from absorbing any moisture from the air. The amount of fat which corresponds to a given weight of soap may be calculated as follows : — C^HsjCOOK — CH2 Ci,H,5COOCH2 C^HasCOOK - 3K + — CH - Ci,H3sCOOCH Ci,H35COOK — CH2 Ci,H35COOCH2 Soap Fat From the above equation it will be seen that in order to convert three molecules of soap into one molecule of fat, three atoms of potassium, 3 X 39-1, have to be withdrawn from three molecules of soap, and have to be replaced by 41 parts of CH2 . CH . CH2 ; this is equivalent to deducting 39-1 from one molecule of soap and adding V, or I3"6 ; or, in other words, deducting 25-5. Hence, if " n " is the number of centimetres of N/io caustic potash required for the titration, and since i c.c. N/io KOH = -00391 gram K = -00136 gram C3H5, we have to deduct from the weight of the soap Wg n X 00391 and add n x -00136 which is equivalent to deducting n X -00255. Also, since i c.c. of phenolphthalein solution on evapora- tion would leave 0-0 1 gram of solid, this quantity must be deducted from the weight of the soap. Hence the percentage of fat may be calculated from the relation F = rW.--OT-(nx -00255)1 ^ I m J in which " m " is the weight of the sample taken. In estimating fat in flour or farinaceous grain by this method, it is best to subject the substance to a preliminary treatment by heating 5 grams of the sample for half an hour with 30 c.c. of dilute sulphuric acid (i : lo), the mixture is 20 FATS, OILS, AND WAXES then diluted with 50 c.c. of 50 per cent caustic potash. Finally, the liquid is acidified with 60 c.c. of sulphuric acid (sp. gr. 1-3), as described above. After the shaking with light petroleum is completed, 50 c.c. of 94 per cent alcohol are added instead of the salt solution ; this has the effect of accelerating the separation of the petroleum layer which otherwise might take a long time. Owing to the relatively small solubility of stearic acid in light petroleum the method may give too low a result in the case of substances very rich in stearin ; the result should, therefore, be checked by a second estimation in which the number of shakings with petroleum are increased two or three fold. Leathes * has modified and considerably improved this method. Kumagawa and Suto f have found that the following method gives good results : Two to five grams of the dry sub- stance J are heated on a water bath for two hours with 25 c.c. of 5 N sodium hydroxide (20 grams in lOO c.c.) in a covered beaker. The mixture is then transferred to a separating funnel and acidified with 30 c.c. of 20 per cent hydrochloric acid. The fatty acids set free are taken up with ether, and the ethereal solution is filtered through asbestos and evaporated. The residue, which contains colouring matter, lactic acid, and other substances as well as fatty acids, is dried for some hours at 50°, and then taken up with light petroleum, where- upon the impurities separate out in resinous form. After filtering through asbestos the petroleum is distilled off and the residue, consisting of almost pure fatty acids, is dried at 50° to constant weight. QUANTITATIVE METHODS EMPLOYED FOR THE CHARACTERIZATION OF FATS. The following estimations are in common use for the characterization of fats : — * Leathes : " The Fats," London, 1926. t Kumagawa and Suto : " Biochem. Zeit.," 1908, 8, 212. I Yoshitaka Schimidzu ("Biochem. Zeit.," 1910, 28, 237) recommends using undried material since drj'ing leads to a loss of fat, probably from oxidation. QUANTITATIVE METHODS 21 (i) The Acid Number. This is the number of minigrams of potassium hydroxide re- quired for the neutrahzation of the free acids in a sample of fat. This number is determined by dissolving i or 2 grams of the sample in 15 or 20 c.c. of a mixture of I part of alcohol with 2 parts of ether, and titrating the solution with N/io alcoholic potash in the presence of phenolphthalein. (2) The Saponification Value. This is the number of milligrams of potassium hydroxide required for saponifying I gram of the fat. From I to 2 grams of the sample are weighed out into a 250 c.c. conical flask ; 25 c.c. of approximately seminormal alcoholic potash are then added, and the flask is attached to a reflux condenser and heated over a water bath for about half an hour ; the solution is then diluted with 25 c.c. of water and cooled, then the excess of potash is titrated back by means of N/2 hydrochloric acid. In order to determine the strength of the alcoholic potash, 25 c.c. of it are heated at the same time under exactly similar conditions in a second conical flask, but without any fat ; in this way any error due to the effect of the alkali on the glass vessel is eliminated. The difference in the two titration readings gives the amount of acid equiva- lent to the potash used up in saponifying the fat, from which the number of milligrams of alkali required for I gram of fat may be calculated. Since one molecule of any monobasic acid requires one molecule of potash, the magnitude of the saponification value is inversely proportional to the molecular weight of the acids contained in the fat. Molecular Saponification Weight. Value. Butyrin .... 302 557-3 Palmitin 806 2o8-8 Stearin 890 189-1 Olein . 884 190-4 Coco-nut oil . — 246-260 Palm-kernel oil * — 242-250 Palm oil t — T 96-202 Olive oil — 185-196 * The oil contained in the kernel of the palm fruit, t The oil contained in the pericarp of the fruit. 22 FATS, OILS, AND WAXES (3) U?isaponifiable Residue. The following method, originally due to Allen and Thomson, is recommended by Lewkowitsch for the estimation of the unsaponifiable residue. Five grams of the fat or oil are saponified by boiling under a reflux condenser with 25 c.c. of alcoholic potash containing 1 1-2 per cent of caustic potash for half an hour. The alcohol is then evaporated off and the residual soap is dissolved in 50 c.c. of hot water and transferred to a separating funnel of about 200 c.c. capacity, about 20-30 c.c. of water being used to rinse out the dish. After cooling, the mixture is shaken with 50 c.c. of ether and set aside until the ethereal layer has separated. The separation is accelerated by the addition of a little alcohol. The soap solution is then run off from below into a second separating funnel and shaken once more with a fresh quantity of ether. Two extractions should suffice, but it is safer to extract a third time. The ethereal extracts are then united, washed three times with 20 c.c. of water to remove any soap, and transferred to a weighed flask ; after evaporating oft" the ether, the flask is weighed again ; the increase in weight gives the amount of unsaponifiable residue in 5 grams of the sample. The isolation and identification of the unsaponifiable residue may be carried out for the purpose of establishing whether a given sample of fat or oil is of animal or vegetable origin, since animal fats contain cholesterol, while vegetable fats contain phytosterol (see p. 48). Fat. Unsaponifiable Residue Castor oil ... 033 P^r cent Linseed .... . G-42-II „ Olive oil . 0-46-I-0 ,, „ Maize oil . . I-35-2-86,, Oil from Pelvetia canaliculata * III „ Human fat 0-33 „ Lard .... 0-35 „ „ Beeswax .... 52-56 „ Authors' observations hitherto unpublished. QUANTITATIVE METHODS 23 (4) Iodine Value. It was first observed by Hiibl that an alcoholic solution of iodine containing mercuric chloride reacted at ordinary temperatures both with the free unsaturated acids and with their glycerol esters the fats. By elaborating this reaction, Hubl formulated the so-called " iodine value " which provides a method of characterizing a fat. For the determination of the iodine value of a fat the following solutions are required : — - [a) An iodine solution made by mixing together equal volumes of two solutions containing respectively 25 grams of iodine and 30 grams of mercuric chloride in 500 c.c. of 96 per cent alcohol. The two solutions should be mixed together about twenty-four hours before use, as the resulting mixture alters its strength considerably during the first few hours after it has been made. {h) A sodium thiosulphate solution containing roughly 48 grams of crystallized salt in i litre of water ; the strength of this solution is accurately determined as follows : 20 c.c. of a potassium bichromate solution containing 3"8657 grams of the pure salt dissolved in i litre of water are run into a stoppered bottle containing 10 c.c. of a 10 per cent solution of potassium iodide and 5 c.c. of concentrated hydrochloric acid. The re- sulting brown solution, if carefully made, should contain exactly 0-2 gram of iodine ; it is at once titrated by means of the thiosulphate solution, and, supposing x c.c. were required to decolorize it then it follows that l c.c. of thiosulphate is equivalent to — gram of iodine. [c) Chloroform or carbon tetrachloride, the purity of which should be tested by mixing 20 c.c. of it with 20 c.c. of the iodine solution and titrating the free iodine two or three hours after ; the amount found should be exactly the same as that contained in 20 c.c. of the iodine solution to which no chloro- form or carbon tetrachloride has been added. [d) A 10 per cent solution of potassium iodide made by dissolving i part of the iodide in 10 parts of water. 24 FATS, OILS, AND WAXES [e] A starch solution freshly prepared by boiling up a suspension of 0-5 gram of starch in 50 c.c. of water. The determination of the iodine value is carried out as follows : — From 0-15 to o-i8 gram of a drying or marine animal oil, 0-2 to 0'3 gram of a semi-drying oil, 0-3 to 0-4 gram of a non- drying oil or 0-8 to l-o gram of a solid fat are accurately weighed from a weighing bottle by difference into a 500-800 c.c. bottle, provided with a well-ground stopper, and dissolved in 10 c.c. of the chloroform {c) ; 25 c.c. of the iodine solution (a) are then run in, and the stopper, which is moistened with potassium iodide solution {d) to prevent loss of iodine by volatilization, is inserted. If a clear solution is not obtained more chloroform must be added. The bottle is then left to stand in the dark, and if the dark brown colour should disappear after two hours or less, another 25 c.c. of the iodine solution must be added, as it is essential that there should be a con- siderable excess of iodine. In the case of solid fats and non- drying oils the reaction can be considered as being complete after six to eight hours, but in the case of drying oils or fish oils twelve to eighteen hours are necessary. After the com- pletion of this time from 15 to 20 c.c. of the potassium iodide solution (d) are added, and, after thorough shaking, the mix- ture is diluted with 400 c.c. of water. If a red precipitate of mercuric iodide is produced, more potassium iodide solution should be added. The excess of free iodine, part of which is dissolved in the chloroform and part in the potassium iodide solution, is then titrated by shaking with the standardized sodium thiosulphate solution until only a faint yellow colour remains. A little of the starch solution is now added, and the titration is continued until the dark blue colour is destroyed. Twenty-five c.c. of the original Hiibl iodine solution, which had been left in a stoppered bottle with 10 c.c. chloroform and kept in the dark for the same length of time as the bottle containing the sample of the fat, are then titrated in a similar way with the sodium thiosulphate, and the difference in the two results gives the amount of iodine absorbed. The amount QUANTITATIVE METHODS 25 of iodine thus absorbed by lOO grams of the fat gives the iodine value. The values obtained by the Hiibl method are generally considered to be very reliable and concordant, but the method is somewhat tedious, and for this reason the more rapid method of Wijs * is preferable. The iodine solution required for this method is obtained by separately dissolving 9-4 grams 0/ iodine chloride and 7-2 grams of finely powdered iodine in separate flasks in about 200 c.c. of gently warmed glacial acetic acid. The two solutions are then united in a I litre graduated flask and made up to the mark with more glacial acetic acid. This solution should be standardized on the following day by mixing 20 c.c. of it with 10 c.c. of lO per cent potassium iodide solution and titrating the free iodine by means of the standard thiosulphate. The actual determination of the iodine value is carried out as follows : — From 0-2-0-4 gram of fat should be carefully weighed and dissolved in 10 c.c. of pure carbon tetrachloride (which has been shown by a blank test not to absorb iodine) ; 25 c.c. of the iodine solution are then added, and the flask is stoppered and set aside in the dark for one or two hours. The liquid is then transferred to a larger flask, the smaller flask being washed out thoroughly by means of 10 c.c. of potassium iodide solution and water until the total volume is about 300 c.c. The solution is then titrated with the thiosulphate. The difference between this reading and the amount required by 25 c.c. of the iodine solution is a measure of the iodine absorbed by the amount of fat. The values obtained by Wijs's method are, as a rule, rather higher than those obtained by the Hubl method. Appended is a list of iodine values of some important fats : — (a) Drying Oils — Linseed oil . * 173-201 Hemp-seed oil . . 148 Sunflower oil . . • 119-135 Pine-seed oil . • • 101-103 * Wijs : " Zeit. anal, Chem.,' ' 189S, 277: " Zeit. Unters. Nahr. Genussm.," 1902, 497. I04 -III io8 -no I03- •108 94- ■102 93-97 79- 88 96- 142 83- 90 32- 41 13- •17 8- •xo 26 FATS, OILS, AND WAXES (fe) Semi-Drying Oils — Beech-nut oil ... Cotton-seed oil . . . Sesame ..... Rape oil (colza) (c) Non-Drying Oils — Almond oil . Olive oil .... Grape-seed oil . . . Castor oil .... , {d) Vegetable Fats — Cacao butter .... Palm-kernel oil * . Coco-nut oil * (5) The Reichert Meissl Value. This represents the number of cubic centimetres of N/lO, caustic potash required for neutralizing the volatile acids liberated from 5 grams of a sample of fat under certain special conditions. The determination is carried out as follows : Five grams of the sample are weighed into a 200 c.c. flask and saponified by warming with 70 c.c. of 10 per cent alcohol and 2 grams of caustic potash. The excess of alcohol is then evaporated off and the residue, after dissolving in lOO c.c. of water, is acidified with 40 c.c. of sulphuric acid (l : 10) ; a few chips of asbestos are then dropped into the flask and the liquid is dis- tilled through a Liebig condenser at such a rate that exactly no c.c. of distillate pass over in an hour ; 100 c.c. of the dis- tillate remaining after filtration are titrated with N/io caustic potash in the presence of phenolphthalein. Appended are the numbers obtained for several different fats : — Palm oil 5-6-8 Lard . 0-68 Coco-nut oil . 6-6-7-0 Tallow 0-5 Linseed oil . o-o Goose fat 0-2-0-3 Olive oil 0-6 Butter fat , . 20-6-33-I The determination of the Reichert Meissl value is of considerable value for the detection of adulteration in butter, since any adulterant will at once lower the value. * Though described as oils, these substances are both solid at ordinary temperatures, melting at about 25°. QUANTITATIVE METHODS 27 (6) The Acetyl Value. This is a measure of the amount of hydroxyl groups which a fat contains ; its value depends upon the fact that compounds containing an alcoholic hydroxyl group react with acetyl chloride or acetic anhydride so as to replace the hydrogen of the hydroxyl by the acetyl group (CH3CO — ) as shown by the equation — ROH + ch'co>^ ^ ROCOCH3 + CH3COOH If the resulting acetyl derivative is saponified by means of caustic potash it breaks up as follows :■ — ROCOCH3 + KOH = ROH + CH3COOK, and it is possible to determine the number of milligrams of caustic potash which are thus utilized in combining with the acetyl groups to form potassium acetate. The number of milligrams of potash required for the saponification of the acetyl derivative obtained from i gram of the fat is termed the acetyl value of that fat. Castor oil and grape-stone oil have particularly high acetyl values which in the castor oil is due to the presence of the hydroxy acid known as ricinoleic acid. The following are the acetyl values of some of the more important oils, fats, and waxes : — Linseed oil . 398 Castor oil 153-156 Olive oil 10-64 Grape-seed oil 144 Rape-seed oil 14-7 Carnauba wax 55-24 Palm oil i8-o Lard . 2-6 Palm-nut oil . I-9-8-4 Butter I-9-8-6 The following method, due to Lewkowitsch, has been adopted as the standard process. About 10 grams of the fat are boiled in a round-bottomed flask under a reflux condenser for two hours with twice their weight of acetic anhydride. The mixture is then poured into a litre flask and boiled for half an hour with 500-600 c.c. of water, a slow stream of carbon dioxide being conducted into the liquid all the while to prevent bumping. After cooling, the upper layer of water is siphoned off and the lower oily layer is again boiled with water as above, the whole process 28 FATS, OILS, AND WAXES being repeated three times. The oil is finally filtered and washed on the filter paper with boiling water until the filtrate is no longer acid, whereupon it is dried in an oven and weighed. About 5 grams of the acetylated product are next saponi- fied by boiling with alcoholic potash * as described under the determination of the saponification value. The alcohol is then evaporated off, and the resulting soap is dissolved in water. Dilute sulphuric acid (i : lo) is then added in excess and the solution is steam distilled until 600-700 c.c. of water have passed over. The distillate is titrated with N/io caustic potash using phenolphthalein as indicator ; the number of cubic centi- metres required for neutralization multiplied by 5'6i and divided by the weight of fat taken gives the acetyl value. Further information regarding the nature of a given fat may be obtained by investigating the relative amounts of the saturated and unsaturated acids. This may be effected by saponification and conversion of the resulting soap into lead soaps by means of lead acetate ; making use of the greater solubility of the lead soaps of unsaturated acids in ether, these may be separated from the lead soaps of the saturated acids. The saturated and the unsaturated acids respectively may then be set free from their lead soaps and examined. SPONTANEOUS CHANGES IN FATS. Rancidity. — Most fats when exposed to air and light sooner or later become rancid, acquiring an unpleasant taste and smell. The actual cause of this change is as yet but little understood, though it appears probable that it is the result of the combined action of a number of different factors such as oxygen, light, moisture, bacteria and enzymes ; the complex fats, and possibly also the small quantities of proteins and other impurities contained in them, are thereby broken down into simpler bodies such as the lower volatile fatty acids and aldehydes. Similarly, but little is known as to the chemical changes involved in the process of becoming rancid ; it is frequently true that a considerable quantity of free acid is * Prepared by dissolving about 32 grams of go per cent stick potash in the least quantity of water and diluting to i litre with 96 per cent alcohol ; the solution should be filtered after standing for twenty- four hours. SPONTANEOUS CHANGES 29 liberated in fats which have become rancid, and this is especi- ally so in the case of fats such as butter, which contain acids of low molecular weight, as butyric acid, the smell of which recalls that of rancid butter. It is, however, a fact that a fat may be acid without being rancid ; * coco-butter, for instance, has usually an acid reaction, but very rarely becomes rancid. With regard to other constituents found in rancid fats, various authors have from time to time observed the presence of hydroxy-acids, aldehydes, alcohols, and of esters of lower fatty acids, and peroxides, but there appears to be a general consensus of opinion that glycerol does not occur. According to Fierz,f in the case of unsaturated fats, oxida- tion may take place at the double bond, in the absence of micro-organisms, with the formation of aldehydes and acids of lower molecular weight which, like butyric acid, have an offensive odour and taste. On the other hand, saturated fats become rancid under the action of Penicilliuni glaucum and Aspergillus niger with the liberation of various odoriferous ketones. This is due to the oxidation of the j8 carbon atom according to the scheme — RCH2CH2 . CH2 . COOH + O -^ RCH^CHOH . CH^COOH + O -> RCHjCO CH2COOH + HjO the latter acid by loss of carbon dioxide giving a ketone— RCH2 COCH2COOH = RCH2 COCH, + CO.. By this means caproic, caprylic, and myristic acid, which occur as esters in coco-butter, may be regarded as the precursors of methyl amyl, methyl heptyl, and methyl undecyl ketones respectively, and which have been shown to occur in rancid coco-butter. These compounds have been experimentally produced from their respective precursors by growing Penicilliuni glaucum and Aspergillus niger upon the ammonium salts of the relative acids. Methyl heptyl ketone has been isolated from Roquefort cheese. Drying and Resinification. — Most fatty oils on exposure to * Vintilesco and Popesco : " J. Pharm. Chim.," 1915 [iv.], 12, 318. f Fierz : " Z. angevv. Chem.," 1925, 38, 6. 30 FATS, OILS, AND WAXES the air tend to thicken, owing partly to polymerization and partly to oxidation ; in some cases the oil actually dries up, leaving a more or less hard mass or a thin elastic film. Those oils which only thicken, without actually becoming hard or dry, are called non-drying oils. They are composed for the most part of triolein (cf. p. lo), and contain only small quantities of solid fatty acids ; to this class of oils belong the following : olive oil, almond oil, arachis or pea-nut oil, quince oil, cherry-, plum-, peach-, and apricot-kernel oil, wheatmeal oil, rice, tea-seed oil, and hazel-nut oil. Two further oils, namely, castor oil and grape-seed oil, are also included in this group of non-drying oils, but they have a slightly different composition from the other members of this group. They are characterized by possessing a consider- able percentage of glycerides of hydroxylated fatty acids, such as dihydroxystearic acid, a fact which is brought out clearly by their high acetyl values (p. 27). In contrast with these non-drying oils are the so-called drying oils, among the more important of which are the follow- ing : linseed oil, cedar-nut oil, hempseed, walnut, poppy-seed, and sunflower oil. These oils exhibit to a greater or less degree the tendency to absorb oxygen from the air, thereby drying up and leaving an elastic skin, a property which is made use of industrially in the manufacture of oil paints. These drying oils are composed chiefly of the glycerides of the unsaturated acids of linolic and linolenic series and contain only relatively small quantities of oleic acid. Owing to the large amount of unsaturated acids which they contain, their iodine value (p. 23) is very high (120-200). In addition to the above there is also a third group of vegetable oils, known as the semi-drying oils, whose iodine value and drying properties lie midway between those of the drying and non-drying oils. They differ from the true drying oils in containing no acids of the linolenic series, and from the non-drying oils in containing linolic acid. The oils belonging to this category fall naturally into two sub-groups : — I. The cotton-seed oil group, to which belong Soja-bean oil, maize oil, pumpkin, water-melon, and melon-seed oils, INDUSTRIAL USES 31 beech-nut oil, cotton-seed, sesame and croton oils, and the lesser-known oils of the apple, pear, orange, barley, and rye seeds. 2. The rape oil group comprising garden cress, hedge mustard, wild radish, black mustard seed, white mustard seed, radish seed, and rape or colza oil. The oils of the latter sub-group have a lower saponification value (p. 21) than any other vegetable oils, and arachidic acid seems to be a normal constituent of them all. To determine whether an oil is a drying one or not, a drop is spread on a glass plate, such as a microscope slip, and left for several days at atmospheric temperature. Non-drying oils such as olive and castor oils are unaltered after about eighteen days ; semi-drying oils such as cotton-seed, sesame, and rape oil are more or less dry, but still sticky in from seven to eight days, whereas real drying oils, like poppy and especially linseed, are quite dry in from three to six days. The mechanism of the process of drying is very imperfectly understood ; it would appear to be in part a chemical change involving oxidation, with the resulting formation of a substance known as Linoxyn,* and partly a physical change. f INDUSTRIAL USES OF VEGETABLE FATS AND OILS. Economically, fats are of considerable value, being used for food, illumination, lubrication, soap manufacture, and for a variety of other purposes. The following is a brief consideration of some of the more important industrial uses of the commoner fats and oils of vegetable origin. Olive Oil is extracted from the fleshy pericarp of the fruit of the olive, Olea europcsa, by pressure. The best quality oil, which is expressed without the application of heat, is used for food ; lower grade oils, obtained by extracting the residues from the presses with fat solvents, such as carbon disulphide or light petroleum, arc used in the manufacture of soap (see P- 33)- * Holden : " J. Soc. Dyers and Col.," 1927, 43, 157. t Wolff : " Chem. Zeit.," 1924, 48, 897. 32 FATS, OILS, AND WAXES Cotton-seed Oil is extracted from the seeds of Gossypium herbaceum by pressing them at a temperature of about 90° ; the crude brown oil is purified by treatment with caustic soda, which removes the free fatty acids, colouring matter, and other impurities. After purification the oil is light yellow in colour. It is used for the manufacture of soap and rubber substitutes. Coco-nut Oil is obtained from the ripe seeds of Cocos nucifera and Cocos butyracea by pressure ; the dried endo- sperms, known as Copra, are imported into Europe, and the oil extracted from them is commonly known as Copra oil. Soaps made from coco-nut oil have the property of absorbing large quantities of salt solutions, and can therefore be used for washing with sea water. Palm Oil which occurs in the fruit of Elaeis guineensis is, when pure, a colourless substance of the consistency of lard ; on exposure to air it readily turns yellow, but the colour can be removed by oxidation by means of a current of air. Both coco-nut oil and palm oil in the crude state contain free fatty acids which can, however, be removed by treatment with alcohol. When so purified they are employed in the manu- facture of margarine. Rape Oil or Colza Oil is a thick, yellowish oil obtained from the seeds of Brassica Rapa and Brassica Napus which is used as an illuminant. By drawing a current of air through the oil heated to 70° a so-called " blown " oil is produced, the specific gravity of which becomes almost equal to that of castor oil, namely 0'97 ; in this condition it is miscible with mineral oils. The mixture which is known as marine oil is used for lubricating marine engines. Linseed Oil is obtained by pressing the seeds of Linum usitatissimum either with or without the application of heat ; the residues after compression are made up into cattle food. The drying vegetable oils, particularly linseed oil, are used in the manufacture of oil paints as vehicles for the pigments ; for artist's white paints, walnut and poppy-seed oils are some- times used. The drying properties of linseed oil used for the manufacture of paint are greatly increased by boiling with lead INDUSTRIAL USES 33 oxide ; such oil is known as boiled oil. A similar effect may be produced by dissolving in it certain salts known as " driers," such as lead linoleate or the metallic salts of resin acids, etc. Varnish consists of a mixture of boiled oil with gum resins and oil of turpentine. Castor Oil is obtained by compressing the seeds of Ricinus communis either with or without the application of heat. The seeds contain a fat-splitting enzyme * or lipase which is employed commercially for the hydrolysis of fats ; they also contain a very poisonous toxalbumin, known as Ricin, which remains in the residues after the expression of the oil. Castor oil is a thick viscid colourless liquid ; when heated above 280° it decomposes with the formation of oenanthol, a substance having a very unpleasant odour. Castor oil is largely used in the dye industry ; for this purpose it is converted into the so-called turkey red oil, used for alizarin dyeing, by treatment with sulphuric acid and neutralization of the resulting sulphonic acid with soda. For the manufacture of hard toilet soap the following fats and oils are used : tallow fat, palm oil, palm-kernel oil, coco-nut oil, and olive oil ; the fats are boiled with caustic soda until saponification is complete, whereupon the mixture is saturated with common salt. The soap, being insoluble in strong salt solution, rises to the surface leaving the glycerol and salt in the aqueous layer below ; the latter is then run off and the scum, which is allowed to harden in moulds, is known as hard soap. Soft soaps are prepared by boiling the cheaper oils, such as hemp-seed oil, cotton-seed oil or linseed oil with caustic potash ; when saponification is complete the mixture is allowed to set to a semi-solid without the addition of sodium chloride ; the resulting mixture contains all the gycerol together with the excess of alkali and a quantity of water. Most of the gycerol of commerce is obtained from fats ; it is used largely for the manufacture of dynamite. * The occurrence of a lipase is common to most fatty seeds, but the only one commercially utilized is that of the castor bean, on account of its high concentration and activity. 3 34 FATS, OILS, AND WAXES Hardening of Oils.— Many low-melting fats or oils are nowadays hardened by treating them with hydrogen in the presence of a nickel catalyst ; the process of hydrogenation involves the removal of the double bonds of saturation with hydrogen, the resulting saturated compound having a higher melting-point. PHYSIOLOGICAL SIGNIFICANCE OF FATS. The great function of fats in the economy of the plant is connected with nutrition. They form one of the most im- portant food-reserves of plants and as such may occur in vegetative or in propagative organs. It is, however, not possible to ascribe this function to all instances of fat occurrence. Thus, in the case of the palm Elaeis guineensis, two distinct types of fat occur ; the one in the pericarp, the palm oil of commerce, and the other in the testa adjacent to the embryo. Apart from the fact that these two fats are different, the former being of the nature of tallow and containing palmitic, stearic, and other fatty acids, and the latter containing acids of a lower molecular weight, it is difficult to see what nutritive purpose a fat occurring in the pericarp can serve in view of the fact that it is destroyed before germination actually begins ; it has, moreover, been shown that germination is hastened if the pericarp is removed prior to planting. Similar considerations also apply in the case of the olive. With regard to their origin in plants very little is known ; they first appear as very small vacuoles in the protoplasm which eventually run together forming large drops. In some cases oil has been described as owing its origin to the activity of elaioplasts, which are colourless bodies of various shapes usually grouped around the nucleus, and, like other plastids, of a protoplasmic nature. They are, or have been, supposed to act with regard to oil formation much as leucoplasts do with respect to starch formation. Elaioplasts have been observed in many Monocotyledons such as Vanilla, Funkia, Gagea, OrniOiogalum, etc., in the ffower of a Dicoty- ledon, Gaillardia Lorenziana, and in Psilotum. PHYSIOLOGY 35 The development of the elaioplasts of Gaillardia has been followed by Beer,* who found that they are formed by the aggregation of chloroplasts which then degenerate and give origin to the oil. He considers it is most unlikely that elaio- plasts perform any function of direct importance to the life of the plant, although they may in some cases, the corolla-hairs of Gaillardia, for instance, serve a biological purpose. Elaioplasts are not, by any means, always present. Rivett f from her study of Alicularia scalaris, a liverwort, concludes that in this instance the fat originates as a general proto- plasmic secretion, not from an elaioplast or other special body. It is a secondary product, its production being unaffected by changes in the cultural conditions brought about by variations in illumination, temperature, and nutritive materials. Although elaioplasts may not perform the function origin- ally ascribed to them, it does not necessarily follow that fats, more especially when occurring in the green parts of plants, may not be direct photosynthetic products. Thus Fleissig considers that in the case of Vaucheria, the abundant fat-like substance is a direct photosynthetic product com- parable to the starch and sugar in ordinary green leaves. On the other hand, it is possible that the fats in such cases may have been produced by secondary changes in the original product of photosynthesis. The fat-economy of Vaucheria, however, requires further investigation ; thus Meyer % states that the oil drops are produced by the chloroplasts and result from the photo- synthetic processes ; they are not, however, fats in that they do not give characteristic microchemical reactions. Similarly the oil bodies described as occurring in the mesophyll of- Ilex, Kalmia, Taxus, Tropceohim, and Vinca, which increase in size with the age of the leaf, do not give characteristic fat reactions. Mangenot § describes two kinds of oil drops in Vaucheria : spherical drops of various sizes associated with * Beer : " Ann. Bot.," 1909, 23, 63. t Rivett : id., 1918, 32, 207. J Meyer : " Ber. deut. bot. Gesells.," 1917. 35, 586 ; 1918, 36, 5, 235, 674. § Mangenot : " Compt. rend. soc. biol.," 1920, 83, 982. 3* 36 FATS, OILS, AND WAXES chloroplasts and which he considers to be the first visible product of assimilation, and very much smaller globules, suggesting microsomes, distributed throughout the cytoplasm. In many cases there can be but little doubt that fats are produced from carbohydrates ; the work of Schmidt,* Le Clerc du Sablon,-}" and others has shown that as the carbohydrates disappear so fats appear. For example, in the case of the almond the seeds when they begin to ripen are rich in carbo- hydrates and poor in fats, whereas the reverse is true when they are fully matured. The same holds true for the seeds of Ricinus and Pceonia. The nature of the carbohydrates used up in this process varies in different plants ; thus it is stated that in the olive mannitol replaces the carbohydrate. This statement, due to de Luca, is not accepted by other investi- gators of the same plant ; according to Funaro mannitol does not appear until after the oil has been formed. " In the case of Ricinus seeds the oil is formed from glucose, and in Pceonia principally from starch. The facts that fat may be translocated as such, provided it be an emulsion sufficiently fine, or in the form of fatty acid and glycerol, suggest that the fats in seeds have not been formed in situ, but have been con- veyed there. This may be true to a certain extent, but con- sideration of the fact that fat will appear as the carbohydrates disappear in immature seeds removed from the parent plant, together with the facts relating to the formation of fats in vegetative organs under the influence of cold (p. 3), leads to the conclusion that the substances in question are formed at the expense of carbohydrates. Further, corroborative evi- dence is afforded by well-ascertained facts relating to similar problems in animals. Ivanow,J experimenting with rape seed, has shown that they contain a lipase which may either hydrolyse a fat or may synthesize one from fatty acid and glycerol. Thus, if a glycerol extract of the seed be mixed with oleic acid, fat is * Schmidt : " Flora," 1891, 74, 300. t Le Clerc du Sablon : " Compt. rend.," 1893, "7> 524 ; 1894, 119, 610 ; 1896, 123, 1084 ; " Rev. Gen. Bot.," 1895, 7, 145 ; 1897, 9, 313. X Ivanow : " Ber. dcut. bot. Gesells.," 1911, 29, 595. PHYSIOLOGY 37 synthesized, but, on diluting with water, the fat is spHt up again. This same author * has pubhshed important observa- tions on the synthesis of fats in oily seeds mainly from the carbohydrates glucose, sucrose, and starch. These substances are synthesized in the order given, the last two being first hydrolysed. The initial acids to be formed are characterized by a low iodine value, showing that they are saturated. Further, since the Reichert Meissl value is constant and does not vary with the acid number, it is concluded that the acids first formed belong to the higher members of the fatty series. The saturated acids are followed by the unsaturated. Ivanow gives the following scheme to indicate the essential stages in the synthesis of fat in a typical instance such as the seed of flax : — /Glycerol \ Carbohydrates >Fat. ^Saturated- fatty acid. ■Unsaturated- fatty acid. The iodine value of a fat is not necessarily constant, as is shown by the observations of Eyre "j* who found that this value steadily increased during the formation of the seed of the flax. Days after Percentage of Fat Flowering. in Dry Seeds. ID 2-5 114 14 I5-I 119 17 3I-I 127 23 37 M3 28 37 170 35 39 180 51 363 190 The extracts of young seed have a high content of free fatty acid, which rapidly decreases as the seeds develop. This in- dicates that the glycerol appears later than the fatty acid, or else the combination of the glycerol and fatty acid is impeded by some factor. After the fourteenth day there is a rapid and more or less * Ivanow : " Beih. bot. Centr.," 1912. 28, 159. t Quoted by Armstrong and Allen ; " J. Soc. Chem. Ind.," 1924, 43, 207 T. 38 FATS, OILS, AND WAXES parallel increase in the amounts of carbohydrates, proteins, and fats. During the germination of oily seeds a reversal of this process takes place. The work of Schmidt, Green,* Le Clerc du Sablon, and others, has shown that the first process is that of hydrolysis which splits the fat into a fatty acid and glycerol, lipase being the active agent. Thus in the sunflower Miller f found that less than i per cent of free fatty acid was present in the oil of the cotyledons of the resting seed ; as germination proceeded there was a gradual increase, thus the ether extract of the cotyledons of a seedling in which the plumule was just showing contained 30 per cent of fatty acid. The presence of the acid may be demonstrated in such germinating seeds, but the same statement does not hold for glycerol, probably because it is translocated with great rapidity, and is quickly transformed. There can, however, be no doubt that this substance is formed because if, for example, castor oil be subjected in vitro to the action of lipase obtained from Ricinus seeds, the presence of glycerol may be detected with ease. With regard to other changes which the original fat under- goes during germination, Schmidt found that the iodine number of the unsaturated acids and oils decreased during germina- tion, which indicates that saturation of the acid radicles takes place. This is controverted by von Furth,J who found no change in the iodine value. The observations of Schmidt, however, have been corroborated by Miller, who found that in Helianthus annuus the iodine value decreased from 136-2 for the seed to 67*4 for a seedling with the plumule just elongating. Further corroboration is given by Ivanow § who, for his study on the transformation of fats during germination, selected flax, hemp, rape, and poppy seeds, since each is characterized * Green : " Proc. Roy. Soc, Lond.," 1890, 48, 370. t Miller : " Ann. Bot.," 1910, 24, 693. X Von Furth : " Hofm. Beitr. Chem. Phys. Path.," 1904, 4, 430 ; 1912, 26, 889. § Ivanow : " Jahrb. wiss. Bot.," 1912, 50, 375. PHYSIOLOGY 39 by the possession of fats rich in acids of a specific series. Thus the oil of hemp seed is rich in acids of the unsaturated linolenic series, whilst poppy-seed oil is rich in acids of the saturated fatty acid series. By ascertaining the iodine and other values of the fats of these seeds at different periods of germination, it was found that the acids disappeared in the sequence linolenic, linolic, oleic, and, finally, palmitic ; in other words, the acids were consumed at a rate inversely proportional to their degree of saturation. Ivanow considers that the fall in the iodine value of the fats is due rather to the rapidity with which the more un- saturated fatty acids are used up in the formation of carbo- hydrates rather than to their oxidation. He further found that the saturated fatty acids not uncommonly exist in a free state whilst the unsaturated acids occur in the form of glycerides. Von Fiirth * also found that during germination of Ricinus, the acetyl value decreased from 87-5 in the resting seed to 50-5 in the young seedling, from which he concluded that the normal fatty acid does not change into hydroxy fatty acid. Also, he could find no proof of the fatty acid breaking down into simpler substances as indicated by the molecular weight re- maining practically constant. This hydrolysis is the first action, but it is not the final one since carbohydrates quickly appear during the germination of such seeds. Since the days of de Saussure, who was the first to draw attention to this phenomenon, much evidence relative to this carbohydrate formation has accumulated. In the case of Ricinus le Clerc du Sablon found that the resting seed contained 69 per cent of oil and 4 per cent of sugar, but in a seedling 1 1 cm. high the oil had fallen to 1 1 per cent and the sugar had risen to 14 per cent. It was further found that the sugar contained in the resting seed has a slight excess of non-reducing sugar, which increased more rapidly than the reducing sugar ; finally, how^ever, the latter variety preponderated. * hoc, cit. ^ 40 FATS, OILS. AND WAXES Le Clerc du Sablon also found the same relation between oil and sugar to obtain during germination of rape, hemp, poppy, almond, and walnut. Similar observations have been made by Green and Jack- son,* who found that in the resting seed of Ricinus the most abundant sugar is sucrose, which gives place to invert sugar in the early stages of germination. Subsequently the sucrose increases in amount, and occurs in quantities greater than the invert sugar ; thus there is reason for supposing that the sucrose is a temporary reserve food. The following table which summarizes the changes in the sugar content is taken from Green and Jackson's paper : — Time of germination Invert sugar Cane sugar in hours. in milligrams. in milligrams. O I-I 10-7 45 2-7 5-17 69 2-3 117 6-7 19-4 168 5-2 IO-5 216 19-5 35-7 240 29-01 35-8 312 40-8 52-6 Miller has found that in the sunflower, Helianthus annuus, the amount of ether extract of the cotyledons diminishes gradually from the beginning of germination, the most rapid depletion occurring during the period between the first ap- pearance of the seed-leaves above ground and the point of full expansion. Also, the greatest increase in the hypocotyl and roots coincides with the period of maximum depletion from the seed-leaves. With regard to the sugar content, Miller states that the resting embryo contains about 4 per cent of sucrose, during germination there is a decrease, and this is followed by a gradual increase until the seed-leaves begin to unfold. Up to this stage the cotyledons contain only a non- reducing sugar, but as the seed-leaves assume the functions of foliage leaves a reducing sugar appears, and, in a short time, is the only sugar present. In the hypocotyl and roots * Green and Jackson : " Proc. Roy. Soc, Lond.," B., 1906, 77, 69. PHYSIOLOGY 41 the amount of sugar rapidly increases until in seedlings about 4 inches long it may amount to 20 per cent of the dry weight, then a gradual decrease takes place. There is also a small increase in the amount of starch. The nature of the carbohydrate differs in different plants ; thus in addition to the above-mentioned plants, during the germination of Allium and of Cucumis much glucose makes its appearance ; this is also true, although to a lesser degree, for Cannabis saliva, in which case the glucose is quickly trans- formed into starch. In other instances starch is said to be the carbohydrate formed. The consideration of the formulae of the substances in question shows that fats poor in oxygen give rise to carbo- hydrates rich in oxygen, and vice versa ; but as to how this is accomplished nothing of a definite nature is known. Many suggestions have been put forward, and before mentioning these the reader may be reminded of the large amount of oxygen which is absorbed during the germination of oil-containing seeds. Detmer considered that starch may arise from the free oleic acid according to the equation — C18H34O2 + 27O = 2(C6Hio05) + 6CO, + 7H2O. This change is supposed to be effected by the oxidation of the chain at the double bond setting free two unsaturated groups which by polymerization give rise to sugar. These conclusions are based on the observations that during the germination of the seeds of Arachis the carbo- hydrate increases to 5-6 per cent of the dry weight, whilst in Ricinus the increase is 16 per cent. The glycerol of the fat would be sufficient to form about 5 per cent of carbohydrate ; this roughly was the amount observed in the case of Arachis, whereas in Ricinus the amount of fat was about three times as great. It has already been mentioned that glycerol so far has not been demonstrated in germinating fatty seeds ; this may be owing to its powers of rapid diffusion or to the fact that it is used up in the synthesis of other substances. 42 FATS, OILS, AND WAXES Le Clerc du Sablon has put forward the idea that there might be present an enzyme which acts on the fat without liberating the glycerol. These views are concerned chiefly with the formation of carbohydrates from fats ; a reversal of the process might or might not explain the formation of fats from carbohydrates. The whole question is of considerable difficulty and refuge may be taken in the hypothesis first put forward by Nageli that the fats are products of the disintegration of the proto- plasm. Thus the carbohydrates might be assimilated by the protoplasm which might produce the oil by some catabolic process. With regard to the possible formation of fats from proteins very little information is available. On the animal side there is some evidence to show that substances derived from pro- teins may be so utilized ; a possible connection may be found in the phospholipines (phosphatides) which are compounds of fatty acids containing either nitrogen or phosphorus, or both. Leathes * points out that the fatty acid may be formed from glucose by processes analogous to the synthesis of butyric acid from lactic acid which in turn is formed from the glucose. For the underlying reasons, which are rather too complicated to be dealt with here, Leathes' monograph must be consulted. It may, however, be pointed out in this connection that the investigations of Hanriot are very significant ; he found that, in attempting the oxidation of fat in vitro, 15 per cent of its weight of oxygen was absorbed, and in the products of its oxidation butyric and acetic acids occurred, but no carbo- hydrate. In conclusion brief mention may be made of Schmidt's views regarding the translocation of fats. He considers that in many cases the oil may be transported as such to those organs requiring it, for he found that the amount of fatty acid present in the germinating seeds was smaller than would be supposed if it were hydrolysed before translocation, also that neutral oil appears in regions of the plant removed from the storage organ. * Leathes : " The Fats," London, 1926. PHYSIOLOGY 43 He considers the walls of cells are permeable to oil ; pro- vided it be an emulsion sufficiently fine, and especially if a free fatty acid be present, the permeability being directly propor- tional to the amount of such acid present. It is thought that the acid forms a soap in the walls, and thus facilitates the passage. It is not improbable that both methods are adopted by the plant, viz. the translocation of the products of the dissociation of the fat, and the translocation of oil qua oil. With regard to the significance of fats in the construction of cell membranes, Hansteen-Cranner * has drawn attention to the occurrence of fatty substances in the cell walls of young plants of Ricinus, Vicia, and other plants, which substances he considers to occur in the form of soaps. He regards the cell wall as a hydrogel complex, the more solid phase of which is made up of the colloidal cellulose together with pectin and soap. The matter has been pursued by Priestley f and his fellow- workers who point out that the extent to which fat compounds are held in the cell wall depend on various factors amongst which the relation between calcium, which forms an insoluble compound with soap, on the one hand, and potassium and sodium, which form more soluble compounds with soaps, on the other, appears to be all important. In soils poor in calcium the fats remain in the cell membrane in a more mobile condition and diffuse more freely towards the surface as is indicated by the thick cuticle and more suberized layers of the endodermis growing in acid soils. The deposition of fat within the cell membrane also is conditioned by the reaction of the tissue ; thus in the root, the phloem, on account of its alkaline reaction, would appear to free itself from fat within its membranes as is indicated by the fact that the formation of the cas- parian strip and suberin lamella of the endodermis, both of which structures are formed in part from fatty acids, occur opposite the phloem before they are formed opposite the xylem rays. The position of these deposits, in the cuticle, or in the * Hansteen-Cranner : " Jahrb. Wiss. Bot.," 1904. 53, 536 ; " Ber. deut. bot. Gesells.," 1919, 37, 380. t Priestley : " New Phyt.," 1924, 23, i, and the literature there quoted. 44 FATS, OILS, AND WAXES walls of the endodermis, exodermis, or cork, depend upon a variety of factors amongst which the ratio between calcium and potassium and sodium would appear to be important. Their fate, however, is the same ; the unsaturated fatty acids undergo oxidation and condensation resulting in a waterproof layer, the fat constituents of which are no longer soluble in fat solvents. MICROCHEMICAL REACTIONS. 1. The microscopical appearance of oil when mixed with water is characteristic owing to its immiscibility with water and its different refractive index. 2. Its solubility in ether, chloroform, benzene, or other fat solvents is easily noted. 3. If oil be present in the preparation it will fairly rapidly turn brown and then black when treated with a I per cent solution of osmic acid. This is not absolutely conclusive since osmic acid stains proteins brown. 4. Tincture of alkannin, or a saturated solution of Scharlach R in 75 per cent alcohol, colours oil globules red or pink. The reaction with the first-named reagent is often ill-defined and frequently fails when the alkanna used has been extracted from the root some time. The test is more satisfactory when freshly prepared tincture is used. A similar reaction is given by Sudan III. It is important to note that these and similar reactions are not conclusive of the chemical nature of the substances acted upon. For example, Sudan III not only stains oils red but also resins, latex, wax, and cuticle ; chloroplasts are stained a pale red ; cellulose, lignified walls, gelatinized membranes, starch, and tannin are unstained. The staining tests mentioned above may be employed after extracting the oil with ether or other solvent. WAXES. The chief function of waxes in plants is to form a protec- tive covering against undue evaporation of water. They are found most commonly in or on the cuticle of leaves and fruits where they give rise to the glaucous effect. PROPERTIES OF WAXES 45 As already stated, the waxes resemble the fats in their chemical constitution in so far as they are esters, but they differ in the nature of their alcohol constituent which is not glycerol but is usually a monohydric alcohol such as cetyl alcohol CigHogOH, carnaubyl alcohol C24H49OH, pisangceryl alcohol C24H49OH, ceryl alcohol CaeHjgOH, myricyl alcohol CgoHfiiOH, cholesterol or phytosterol C27H45OH. In addition to the acids already mentioned as occurring in fats, the following are also met with in waxes in the form of esters : ficocerylic acid CigHgeOo, carnaubic acid C24H48O2, and , pisangcerylic acid C24H48O2, as well as acids belonging to series of the general formula Ci)H2„ _ 2O2 and CyHanOs. The term wax used in the chemical sense has reference only to the chemical composition of these substances, regardless of their physical state of aggregation, and consequently both liquid and solid waxes are known. Waxes of the former class are, however, only known in the animal kingdom, they are ordinary sperm oil and arctic sperm oil. Among the better-known vegetable waxes may be men- tioned : — [a] Carnauha Wax obtained from Copernicia cerifera ; this wax contains ceryl and myricyl alcohols, and two acids, cerotic acid CosHgaOg, and carnaubic acid C24H48O2, together with a hydroxy-acid of the formula C21H42O3. This is a very hard wax and is used in the making of gramophone records. {b) Pisang Wax obtained from the leaves of Cera musae is the pisangceryl ester of pisangcerylic acid. The following are some of the more important waxes of animal origin :- — Wool wax, better known as wool fat or lanolin (which is rich in cholesterol), beeswax, spermaceti, and Chinese insect wax. PHYSICAL AND CHEMICAL PROPERTIES OF WAXES. Waxes are soluble in all the ordinary fat solvents such as benzene, ether, chloroform, etc., though they are rather less soluble than the fats. 46 FATS, OILS, AND WAXES Being free from glycerides the waxes, when heated, give 110 sfnell of acrolein ; they do not become rancid like the fats, and are less easily hydrolysed, but they can be decomposed by prolonged heating with alcoholic potash. Owing to the high molecular weight of their constituent acids, the saponification value of waxes is low. Saponification Value. Carnauba wax ...... 79-95 Waxes are further characterized by giving abnormally high values for the unsaponifiable residue. WAXES. As already stated (p. 22) all fats and waxes on saponifica- tion with caustic alkalis yield a certain amount of substance, known as the unsaponifiable residue, which is insoluble in the alkaline solution remaining after hydrolysis and may be ex- tracted therefrom by means of ether. This material in the case of fats is composed chiefly * of a group of alcohols known as sterols, while in the case of waxes it will include in addition the higher saturated alcohols. The sterols may occur in the uncombined state in fats, or combined with fatty acids as esters. The sterols form a group of highly complex hydro-aromatic monohydric secondary alcohols whose constitution has not as yet been completely determined. They fall into two main groups, the cholesterols and the phytosterols which are characteristic of the animal and vegetable world respectively. REACTIONS AND PROPERTIES OF CHOLESTEROL AND PHYTOSTEROL. Cholesterol. Cholesterol is a monohydric alcohol of the formula C27H45OH ; its constitution is still unknown, although a great deal of work has been expended on this question ; * It may be mentioned that the unsaponifiable residue of fats contain also the fat soluble vitamin A when this substance is present. CHOLESTEROL 47 it would appear to be a secondary alcohol containing an un- saturated group. Cholesterol has a constitution probably represented by the formula * — CH., H,C CH I C,,H,9 . CH, . CH(CU,), HC CH I H.,C C CH., 'Ill' HoC CH CH (HO)HC CH Cholesterol occurs in the bile, certain gall stones, brain, blood, and wool fat. It is insoluble in water and crystallizes from chloroform in needles and from ether or alcohol in rhombic plates, m.p. 148-150°. It may conveniently be ob- tained by evaporating the ethereal extract of gall stones to dryness. Reactions. — i. Crystals of cholesterol pressed on a white porcelain surface and moistened with a drop of sulphuric acid (5 parts concentrated acid to i part of water) turn pink. The addition of a drop of dilute iodine causes a play of colours from red to blue or green. 2. A solution of cholesterol in chloroform gently agitated with concentrated sulphuric acid turns red, while the sulphuric acid which forms the lower layer assumes a green fluorescence. 3. On the addition of concentrated sulphuric acid drop by drop to a little cholesterol dissolved in a mixture of 2-3 drops of chloroform and about 10 drops of acetic anhydride, a transient pink colour is at first formed ; on the addition of more acid, however, the colour changes to blue and finally to green. 4. Alcoholic solutions of cholesterol mixed with a few drops of I per cent alcoholic solution of digitonin,t give an immediate white precipitate, C27H46OC54H92O28, a reaction employed in the estimation of cholesterol. J *Windaus: "Annalen," 1926,447, 233. t Panzer : " Chem. Zentr.," 1912 (ii.), 540. J Windaus : " Ber. deut. chem. Gesells.," 1909. 42, 238 ; " Zeit. physiol. Chem.," 1910, 65, no; Salomon: "Ber. deut. pharm. Gesells.," 1914' 24, 1 89. 48 FATS, OILS, AND WAXES Phytosierols. The term phytosterol was at one time employed to designate a definite chemical individual of the formula C27H45OH, but it is now used more as a generic term to include a number of different substances having certain properties in common. Thus Windaus and Hauth * showed that the substance obtained from Calabar beans and commonly known as phytosterol was in reality a mixture of two substances— {a) Sitosterol of the formula C27H45OH, and {h) Stigmasterol C30H47OH, an observation which has been confirmed by Salway. f Similarly Klobb % describes a dextro-rotatory phytosterol of the formula C31H52O, 3H2O occurring in Anthemis nohilis and a number of Isevo-rotatory phytosterols of different formulae obtained from Matricaria Chamomilla, Tilia europaea, Linaria vulgaris, and Verhascum Thapsus.^ All vegetable fats contain phytosterol, the amount varying from about 0-13 to 0-30 per cent and rising in the case of pea fat and the fat of Calabar beans to a considerably higher value. In the case of the wheat, the grain of which contains sitosterol whilst the bran contains a different phytosterol, the amounts of these substances differ in the various parts of the plant. The percentage present in the green parts is higher than the percentage occurring in the grain which is somewhat greater as compared with the percentage in etiolated plants. The fact that the highest percentage occurs in the embryo suggests a function in connection with germination and growth ; not necessarily a direct nutritive function since a starved plant contains as much as the grain. || The sterols are widely distributed in the vegetable * Windaus and Hauth : " Ber. deut. chem. Gesells.," 1906, 39, 4378 ; 1907, 40, 3681. t Salway : " J. Chem. Soc," 191 1. 99' 2154. J Klobb : " Compt. rend.," 1911, 152, 327; "Ann. Chim. Phys.," 1911, viii., 24, 410. § See also Power and Rogerson : " J. Chem. Soc," 1910, 97> i95i ; Rogerson : " Amer. Journ. Pharm.," 191 1, 83, 59 ; " J. Chem. Soc," 1912. loi, 1040. li Ellis : " Biochem. Journ.," 1918, 12, 154, 160, 173. PHYTOSTEROLS 49 kingdom : in addition to the higher plants, they occur in Sphagnum, Pelvetia, Laminaria, Agaricus, Lactarius, and Polyporus* Ergosterol is the name given to a sterol isolated from ergot by Tanret ; f this substance, melting at 154°, to which he assigned the formula C27H42O, HgO, was accompanied by a second sterol which he described as fungisterol of the formula C25H40O, HgO, m.p. I44°- Both these sterols are regarded as belonging to a class characteristic of crypotogams and differing from cholesterol and phytosterol in their reaction in chloroform solution with sulphuric acid ; whereas in the case of the ordinary sterols the chloroform solution acquires a red colour, it is the acid which turns red in the case of the fungus sterols. Yeast has been shown to contain a mixture of sterols — namely ergosterol and zymosterol, m.p. 99-104°.$ The work of Webster and others has shown that the irradia- tion of ergosterol with ultra violet light gives rise to vitamin D. Phytosterols crystallize from alcohol in elongated plates and from ether in slender needles. The melting-point varies somewhat according to the source from which it is prepared; it lies somewhere between 135 and 137° or it may be as high as 144°. The reason for this may be that the various substances obtained from different sources and described as one and the same substance are in reality different substances but all of a phytosterol nature. The colour reactions of phytosterols resemble those of cholesterol. DISTINCTION BETWEEN CHOLESTEROL AND PHYTOSTEROL. In examining the unsaponifiable matter of a fat for sterols, the unsaponifiable residue remaining after evaporation of the ether is dried over a water bath and then dissolved in the least possible quantity of absolute alcohol and allowed to crystallize. The crystals which separate should be examined ♦Ellis: "Biochem. Journ.," 1918, 12, 154. 160, 173. t Tanret : " Compt. rend.," 1889, 108, 98, and 1908, 147, 75. t Smedley-Maclean : "Biochem. Journ.," 1928,22, 22. 4 50 FATS, OILS, AND WAXES under a microscope ; cholesterol crystallizes in four-sided plates and phytosterol in elongated hexagonal plates. Cholesterol and phytosterol cannot with certainty be dis- tinguished by means of their melting-points, owing to the fact that phytosterol may melt at any temperature between 135 and 144° according to the source from which it is prepared. As, however, there is a considerable difference between the melting-points of the acetates of these two substances the following procedure may be adopted. After completely evaporating off the alcohol, the residue is carefully heated with 2-3 c.c. of acetic anyhdride over a free flame until the liquid boils, the remaining acetic anhydride being evaporated off over a water bath. The residue is then re-crystallized two or three times from the least possible quantity of absolute alcohol, and the melting-point of the crystals so obtained is determined. Cholesterol acetate melts at ll4*3-ii4-8°. Phytosterol acetate * melts at 125-137°. Stigmasterol acetate melts at 141°. Since cholesterol and phytosterol are the sterols charac- teristic of animal and vegetable fats respectively the above procedure may be adopted for distinguishing the source of origin of a given fat, or for detecting the presence of vegetable fat in animal fat. For this purpose a melting-point of the sterol acetate up to 116° is taken to imply the absence of vege- table oil, but a melting-point of 117° or more indicates con- tamination with vegetable oil. ESTIMATION OF THE STEROL CONTENT OF AN UNSAPONIFIABLE RESIDUE. The method devised by Windaus f depends upon the formation of an insoluble compound of the sterols with digitonin. The unsaponifiable residue obtained by the method already described, is dissolved in twenty times its weight of alcohol ; it is then warmed to 65° and treated with a I per cent solution * The acetyl derivative obtained by Power and Moore from the root of Bryonia has the melting-point 155-157°. t Windaus : " Zeit. physiol. Chem.." 1910. 65, no. LIPINS 51 of digitonin in 95 per cent alcohol until no further precipitate is formed ; a little chloroform is then added to prevent the separation of any excess of digitonin and the whole is allowed to stand for some hours while the precipitate of the sterol digitonide settles down ; the precipitate is then filtered off on a Gooch crucible, washed with chloroform and finally with ether, dried for 10 minutes in a steam oven, and weighed. The weight multiplied by the factor 0-2431 gives the weight of sterol. LIPINS. The term lipin is applied to a group of glycerol esters which in their physical and chemical properties are closely allied to the fats. The nomenclature of the group has in the past given rise to much confusion, the term lipoid (from the Greek word AtTTos- = fat) having been used somewhat loosely to in- clude a heterogeneous group of substances which were all soluble in the ordinary fat solvents, but were not necessarily esters of glycerol. Like the fats, the lipins are esters of glycerol with saturated fatty acids and with unsaturated acids of the oleic and other series, but they differ from the fats in containing in addition the elements nitrogen and phosphorus, or nitrogen only, as is exemplified by the formula here given for lecithin, one of the best-known representatives of the group : — ■ CHj . O . COC17H35 (Stearyl) :H . O . COC„H33 (Oleyl) P(OH) . O . CH2CH,N(CH3)<,OH II O CH„ . O . As already stated the phospho-lipins in general resemble the fats in being soluble in the same solvents such as ether, petrol, benzene, etc. ; they are, however, generally more soluble in alcohol than the fats, but on the other hand they are insoluble in cold acetone though frequently soluble in hot acetone ; the cerebrosides are practically insoluble in ether. The fact that lipins are themselves soluble in fats and are usually found in close association with true fats in plant and 52 FATS, OILS, AND WAXES animal tissues, adds considerably to the difficulty of their preparation in a pure condition ; moreover, a given lipin which may be extracted by means of ether in admixture with another lipin may, in a purified condition, be practically in- soluble in this solvent. Again, it was first found by Hoppe Seyler * that when egg yolk is extracted with ether until no more extract is obtained, the residue still contains lipins which can be readily extracted by means of warm alcohol ; this has since been found to be a property common to all tissues both plant and animal ; no matter how long the extraction with ether is continued, a considerable quantity of the lipin is retained by the tissue only to be extracted by replacing the ether by alcohol. In general, the first step in the purification of an ether extract from lipin consists in the addition to the concentrated ethereal solution of four times its volume of cold acetone, which will precipitate the phospholipins and probably also the cerebrosides if present. The separation of fat from lipin by this method will only be partial, and repeated solution and precipitation will be required to effect any reasonable amount of purification, f In order to distinguish a Hpin from a fat, recourse is taken to the fact that the former, unlike fats, contain either nitrogen or phosphorus or both. To establish the presence of nitrogen, it is sufficient to heat the purified substance with a little soda lime and to test for the evolution of ammonia by red litmus paper. Phosphorus may be detected by fusing with fusion mixture on a platinum foil until all carbon is burnt away, the residue is dissolved in nitric acid and tested for the presence * Hoppe Seyler ; " Med. chem. Unters.," 1869, 3, 392. t On applying this method to the ether-soluble extract of cabbage-leaf cytoplasm, Chibnall and Channon (" Biochem. Journ.," 1927, 21, 233) claim to have precipitated, not an ordinary phospholipin, but the calcium salt of a diglyceride phosphoric acid, to which they assign the formula — CH2O— CORi i HO— COR2 CHp— P^O 0>Ca LECITHIN 53 of phosphate by ammonium molybdate. The classification of the Hpins is based upon their nitrogen and phosphorus content as follows : — A. Phospholipins which contain both phosphorus and nitrogen. According to the number of atoms of phosphorus, one or two, contained in their molecule, they are classed as mono- or di-phosphatides. To this group belong lecithin and kephalin. B. Galactolipins or Cerebrosides which contain nitrogen but no phosphorus and yield on hydrolysis galactose in addition to fatty acids and glycerol. A. PHOSPHOLIPINS. LECITHIN. Although widely distributed in the vegetable kingdom lecithin usually occurs together with other lipins and in rela- tively small amount ; for this reason the most convenient source for the preparation of lecithin is egg yolk. This sub- stance is extracted with five times its volume of 96 per cent alcohol ; the extract is then cooled to 0°, filtered and pre- cipitated with an alcoholic solution of cadmium chloride ; the precipitated double salt is next washed with alcohol and ether ; it is then decomposed by boiling with eight times its quantity of 80 per cent alcohol and carefully adding a con- centrated solution of ammonium carbonate until all the cad- mium is thrown out of solution ; the solution is filtered whilst hot and on cooling the filtrate to 10° the lecithin is deposited. It may be purified by dissolving in chloroform and precipi- tating from solution by the addition of acetone in which lecithin is insoluble. The following are some of the more characteristic re- actions of lecithin : — 1. If to an alcoholic solution of lecithin an alcoholic solution of cadmium chloride be added, a white precipitate of the cadmium chloride double salt is formed. 2. If a little lecithin is boiled with caustic soda, trimethyl- amine is formed, and may be identified by its characteristic 54 FATS, OILS, AND WAXES smell ; the solution contains sodium salts of fatty acids ; on acidifying with sulphuric acid the fatty acids are precipitated. 3. Lecithin on exposure to light and air absorbs oxygen undergoing a change which reduces its solubility in alcohol or ether and makes it increasingly soluble in water. 4. Mixed with a little water, lecithin, in common with some other lipins, swells up, forming slimy threads known as myelin forms ; with excess of water these gradually produce a sort of emulsion or colloidal solution from which they can be precipitated by the addition of salts of barium or calcium. Lecithin like many other lipins is a yellow or yellowish- white wax-like solid with a peculiar odour ; the lipins are very hygroscopic, but some of them when carefully dried in a vacuum can be obtained in form of powder. Lecithin is readily hydrolysed by boiling with alkalis, notably baryta, and is also broken up by lipase, and, less readily, by mineral acids. The products of its hydrolysis are glycero-phosphoric acid- — CHjOHCHOHCHaOP^ (OH)2 II O choline HON(CH3)3CH2CH20H and fatty acids ; a similar hydrolysis takes place in the germinating seed.* Originally it was considered that the fatty acids of lecithin were either stearic, palmitic, or oleic, but it has since been found that the more highly unsaturated acids, linolic and linolenic, are also present. f The unsaturated arachidonic acid X C20H32O2, containing four double bonds which occurs in lipins of animal origin, has not hitherto been isolated from plant lipins. To examine the products of the hydrolysis of lecithin, this substance is heated with a solution of barium hydrate in excess ; a baryta soap is formed, which may be filtered off. The aqueous solution contains barium glycero-phosphate and choline ; the latter may be extracted as follows : — § * Schulze: " Zeit. physiol. Chem.," 1887, li, 365 ; Schulze and Frank- furt : " Ber. deut. chem. Gesells.," 1893, 26, 2151. t Levene and Rolf : " J. Biol. Chem.," 1925, 62, 759 ; 1926, 68, 285. X Ibid.. 1921, 46, 353 ; 1922, 54, 91. §Leathes: " The Fats," Monographs of Biochemistry, London, 1910. KEPHALIN 55 Treat the solution with a stream of carbon dioxide until no more barium carbonate comes down. Filter and evaporate the filtrate to dryness. Treat the residue with absolute alcohol, which will dissolve the choline but not the barium glycero- phosphate. The alcoholic solution, if treated with an alcoholic solution of platinic chloride, gives a precipitate of the double platinichloride of choline. Green and Jackson * give the following method : Allow the finely-divided material to stand for some days under absolute alcohol. Pour off the extract, and evaporate to dryness ; the residue is again extracted with absolute alcohol, and finally with a mixture of alcohol and ether. These extracts are mixed, and the solvents evaporated off. The choline is con- tained in the residue. In addition to the above products of the hydrolysis of lecithin of animal origin, a number of phospholipins isolated from the seeds of Avena saliva, Lupinus spp, Pinus cemhra as well as from pollen and potato tubers yield glucose, galactose, and pentoses. t KEPHALIN. This is the name given to a phospholipin whose nitrogen base is aminoethyl alcohol, NHgCH^CHaOH, in place of choline ; its chemical constitution is closely allied to that of lecithin but it differs from this substance probably in the nature of the acid radicles it contains. It occurs together with lecithin in most animal and vegetable tissues ; as ex- amples of the latter may be mentioned the soya bean % and yeast. § Kephalin, unlike lecithin, is practically insoluble in alcohol, and the two substances may be separated by making use of this fact. Betaine has likewise been described as replacing choline as the nitrogen base of a phospholipin, by Zlataroff.jl * Green and Jackson : " Proc. Roy. Soc," B., 1906, 77, 69. t Winterstein and Hiestand : " Zeit. physiol. Chem.," 1906, 47, 496 ; 1908, 54, 283 ; Zlatarov : " Biochem. Zeit.," 1925. 161, 399- X Levene and Rolf : " J. Biol. Chem.," 1925, 62, 759. § Daubney and Smedley-Maclean : " Biochem. Journ.," 1927, 31, 373. 11 Zlataroft : " Biochem. Zeit.." 1925. i6l, 379- 56 FATS, OILS, AND WAXES Water-soluble phosphatides obtained from beetroot, soja bean and from Aspergillus oryzce have been described by Hansteen-Cranner and Grafe and his co-workers.* B. CEREBROSIDES OR GALACTOLIPINS. The name cerebrosides was originally applied by Thudichum to a group of substances isolated by him from the brain of animals. They are characterized by being phosphorus free but yielding on hydrolysis a nitrogen base, a saturated fatty acid, and galactose ; for this reason they are better known as galactolipins. Substances of this type of vegetable origin were first isolated from Lycoperdon hovista by Bamberger and Land- siedl t and later from Hyphaloma fasciculare and Amanita muscaria by Zellner,$ while Trier § obtained a small quantity of a cerebroside from rice. Unlike the phospholipins, the galactolipins are, when dry, white powders tending to crystallize. They differ also from the former substances in being insoluble in ether ; they are, however, soluble in hot alcohol, benzene, and pyridine, but, like the phosphatides, they are insoluble in cold acetone. OCCURRENCE. Lecithin-like compounds occur in the grains of cereals, in the seeds of several Leguminosae, Ricinus, and species of Pinus ; in the leaves of Castanea, and in Fungi ; they are also widely distributed in animals. In fact, these substances are stated to occur in small quantities in all living cells, and they appear to be more especially abundant where fats occur. Zlataroff con- siders that light is requisite for the formation of lecithins since he finds that the amount present in seeds increases during germination in the light. I| * Grafe: "Biochem. Zeit.," 1925. I59> 445: 1925. 162, 366; 1926, 176, 266, 177, 16 ; 1927, 187, 102. t Bamberger and Landsiedl : " Monat. f. Chem.," 1905, 26, 1109. + Zellner : id., 191 1, 32. i33. io57- § Trier : " Zeit. physiol. Chem.," 1913, 86, 413. !| Zlataroff : " Biochem. Zeit.," 1916, 75, 200. CEREBROSIDES 57 The approximate amount of lecithin contained in various substances may be seen from the following table : — Egg yolk Liver Blood Leguminous seeds Cereals 9*4 per cent. 2-1 1-8 0-8-I-64 ,, 0-25-0-53 .. Pure lecithin has not as yet been obtained from vegetable sources, the substances isolated by Winterstein * and his col- laborators from wheat flour and from the seeds of Avena saliva, Lupinus albus, L. luteus, Vicia saliva, from the leaves of MschIms hippocastanum, etc., being mixtures which, moreover, contain a carbohydrate complex. For an account of the methods employed in the extraction of these substances the original papers should be consulted. Smolensky f found that wheat germs (i.e. the embryos which are a bye-product of the flour mills) yielded a phosphatide whose composition was much closer to that of ordinary lecithin than was that ob- tained from the flour. Physiological Significance. Lipins are, apparently, universally present in living cells and must, presumably, play an important part in the physio- logy of the organism : but what their function may be is unknown and as a consequence many roles have been ascribed to these bodies. Overton % showed in many instances that those substances which are soluble in lipins readily enter the cell, whilst those which are insoluble in lipins are absorbed by the cell with difhculty. From such observations he concluded that the plasma membrane is composed essentially of lipins and formu- lated his solution theory of permeability. His views at first * Winterstein and Hiestand : " Zeit. physiol. Chem.," 1907, 54, 288 ; Winterstein and Smolensky : id... 1908, 58, 506 ; Winterstein and Steg- mann : id., 1908, 58, 527' See also Schulze and Likiernik : id., 1891, 15, 405 ; Schulze : id., 1895, 20, 228. t Smolensky : id., 1908, 58, 522. t Overton: " Vierteljahschr. Naturf. Ges. Zurich," 1895, 40, 159; 1896, 41, 383 ; 1899, 44, 88 ; " Jahrb. wiss. Bot.." 1900, 34, 669. 58 FATS, OILS, AND WAXES . found acceptance ; it was, for instance, supported by Czapek * in his work on the surface tension of the external limiting membrane, and Green and Jackson f considered that lipins exercise considerable influence on the transport of material from cell to cell. On the other hand, further work on the uptake of inorganic salts and dyes by the vegetable cell and on the surface tension of solutions, indicate the imperfections of Overton's theory. It, however, stimulated investigation on the nature of the plasma membrane and, generally, on permeability, a subject which is without our present province. J Palladin§ suggested that lipins play a part in respiration in that the more these substances are extracted with organic solvents, the more is respiration depressed as measured by the output of carbon dioxide in the presence of water during definite periods of time. This thesis involves many problems. Thus, if respiration be a matter of enzyme action, then, presumably, there must be some essential connection between lipin and enzyme. There is no doubt that fats are utilized in respiratory processes ; are they, after desaturation, built up into lipins which are then oxidized for the liberation of energy ? The evidence available on these points relates either to the animal or to the chemical laboratory. Vernon, 1| working on animal tissues, found that if the material were extracted with organic solvents, in order to remove the lipins, the oxidase reaction rapidly disappeared, which means that oxidase reaction is somehow dependent on the cell lipins. Further, Gallagher ^ isolated from the potato a lipin which in the presence of oxygen acquired the property of immediately oxidizing guaiacum in the presence of oxidase. If this be significant in respiration, it indicates that oxidase plays an essential part in the process, a conclusion which is ♦ Czapek : " Ueber eine Methode zur direkten Bestimmung der Ober- flachenspannung der Plasmahaut von Pflanzenzellen," Jena, 1912. t I.oc. cit. X See Stiles : " Permeability." New Phyt. Reprint, No. 13. 1924- § Palladin : " Ber. deut. bot. Gesells.," 1910, 28, 120. Palladin and Stanevitsch : " Biochem. Zeit.," 1910. 26, 351. II Vernon : id., 1912, 47, 374 ; 1914, 60, 202. *\ Gallagher : " Biochem. Journ.," 1923, 17, 515. PHYSIOLOGY 59 rendered doubtful on other considerations, at any rate for the plant.* The following table, due to Green and Jackson, f shows the relation between the lecithin, fatty acid, and oil of the endo- sperm of Ricinus, expressed in per cent of weight of the seeds at different stages in their germination : — Degree of development. Oil in seeds. Fatty acid in seeds. Lecitbin. Resting seeds . Testa just cracked . Radicle protruding 1-2 cm. Root system established . 82-8 67-5 52-5 23-6 2-2 4-6 II-9 16-89 •236 •17 •475 •873 From this it appears that lecithin is formed during germina- tion ; although there is, during the early stages of germination, a diminution in the quantity present. It was found when once the maximum was reached that this amount remained constant until the whole of the endosperm was used up. FURTHER REFERENCE. Maclean and Smedley-Maclean : " Lecithins and Allied Substances," Monographs of Biochemistry, London, 1927. * See Vol. TL, chapter on " Respiration." j Green and Jackson, loc. cit. SECTION II. ALDEHYDES AND ALCOHOLS. In view of the important part played by aldehydes and alcohols in questions relating to the carbohydrates and other compounds, it appears desirable here to draw attention to the chief properties of these substances. It is well known that the aldehydes are the first products of the oxidation of primary alcohols : — CH3OH + O = HCHO + H.O Methyl alcohol Formaldehyde CH3CH2OH + O = CH3CHO + H2O Ethyl alcohol Acetic aldehyde The reconversion of formaldehyde into the alcohol can be effected by means of nascent hydrogen obtained by sodium amalgam and water. Chemically, the aldehydes are very active, undergoing a number of reactions, some of which are of biological signifi- cance, whilst others serve as valuable means of isolation or identification. I. Aldehydes are readily oxidized to the corresponding acids by even such mild oxidizing agents as ammoniacal silver hydroxide or Fehling's solution, or even atmospheric oxygen, as is shown by the following experiments :— {a) A few drops of caustic potash are added to some silver nitrate solution in a test tube, ammonia is then care- fully added, drop by drop, until the brown precipitate has just redissolved. A little dilute acetaldehyde solution is poured in and the mixture is warmed gently ; if the solution be sufficiently dilute, a silver mirror will be deposited on the side of the test tube ; otherwise a black precipitate will be formed : — - CH3CHO + AgaO = CH^COOH + 2Ag 60 REACTIONS 6 1 {b) A little Fehling's solution is gently warmed with a few drops of dilute aldehyde solution ; a change in colour takes place, from blue to green and yellow ; finally the solution becomes colourless and a red precipitate of cuprous oxide (CuaO) comes down. The readiness with which aldehydes are oxidized to acids accounts for the fact that most samples of aldehydes, unless freshly prepared, contain varying amounts of free acid. 2. Aldehydes are readily reduced by nascent hydrogen to the corresponding primary alcohols, according to the equation CH3CH0 + 2H = CH3CH2OH Acetic aldehyde Ethyl alcohol 3. Aldehydes restore the colour to Schiff's Reagent (a solution of magenta decolorized by sulphurous acid). 4. Aldehydes when warmed with caustic potash are con- verted into resinous substances of unknown composition. This can be readily shown with acetaldehyde ; formaldehyde, how- ever, when treated with potash undergoes a different change, being converted into a mixture of methyl alcohol and potassium formate, according to the equation 2HCHO + KOH = CH3OH + HCOOK Potassium formate 5. Aldehydes react with ammonia to form additive compounds ; thus acetic aldehyde undergoes the following reaction : — - CH3CH0 + NH3 = CH3CHOHNH2 Acetic aldehyde Aldehyde ammonia Here again formaldehyde behaves differently ; if ammonia is added to a formaldehyde solution, it is neutralized quantita- tively according to the equation : — 6CH2O + 4NH3 = (CHj^sN^ + 6H2O Formaldehyde Hexamethylene tetramine with the formation of a crystalline solid which is used in medicine under the name of urotropine. The reaction can be employed for estimating * the amount * For another method of estimating formaldehyde by weighing the mercury produced by the reduction of an alkaline solution of mercuric sulphite, see Feder : " Archiv. d. Pharm.," 1907, 245, 25. 62 ALDEHYDES AND ALCOHOLS of formaldehyde in a solution by adding a known excess of standardized ammonia solution, and after some time titrating back the excess of ammonia by means of standard acid, using litmus as indicator. Thus, for example, if 25 c.c. of the formaldehyde solution, after shaking with 50 c.c. of N/2 ammonia, required for neutralization 20 c.c. N/2 hydrochloric acid, then the amount of ammonia used up by the formaldehyde would be 50 — 20 = 30 c.c. ^0 17 But ^0 c.c. N/2 ammonia contain — — X -^ = -255 gram ■^ ' 1000 2 NH3, and since from the equation 4NH3 (68) are equivalent to 6CH2O (180) .-. -225 gram NH3 = -68 gram CHgO, .-. 25 c.c. of the solution contained 0-68 gram formaldehyde. 6. With sodium bisulphite aldehydes form crystalline addition compounds which, being sparingly soluble in water, can be used for isolating aldehydes from mixtures. Thus if some saturated sodium bisulphite solution be added to a fairly strong solution of aldehyde and the mixture shaken vigorously, a rise in temperature takes place accompanied by the formation of a white crystalline precipitate :— CH3CHO + HNaSOg = CHjCHOHSOsNa 7. Aldehydes also form additive compounds with hydrogen cyanide ; these compounds are known as hydroxycyanides or cyanohydrins : — ■ CH3CHO + HCN = CHsCHOHCN Acetic aldehyde cyanohydrin 8. Aldehydes form crystalline compounds with hydro- xylamine, phenylhydrazine, and semicarbazide ; in all cases water is split off between the two reacting substances : — CH3CHO + NH2OH = CH3CH : NOH + H2O CH3CHO + CgHjNHNH, = CH3CH : N . NHCeHj + HjO The resulting compounds, which are known as oximes, hydra- zones or semi-carbazones, are usually substances with a charac- teristic crystalline form and melting-point, which may be REACTIONS 63 employed for the identification of the corresponding aldehydes. The use of phenylhydrazine for the identification of the sugars has already been described. 9. The aldehydes are able to react with alcohols with the formation of condensation compounds known as acetals ; thus, for example, acetic aldehyde reacts with ethyl alcohol as fol- lows : — + H,0 ( ( ■'Nh + CH3 HOC.Hs = /OC,H, HOC2H5 C^OQHs ^H al Acetic dehvde Ethyl alcohol Acetal By analogy, acetic aldehyde should also be able to react with water as follows : — CH3 CH, HOH ^0 + Cf HOH ( \h /OH + :^OH H^O \h This substance does not, however, actually exist, since a compound having two or more hydroxyl groups attached to the same carbon atom is, as a rule, unstable, and at once loses water. Exceptions to this rule are, however, occasionally met with ; for example, chloral CCI3CHO forms a stable com- pound, chloral hydrate, of the formula : — OH CCI3— C^OH ^h 10. Aldehydes exhibit a tendency to polymerize, that is, for two or more molecules to combine together to form new compounds of higher molecular weight. Thus two molecules of formaldehyde will combine together, forming a compound known as paraformaldehyde {C\l.fi)2 ', this substance, which is a white solid, is obtained by evaporat- ing an aqueous solution of formaldehyde. A second polymer formed from three molecules of formal- dehyde is known as metaformaldehyde or trioxymethylene 64 ALDEHYDES AND ALCOHOLS (CH20)3. This substance is produced by the spontaneous polymerization of anhydrous formaldehyde. In the case of both the above polymers the molecules of formaldehyde are probably connected together through oxygen atoms as under : — CH2 CHj /\ /\ O O and O O \ / / \ CHj CHg O CHj Paraformaldehyde Trioxymethylene or Metaformaldehyde which accounts for the fact that they are readily broken up into the simple molecules of formaldehyde by heating. II. A different type of polymerization, involving the link- ing together of molecules of formaldehyde through carbon, is also known ; this type of polymerization, which is sometimes known as aldol condensation, results in the formation of a more stable complex which cannot be reconverted into the simple substance. The reaction takes its name from the substance produced by the action of dilute hydrochloric acid or zinc chloride on acetic aldehyde : — CH3CHO + CH3CH0 = CH3CHOH . CHo . CHO Aldol ' The analogous reaction with formaldehyde is, however, brought about by dilute alkalis ; in this way two molecules of formal- dehyde give rise to glycollic aldehyde, HCHO + HCHO = CH2OH . CHO Glycollic aldehyde or three molecules may combine together to produce glyceric aldehyde, HCHO + HCHO + HCHO = CH.OH . CHOH . CHO Glyceric aldehyde By repeatedly shaking a 4 per cent solution of formal- dehyde for half an hour with an excess of lime water, and then filtering the solution and setting it aside for some days until the odour of formaldehyde had disappeared, Loew * was able * Loew : " Bar. deut. chem. Gesells.," 1887, 20, 142, 3039 ; 1888, 31, 270 ; 1889, 22, 470, 878. FORMALDEHYDE 65 to obtain a crude mixture of sugars called formose, from which true reducing hexose sugars have been isolated. This change may be represented by the equation — 6HCH0 = CgHijOg Similarly H. and A. Euler * have shown that when a 2 per cent solution of formaldehyde is heated for some hours with calcium carbonate, a pentose sugar — arabinoketose — is produced ; in addition to this substance, glycollic aldehyde and dihydroxyacetone are produced, but in smaller quantity. FORMALDEHYDE. From the point of view of photosynthesis formaldehyde is of outstanding interest ; as is well known, it is at ordinary temperatures a colourless gas with a pungent odour ; when cooled to — 21° it condenses to a liquid. It is usually met with in the form of an aqueous solution, commercial formalin, which contains about 40 per cent of the gas dissolved in water and is used as a disinfectant or as a hardening medium for pathological and other specimens and occasionally as a pre- servative for milk. It undergoes most of the general reactions for aldehydes which have been mentioned above. Its peculiar behaviour towards ammonia, resulting in the formation of hexamethylene tetramine, has already been mentioned ; this substance, which is used under the name of urotropine, is a crystalline base which dissolves in hot or cold water ; with bromine it forms an additive compound — tetra- bromo-hexamethylene tetramine (CH2)6N4Br4 — which has been used for detecting small quantities of formaldehyde in solution. Formaldehyde also reacts with ammonium salts as well as with free ammonia, as follows :- — 6CH.p + 4NH4CI = (CH2)gN4 + 6HijO + 4Ha Hexamethylene tetramine This reaction has been made use of as a means of estimating ammonium salts in solution by titrating the amount of free acid liberated according to the above equation on adding suffi- cient formaldehyde to a solution containing ammonium salts. * Euler, H. and A. : " Ber. dent. chem. Gesells.," 1906, 39, 36, 39. 5 66 ALDEHYDES AND ALCOHOLS For this purpose both the formaldehyde solution and the solu- tion to be analysed must be previously neutralized, if necessary. An excess of the neutralized formaldehyde solution is then added to a known volume of the solution containing the ammonium salts, and after thoroughly shaking for one or two minutes the amount of acid set free is determined by titration with standard caustic soda, using methyl orange as indicator ; the amount of ammonia can be calculated from the fact that each 36*5 grams of hydrochloric acid liberated correspond to 17 grams of ammonia. The reactions most suitable for characterizing small quan- tities of formaldehyde are as follows : — Rimini's test consists in adding 2 drops of phenylhydra- zine hydrochloride, 2 drops of sodium nitroprusside solution and I c.c. of sodium hydroxide to i c.c. of the liquid to be tested. A blue colour is formed, which changes rapidly through green and brown to red. Schryver * has modified this test and made it much more sensitive ; he recommends the following method : to 10 c.c. of the liquid to be tested add 2 c.c. of a I per cent solution of phenylhydrazine hydro- chloride freshly made up and filtered ; then add i c.c. of a 5 per cent solution of sodium ferricyanide, also freshly made up, and 5 c.c. of hydrochloric acid ; a brilliant magenta colour is produced. The test is a very delicate one and will detect quantities of formaldehyde varying from I part in 1,000,000 to I part in 100,000. Acetic aldehyde gives no colour with this reagent. The following test, due to Deniges,f is sensitive for formal- dehyde, even in presence of acetic aldehyde up to 2 per cent ; 5 c.c. of an aqueous solution of formaldehyde are mixed with 1*2 c.c. of pure sulphuric acid (sp. gr. 1-66) and 5 c.c. of Schiff's reagent. An intense violet colour having an absorption band in the orange is produced. Schiff's reagent may be prepared by adding a litre of o-oi per cent of solution of magenta to 20 c.c. of sodium hydrogen sulphite solution (sp. gr. 1*3), and * Schryver : " Proc. Roy. Soc. Lond.," B., 1910, 82, 226. t Deniges : " Compt. rend.," 1910, 150, 529. FORMALDEHYDE 67 after five minutes adding 20 c.c. of hydrochloric acid (sp. gr. i-i8). Kimpflin * tested for formaldehyde in the leaf of Agave tnexicana by injecting into it, by means of a capillary tube, a concentrated solution of sodium hydrogen sulphite, contain- ing an excess of ^-methylamino-m-cresol. The presence of formaldehyde was indicated by the formation of a red pre- cipitate on exposure to light. The precipitate is best seen by examining a section of the leaf which has been dipped in absolute alcohol. Formaldehyde is the only aldehyde giving a stable red colour with the above reagent, but other aldehydes give unstable green, yellow, or reddish-brown colours. Occurrence in the Plant. — Since the work of Reinke, many have reported the occurrence of formaldehyde in the plant, f and its presence has been accepted as evidence of the truth of Baeyer's hypothesis of photosynthesis. It has, however, since been shown that this formaldehyde is a degradation product of chlorophyll. Thus Warner % has found that formaldehyde is produced when chlorophyll is exposed to sunlight or electric light in air ; since this substance is produced both in the presence and in the absence of carbon dioxide, it would appear that the latter plays no part in the production of formaldehyde by photo- synthesis outside the plant, and that the formaldehyde is in reality an oxidation product of the chlorophyll. The above-mentioned investigations were carried out with impure chlorophyll, Jorgensen and Kidd,§ on the other hand, used chlorophyll a and h (see p. 313) in a state of purity which satisfied Willstatter and StoU's criteria. They experimented with a chlorophyll sol with water as the dispersion medium. On exposing this sol, contained in glass vessels and in contact * Kimpflin : " Compt. rend.," 1907, 144, 148. t Curtius and Franzen : " Ber. deut. chem. Gesells.," 1912, 45, 1715 ; Reinke : " Bar. deut. bot. Gesells.," 1883, i, 406 ; Curtius and Reinke : " Ber. deut. chem. Gesells.," 1897, 30, 201 ; " Sitz. Heidelberger Akad. Wiss. Math. Nat.," 1915, Abt. A. ; PoUacci : " Atti. Inst. Bot. Pavia.," 1900, 6, 1902 ; 1904, 8, 10 ; Usher and Priestley : " Proc. Roy. Soc, Lond.," B., 1906, 77, 369. X Warner : id., 1914, 87, 378. § Jorgensen and Kidd : id., 1916, 89, 342. 5* 68 ALDEHYDES AND ALCOHOLS with various gases, to light, they found that formaldehyde was only produced in the presence of oxygen. In the case of con- tact with carbon dioxide, phseophytin (see p. 319) was pro- duced, and there was no further change. In oxygen the chlorophyll turned yellow, due to the presence of phaeophytin, and ultimately was bleached ; when the bleaching is in pro- gress, formaldehyde occurs in but small quantity, but when the bleaching is complete, there is an increase in the amount of formaldehyde. They suggest that the formaldehyde arises chiefly from the phytol which probably is split off from the chlorophyll under the action of light and oxygen. In conclusion, mention may be made of a simple way of demonstrating the production of formaldehyde from chloro- phyll, due to Osterhout.* A solution of chlorophyll is made with carbon tetrachlor- ide, and in it filter paper is soaked. The filter papers are dried, moistened with water, and placed on the inner surface of a glass bell-jar. The bell-jar is inverted over a dish of water, sealed from the air, and exposed to sunlight. The chlorophyll papers are gradually bleached ; when pale green in colour the water in the dish gives a positive reaction for aldehyde. The same result was obtained when the carbon dioxide was excluded or increased to 10 per cent, which indi- cates that the aldehyde is due to the colouring matter rather than the carbon dioxide. Like results obtained when various aniline dyes, notably methyl green and iodine green, were used in place of the chlorophyll. To summarize, while there is much experimental proof for the presence of formaldehyde and higher aldehydes in plants, this is not evidence in support of the formaldehyde hypothesis of carbon assimilation, since it has been repeatedly shown that formaldehyde is produced by the decomposition of chlorophyll itself. The whole question is considered in greater detail in the second volume. * Osterhout : " Amer. J. Bot.," 1918, 5. 5"- ALCOHOLS 69 ALCOHOLS. OCCURRENCE OF ALCOHOLS IN PLANTS. Methyl Alcohol has been found to occur in the aqueous distillates and in the essential oils of a very large number of different plants, amongst which might be mentioned Juniperus Sabma, Zea Mais, Lolium peremie, Iris germanica, Euonymus europaea, Thea sinensis, Eugenia caryophyllata, Carum carvi, Anthriscus cerefolium, etc. Ethyl Alcohol is not quite so widely distributed as methyl alcohol, but occurs in distillates from Cananga odorata (Ylang Ylang), Pyrus Malus, Mespilus germanica. Eucalyptus, Anthris- cus cerefolium, Pastinaca sativa, Vacci?iium Myrtillus, Betula alba, etc. Mention also should be made of the occurrence of this alcohol, together with lactic acid and acetone * in some cases, in the higher plants especially during anaerobic respiration. Stoklasa.t for instance, found that this substance together with acetic and formic acids, was produced during anaerobic respiration of potatoes and seeds. Indeed, many consider that alcoholic fermentation is the first expression of respiration, and whether alcohol is formed or not depends upon the conditions ; thus under normal conditions in the presence of oxygen the first products are oxidized before the alcohol stage in the process is reached, or the alcohol may be used up in anabolic processes as soon as it is formed, or it may be oxidized to water and carbon dioxide — the normal end products of aerobic respiration. f Amyl Alcohol has been identified in the essential oils of geranium, eucalyptus, lavender, peppermint, and chamomile. Several unsaturated alcohols, such as citronellol CmHaoO, geraniol, and linalool, both of the formula CxoHjsO, occur in essential oils, such as rose oil and oil of bergamot, while * Palladin and Kostvtschevv : " Ber. deut. bot. Gesells.." 1906, 24, 273. t Stoklasa : id., 1904, 22, 358 ; " Centr. f. Bakter. u. Parasit.," 1905, II., 31, 86. Godlewski and Polzeniusz : " Bull. Acad. Sci., Cracow," 1901, 227 ; Stoklasa, Jelinek, and Vitek : " Beitr. z. chem. Phys. u. Path.," 1903, 3. 460. { See Kostytschew : " Ber. deut. bot. Gesells.," 1908, 26, 565. 70 ALDEHYDES AND ALCOHOLS amongst the alcohols belonging to the aromatic series must be mentioned cinnamic alcohol, benzyl alcohol, menthol, borneol, etc. Other monohydric alcohols, with the exception of phyto- sterols and allied substances are of rare occurrence. Examples of polyhydric alcohols occurring in plants are mannitol, sorbitol, and dulcitol, isomeric substances of the formula — CH.OH CHOH CHOH CHOH CHOH CH^OH Mannitol occurs to the extent of about 40-50 per cent in manna, the dried sap of Fraxinus ornus, and up to 20 per cent of the dry weight of Agaricus integer consists of mannitol ; it also occurs in many other fungi and in leaves, twigs and unripe fruits of the olive tree and has been found in Rhinanthus, celery, Syringa vulgaris, asparagus, cauliflower, carrot, pulse, etc. Furthermore, it occurs in various fucoids where it may possibly replace sugars in the metabolism of the plant. Tutin * has shown that apple juice fermented by the bacillus responsible for " cider sickness " results in the reduction of some of the sugar to mannitol. Sorbitol occurs in the berries of Pyrus aucuparia and also in apple juice from which it may be obtained by fermenting away the sugars and acetylating the residue with acetic anhydride in the presence of pyridine ; the resulting hexa- acetyl sorbitol is hydrolysed with 2 per cent sulphuric acid and the regenerated sorbitol is crystallized from alcohol. f Dulcitol occurs in the cortex of Euonymus europaea and in the bark of Euonymus atropurpurea. It has also been found to occur in Melampyrum arvense and M. pratense. It is suggested by Braecke % that the alcohols mannitol and dulcitol are the predominant nutritive compounds for the genera Rhinanthus and Melampyrum respectively, since sucrose is not found at any time as a reserve carbohydrate in Rhinanthus crista galli, Melampyrum pratense or M. arvense. Adonitol is a pentahydric alcohol occurring in Adonis * Tutin : " Biochem. Joum.," 1925, 19, 418, t Tutin : id., 1925, 19, 416. % Braecke : " Bull. Soc. Chim. Biol.," 1925, 7, 155. INOSITOL 71 vernalis. According to Treboux,* it is converted by the plant into starch. Adonitol has a sweet taste, and is used in bacteriological media. Of recent years a number of dihydric alcohols of high molecular weight have been found to occur in plants. They belong to different series whose general formulae are :— CnH2„_s04, CuHan-gO,, and CnH.ju-ioOi- Trifolianol, C2iH3402(OH)2, isolated by Power and Sal- way,t from red clover leaves, may be taken as an example of the first group, while Bryonol, C22H3402(OH2), obtained by Power and Moore J from Bryony root, and Calabarol, C23H3402(OH)2, isolated by Salway § from Calabar beans, are representatives of the second and third groups respectively. Of the polyhydric alcohols. Inositol is of particular interest, and may, therefore, receive more detailed consideration. INOSITOL. Inositol, which has the formula C6H12O6, is isomeric with the hexoses, and, like these substances, has a sweet taste ; for these reasons, it was at one time thought to be a true sugar and was called muscle sugar owing to its occurring in muscle. Inositol is, however, not a carbohydrate at all but a polyhydric alcohol derived from benzene and having the constitution — CHOH— CHOH CHOH CHOH \ / CHOH— CHOH Besides being found in muscle, inositol is of common oc- currence in plants, in the leaves, especially when young, of Vitis, Juglans, etc. ; in the roots and rhizomes of very many plants ; in various seeds and fruits, e.g. Phaseolus, Pisum, and other leguminous seeds, Vitis, various cereals, and oily seeds, such as mustard, and flowers and bracts of Cornus flonda.\\ * Treboux : " Ber. deut. bot. Gesells.," 1909, 27, 428. t Power and Salway : " J. Chem. Soc, Lond.," 1910. 97» 249. X Power and Moore : id., 1911, 9% 943- § Salway : id., 1911. 99. 2155. II Sando : " J. Biol. Chem.," 1926, 68, 403. 72 ALDEHYDES AND ALCOHOLS It may be looked upon as a plastic substance since Maquenne has found that it disappears from the young fruits of Phaseolus as ripening proceeds. Preparation. The separation of inositol from the plant juices is effected as follows : — The sap is expressed from the organ, or, if this be impractic- able, the parts are ground up very thoroughly with water. The liquid is then filtered and, if it gives an acid reaction, is neutralized by the addition of baryta water. A solution of basic lead acetate is then added until no more precipitate comes down. The precipitate is filtered off, then washed and suspended in water, and saturated with a current of sulphuretted hydrogen. The lead sulphide is filtered off and the filtrate evaporated on a water bath to the consistency of a syrup. On the addition of alcohol, containing one-tenth of its volume of ether, inositol is deposited in prismatic crystals. Inositol has a sweet taste, is soluble in water but insoluble in alcohol and ether. It crystallizes in prisms, it is not fermentable and it does not reduce Fehling's solution. Identification. 1. When moistened with a little dilute nitric acid, then evaporated almost to dryness, and made alkaline with am- monia, the addition of a few drops of calcium chloride produces a rose-red coloration. 2. A solution of inositol evaporated to dryness with a few drops of mercuric nitrate produces a yellow stain which on heating turns red. 3. Solutions of inositol are not optically active. With regard to its significance in the plant there is evidence to show that inositol is a transitory substance and is used up in the synthesis of other substances. Inositol also occurs in combination with phosphoric acid. This compound, known as phytin, appears to be an acid ETHYL ALCOHOL . -Ji calcium and magnesium salt of inositol phosphoric acid which is a condensation compound of inositol with six molecules of phosphoric acid.* Phytin occurs especially in seeds ; Arbenz t gives the following percentages of phytin, calculated as phytic acid, of the dry weight : Rice bran, 4-232 ; rice flour, o-2i6 ; wheat bran, 5-073 ; whole meal, 0-572 ; wheat flour, 0-208 ; maize flour, 0-857 ; lentils, 0-326 ; peas, 0-561 ; oatmeal, 0-506 ; cocoa, 2-230. In vegetative organs it would appear to be absent for none was found in carrots, turnips, cauliflour, cabbage, spinach, and asparagus ; also none was found in apples, pears, and figs. According to Posternak,$ a large amount, 80-90 per cent, of the phosphorus of certain seeds exists in the form of phytin ; it occurs, for instance, in the globoid portion of aleurone grains, and the seeds, which contain it also possess an appropriate enzyme phytase for its decomposition into phosphoric acid and inositol. § Quebrachitol is the name given to a monomethyl ether of inositol which occurs together with this substance in rubber latex. MANUFACTURE OF ETHYL ALCOHOL. The action of yeast on sugar is made use of in the manu- facture of ethyl alcohol, which substance is prepared from potatoes, rice, and other grains rich in starch. The manu- facture from potatoes is carried out as follows : Potatoes are heated in closed vessels to 125-135° by means of super- heated steam under a pressure of about 3 atmospheres ; by suddenly releasing the pressure the potatoes are burst, and are thus obtained in a finely divided state. The whole mass is then thoroughly stirred up with malt at a temperature of * Cf. Neuberg : " Biochem. Zeit.," 1908, 9, 557 ; 1914. 6'. 187 ; Winter- stein : " Zeit. phvsiol. Chem.," 1908, 50, 118. See also Plimmer : " Bio- chem. Journ.." 1913, 7, 43 ; Boutwell : " J. Amer. Chem. Soc," 1917, 39, 491 ; Posternak : " Compt. rend.," 1919. i69» 37. 138- t Arbenz : " Chem. Zentr.," 1922, 93, iv., 67. J Posternak : " Compt. rend.," 1903, 137. 202. 337, 439. §Cf. Suzuki, Yoshimura, and Takaishi : " Bull. Coll. Agric, Tokyo," 1907, 7, 503. See also Rose : " Biochem. Bull.," 1912, I, 428. 74 ALDEHYDES AND ALCOHOLS about 60°, whereby the starch undergoes hydrolysis with formation of maltose and dextrin : — (CeHioOsln + H,O^Ci,H,,0„ + (QHioO.^x Starch Maltose Dextrin After about one and a half hours the mixture is rapidly cooled to 15° and mixed with yeast; fermentation at once sets in, accompanied by a considerable evolution of heat ; the mixture is therefore cooled artificially, so that the temperature is maintained steady at about 27-5-30°. During this time the maltose is converted first into dex- trose and then into alcohol and carbon dioxide according to the equations — CfiHiPa = 2C,H50H + 2CO2 In order to convert the dextrin, which would otherwise be lost, into a fermentable substance, the temperature towards the end is maintained at about 26-29° in order to give the malt a further opportunity of hydrolysing the dextrin to glucose, and so rendering it capable of being fermented by yeast. When the fermentation is completed after about three days, the mixture contains about 13 per cent of alcohol by volume ; by distilling the mixture through a fractionating column, so much of the water is removed that the distillate contains about 80 to 95 per cent of alcohol.* No amount of fractional distillation without dehydrating agents will produce alcohol containing less than 4-43 per cent by weight of water, since such alcohol gives a constant boiling mixture. Alcohol containing 0*5 per cent or less of water is, in commerce, known as absolute alcohol, although in a scientific laboratory the term is only correctly applied to alcohol which is quite free from moisture ; such alcohol can only be obtained by careful fractionation from freshly burnt quicklime. f If * The residue remaining after distillation contains, in addition to the solid unfermentable materials, a certain amount of other soluble products of fermentation, such as glycerol and succinic acid ; it is used as a cattle food. t Occasionally the last traces of moisture are removed by treating the alcohol with sodium wire. ETHYL ALCOHOL 75 the alcohol is dehydrated over quicklime to which a little barium oxide has been added, complete dehydration is marked by the formation of a yellow colour due to the production of barium ethylate, which can only be formed in the absence of any trace of moisture. A delicate test for the detection of traces of moisture in alcohol consists in adding a few drops of the sample to a solution of liquid parafhn in anhydrous chloroform ; if there is any moisture present, a turbidity will be at once produced. SECTION III. THE CARBOHYDRATES. The importance of carbohydrates becomes obvious when once it is reahzed that the metabolism of the green plant is essentially a carbohydrate metabolism. Carbohydrate, an essential in the food of the plant and of the animal, is synthesized from raw inorganic material only by the green plant, wherefore the maintenance of life is entirely dependent on the plant. Glucose, perhaps the simplest carbohydrate expression, is the all-im- portant respirable material both in the animal and in the plant and it, together with other simple sugars, forms a raw material for the making of more complex carbohydrates, of proteins, of fats, and of other substances. With but few exceptions, carbohydrate in the form of cane sugar, starch, inulin, glycogen, and hemicellulose are the most significant reserve food materials of plants, whilst in the animal, glycogen alone forms a temporary reserve. In the plant, carbohydrate in the shape of cellulose, ligno-cellulose, hemicellulose, and pento- sanes, form entirely or in part the structural basis of the cell wall when present,* and thus plays an important role in the structural mechanism. In the animal, on the other hand, carbohydrates are seldom thus employed ; chitin, spongin, and chondro-mucoid, which to a limited extent enters into the composition of muscle, may be cited. Indeed, the synthesis of carbohydrate in the animal is for the most part restricted to the production of lactose, or milk sugar, from pre-existing glucose. Notwithstanding the differences in the physiological * It will be remembered that the Myxomycetes, many Chrysophyceae, Euglenineae, and other members of the lower Protophyta, together with the gametes of the majority of plants, both high and low, are naked structures with no cell wall. 76 CLASSIFICATION TJ significance of the various types of carbohydrates in the plant, these substances are all closely related chemically, being composed of the same elements — carbon, hydrogen, and oxygen — united together in a similar fashion. The term carbohydrate originated through the erroneous conception that these substances were compounds of carbon with water, since the proportion of hydrogen to oxygen in most of them is the same as in water, as may be seen from the formula for grape sugar, which is CgHioOe, but which might be written Cg . 6H2O. The discovery of methyl pentoses of the formula CgHiaOj shows, however, that the maintenance of this proportion of hydrogen to oxygen is not an essential characteristic of this group of compounds. CLASSIFICATION OF CARBOHYDRATES. On purely physical grounds such as appearance, solubility in water, taste, etc., the carbohydrates may be roughly divided into sugars and non-sugars ; the systematic classification of the carbohydrates is, however, based upon their behaviour towards hydrolytic agents, such as mineral acids or enzymes. Thus there are a considerable number of naturally occurring sugars containing five and six carbon atoms which cannot be hydrolysed ; such sugars form a group known as monosacchar- ides. On the other hand, many sugars are known which on hydrolysis break up into two molecules of monosaccharide according to the equation — C12H22O11 + H3O - 2CeHi20g Such sugars are known as disaccharides. Similarly sugars which on hydrolysis give three molecules of monosaccharide as follows — C18H32O18 + 2HjO = 3C«H,20s are termed trisaccharides. Finally, carbohydrates, such as starch and cellulose, which on hydrolysis yield an unknown number of molecules of mono- saccharides are classed as polysaccharides. The nomenclature of the monosaccharides is based on 78 THE CARBOHYDRATES the number of carbon atoms in their molecules, those contain- ing five being called pentoses, while those containing six atoms are known as hexoses. For this reason the use of the terms monose and biose in place of monosaccharide and disaccharide is to be deprecated owing to the confusion which is liable to result therefrom. A scheme for the classification of the carbohydrates is given below: — Trioses (CgHgOg) Tetroses (C4H8O.J) Pentoses (C5H10O5) bose, Apiose. Methyl pentoses (CgHijO^) Quinovose. Arabinose, Xylose, Ri- Rhamnose, Fucose, Monosaccharides • Hexoses (CgHjjOg) f Aldoses : Glucose, Man- j nose. Galactose. ] Ketosea : Fructose, Sor- ( bose Sedoheptose, Mannoketo- Sugars Disaccharides ■ Heptoses (C,Hi40,) heptose. Glucoxyloses (CiiHjnOio). Primeverose, Strophanthobiose, Vicianose. (C12H22O11). Sucrose, Lactose,* Maltose, Iso- maltose, Gentiobiose, Cellobiose, Treha- lose, Melibiose, Turanose. Trisaccharides (C^gH^gOig). Raffinose, Melicitose, Gentianose. Tetrasaccharides (€,41142021). Stachyose. Unknown constitution : Agavose, Lupeose. Non-sugars or Polysaccharides Pentosans (C5Hg04)n Hexosans (CgHuOs)!! Araban, Xylan. Glucosans : Starch, Dextrin, Gly- cogen, Lichenin, Cellulose. Fructosans : Inulin, Graminin, Phlein, Triticin. Mannans. Galactans. T^ . , T, u J i ^ Hemicelluloses. Derived carbohydrates ^ _ containing -COOH Mucilages. and other groups ( p^^^.^ Lbstances. SOLUBILITIES OF THE CARBOHYDRATES. As might be expected of a group of substances of such varying complexity and widely different function in the plant, the solubilities of the carbohydrates present a considerable range of variation, some idea of which can be obtained from the following facts : — I. Readily soluble in water or dilute alcohol, but insoluble in absolute alcohol and other organic solvents, e.g. sugars, inulin. * Not found in the vegetable kingdom. SOLUBILITIES 79 2. Sparingly soluble in cold water but more so in hot, and precipitated from solution by alcohol, e.g. gums, mucilages, starch, and pectins. 3. Insoluble in water but soluble in dilute caustic alkali, and precipitated from solution by the addition of acid or alcohol, e.g. hemicelluloses. 4. Insoluble in water, alkali, and organic solvents, but soluble in cuprammonia, e.g. cellulose. GENERAL TEST FOR CARBOHYDRATES AND THEIR DERIVATIVES. In attempting to characterize an unknown organic sub- stance, there is one test which should always be employed at the outset, and that is Molisch's reaction. This test is extremely delicate, and may be applied to a substance in aqueous solution or, if the substance is insoluble in water, to a little of the liquid obtained by boiling the solid with dilute sulphuric acid. By this treatment the substance, if it contains a carbohydrate, will be hydrolysed and then will yield suffi- cient monosaccharide to give the test which is carried out as follows : — A few drops of 15 per cent alcoholic solution of a-naphthol are added to about a third of a test tube full of the solution to be tested and concentrated sulphuric acid is carefully poured down the side of the tube. At the junction of the two liquids a green ring is produced * and over this a red zone ; on gently agitating the colour changes to purple. Alternatively, 1-2 drops of the solution are mixed with about 4 drops of a 4 per cent alcoholic solution of a-naphthol ; about I c.c. of concentrated sulphuric acid is then added and the whole is gently agitated. A purple colour indicates the presence of carbohydrate. The reaction depends upon the production of furfural, by the action of the sulphuric acid on the carbohydrate, and its condensation with the a-naphthol. This reaction is given by all true carbohydrates and all * No attention should be paid to the production of a green colour, which is given by the action of sulphuric acid on alcoholic a-naphthol, even in the absence of carbohydrate. 8o THE CARBOHYDRATES substances which contain a carbohydrate complex, such as glucosides and proteins. The further tests employed for the characterization of carbohydrates depend upon the indica- tions obtained from the solubilities of the substance under examination, and these will be given under their respective headings in the following pages. CONSTITUTION AND ISOMERISM OF SUGARS. The analysis of any one of the hexose sugars, such as dextrose, levulose, galactose or mannose, would yield the same result, viz. 40 per cent of carbon, 6-6 per cent of hydrogen, and 53'3 per cent of oxygen ; and this notwithstanding the fact that these sugars are different substances. From the results of an analysis, it is possible to determine the simplest ratio of the atoms to each other in the molecule by dividing each percentage by the atomic weight of the corresponding element, and then determining the simplest numerical ratio between the resulting numbers : — „ 40-0 . TT &'^ ^ > r^ 53"3 C = ^ = 3*3 ; H = — = 6-6 ; O = ^ = 33 .-. C : H : O = 33 : 6-6 : 33 — 1:2:1. The formula CHgO thus arrived at is known as the Empirical Formula ; it indicates the ratio of the number of different atoms in the molecule, but does not indicate their actual number. The formula which, while maintaining the above ratio, also shows the actual number of atoms present in the molecule, is known as the Molecular Formula ; and it can only be assigned correctly when the molecular weight is known. Now the molecular weight of all these sugars is 180, hence their molecular formula must be (CH20)6 or CgHiaOe. Compounds such as the various hexoses which have the same molecular formula and yet are not identical are said to be isomers. The carbohydrates exhibit two kinds of isomerism, known respectively as structural and stereo-isomerism. Structural isomerism is well illustrated by the two sugars dextrose and levulose. A study of their reactions, which need not here be detailed, leads to the conclusion that they both ISOMERISM 8 1 contain five hydroxyl (OH) groups ; that dextrose belongs to the class of compounds known as aldehydes, which are charac- terized by the group — CHO ; and that levulose is a ketone and therefore contains the group =C0. These facts are all explained by the following constitutional formula : — Dextrose Levulose CH.OH CH.OH I I CHOH CHOH CHOH CHOH HOH CHOH J: CHOH CO I I (JHO CHjOH Stereo-isomerism is the second type of isomerism, and is exhibited by the three sugars dextrose, mannose, and galac- tose, all of which are aldehydes, and have therefore the same structural formula. The possibility of isomerism in this case is accounted for by the presence in these molecules of what are known as asymmetric carbon atoms. Writing the formula for dextrose once more in a slightly different way, it will be seen that the carbon atom printed in " clarendon " (C) has its four valencies attached respectively to the groups (CH2OH . CHOH . CHOH . CHOH)-, H-, -OH, and -CHO :— H CH2OH . CHOH . CHOH . CHOH— C— CHO oi Any carbon atom whose valency is satisfied by four different groups or elements, whatever their nature may be, is said to be asymmetric, since it is possible to represent it by either of two solid models which are not super-imposable, the one being the mirror image of the other ; there exists, therefore, between two modifications of such an asymmetric carbon atom a dif- ference due to the different spacial distribution of the four substituting groups around it. Now the isomerism existing between glucose and mannose is accounted for by their each containing one of the two possible modifications of this same asymmetric carbon atom. Similar considerations will show 6 82 THE CARBOHYDRATES that each of the three carbon atoms marked with a star is also asymmetric, and it is therefore not surprising that it is possible to account for no less than sixteen different isomeric aldehyde sugars or aldoses ; of these, however, relatively few have been found in nature. The constitution of glucose is ordinarily represented by the formula CHgOH CHOH CHOH CHOH CHOH CHO, which shows it to be a pentahydric alcohol and an aldehyde at the same time. When dissolved in water, however, it behaves in a peculiar manner, exhibiting the phenomenon of muta-rotation, that is to say, the optical activity of the resulting solution does not attain a steady value until some time after the solution has been made up. ^ The change is supposed to be connected with some altera- tion in its molecular configuration which may be explained by assuming that the compound CHjjOH CHOH CHOH CHOH CHOH CH<^ ^OH is temporarily formed,* but that water is thereupon split off again between one of the hydroxyl groups of the terminal carbon atom and the hydroxyl attached to the fourth carbon atom as follows : — OH I /OH CH^OH CHOH CH CHOH CHOH CH/ -> 6 5 4 3 2 I \oH 1 ° ^1 CH^OH CHOH CH CHOH CHOH CHOH + H.O 6 5 4 3 2 I It will be seen that in this formula, sometimes known as the lactone or butylene oxide formula, the terminal carbon atom (which is conventionally regarded as carbon atom i) has now become asymmetric, whereas it was not so before ; this method of writing the formula involves the possible existence of two optically isomeric varieties of ordinary glucose, both of which are in fact known. f When glucose * Compare the formation of similar compounds from other aldehydes (p. 63). t Tanret : " Compt. rend.," 1895, 20, 1060 ; Lowry : " J. Chem. Soc," 1899. 75. 213 : 1903. 83, 1314. CONSTITUTION 83 is crystallized from 70 per cent alcoholic solution at ordinary temperatures, a modification known as a-glucose is obtained whose specific rotation is a^ = + 110°; if crystallized from water at a temperature above 98°, another variety, known as j8-glucose (a^ = + I9°), is obtained ; if either a-glucose or jS-glucose is dissolved in water, a gradual change in rotation is observed until a steady value of a^ = 52"5° is attained, which is regarded as the specific rotation of an equilibrium mixture of a- and j3-glucose. The attainment of the stable condition is accelerated by acids, and is practically instan- taneous in presence of traces of alkali. It will be readily understood that such a bridge or ring structure as is represented by the y-lactone may also be de- scribed as a butylene oxide formula, seeing that four carbon atoms are involved in the ring. Theoretically isomeric sugars possessing an ethylene, propylene, amylene, or hexylene oxide formula should also be a possibility : — 1 CHOH— , I o 2 CH 1 I CHOH I CHOH CHOH I CH.,OH Ethylene oxide I CHOH— I CHOH O I I 3 CH 1 CHOH I CHOH I CH2OH Propylene oxide I CHOH- CHOH CHOH O CHOH 5 CH CH2OH Amylene oxide I CHOH- I CHOH I CHOH I CHOH I CHOH O 6 CH2 Hexylene oxide It has been the task of Irvine and his collaborators to investigate this aspect of the isomerism of the sugars. Irvine's resume * gives an account of these important investigations which cannot be further considered here owing to exigencies of space. The work of Irvine appeared to have firmly es- tablished the butylene oxide formula for glucose, but in the light of subsequent work the amylene oxide formula is now generally accepted.f This implies the recognition of the fact that glucose and the other hexoses are six-membercd hetero- cyclic compounds whose constitution may be represented as follows : — * Irvine : " J. Chem. Soc," 1923, 123, 898. t Charlton, Haworth, and Peat : id., 1926, 89, 1858. 84 THE CARBOHYDRATES H OH ^/ OH OH J I* /I l\ H —OH o H / HO ■HH— or , ._. -CH.OH HO\oH H / \ / \l !/ I > /\ H CHjOH H OH in which the reducing group is marked by a star and in which the thickened hnes are all in the same plane : a discussion of this question, together with some important deductions there- from, is given by Haworth,* and in consequence the inter- pretation of the constitution of all the polysaccharides and glucosides derived from glucose has been modified. OXIDATION PRODUCTS OF SUGARS. Before proceeding to a description of the methods employed for the identification of individual sugars, a brief consideration of some of their products of oxidation is appropriate in view of the fact that some are important constituents of natural products. Oxidation by means of nitric acid under carefully controlled conditions attacks both the terminal carbon atoms of alde- hydic sugars,! leaving the intermediate secondary alcohol groups unaltered. CH2OH— (CHOH)4— CHO->COOH— (CHOH)j— COOH In this way glucose, mannose, or galactose yield the di- carboxylic acids saccharic, mannosaccharic, and mucic acids respectively. An intermediate stage of oxidation, in which the aldehyde group remains unaltered and only the terminal primary alcohol group is oxidized to carboxyl, is represented by the substances glucuronic and galacturonic acids of the formula COOH . (CH0H)4 . CHO derived respectively from glucose and galactose. * Haworth, " J. Soc. Chem. Ind.," 1927, 46, 295. + Ketonic sugars are broken down to compounds containing fewer carbon atoms. OXIDATION PRODUCTS 85 Although it has been found possible to produce in vitro a small quantity of glucuronic acid from glucose by the action of hydrogen peroxide, this is not a practical method. In the animal and vegetable world, however, conditions appear frequently to arise in which the aldehyde group of the sugar is protected from oxidation by coupling with some other group as a glucoside, leaving the primary alcohol at the other end of the molecule open to attack. Such coupled glucuronic acids occur normally in the urine of animals, but may be increased in quantity by the administration of certain sub- stances. In the plant world glucuronic and galacturonic acids appear similarly combined with other complexes ; the former has been reported as a constituent of glycyrrhizin * and scutellarin f while the latter occurs in pectins. These aldehyde acids are known collectively as " uronic " acids ; when heated with hydrochloric acid they are con- verted into furfural with evolution of carbon dioxide. f A method for their estimation based upon the measurement of this carbon dioxide has been devised by ToUens and Lefevre,J and modified by Nanji and Norman. § When heated with Bial's reagent, glucuronic acid gives the same colour as pentoses and methyl pentoses, the colour, however, develops rather more slowly. When boiled with an equal volume of hydrochloric acid and a small quantity of i per cent solution of naphtho- resorcin in alcohol, the solution darkens, and on shaking up the warm solution with benzene the latter acquires a reddish- violet colour which shows an absorption band at the D line.|l Solutions of pentoses, hexoses or disaccharides under the same conditions yield no colour or at most a faint yellow to the benzene. A point of some importance arises in connection with the possibility of the uronic acids acting as intermediate stages * Tschirch and Gauchmann : " Archiv. d. Pharm.," 1908, 246, 550. t Goldschmiedt and Zerner : " Monat. d. Chem.," 1910, 31, 441, 476. X Tollens and Lefevre : " Ber. deut. chem. Gesells.," 1907, 40, 4519. § Nanji and Norman : " J. Soc. Chem. Ind.," 1926, 45, 337 T. li Neuberg and Saneyoshi : " Biochem. Zeit.," 191 1, 36, 56 ; van der Haar : id., 1918, 88, 203. 86 THE CARBOHYDRATES in the production of pentoses from hexoses. Thus, assuming a glucose molecule to have its aldehyde group protected from attack, it would, on oxidation, give glucuronic acid which by loss of carbon dioxide would yield xylose : — CH20H(CHOH)4 . CHO (protected) -> COOH (CHOH), . CHO Glucuronic acid COOH . (CHOH)4 CH0-C02-> CUfiU {CUOH)^ . CHO Glucuronic acid Xylose On the other hand, if a given glucose molecule were not so protected and were susceptible to oxidation at both ends, it could give rise to an isomeric glucuronic acid whose aldehyde and carboxyl groups were at the opposite ends by comparison with the previous one : — CH2OH (CHOH) CHO (unprotected) -> CHO (CHOH) . COOH CHO . (CHOH)4 COOH— CO, -> CHO (CHOH)3 . CH,OH Arabinose The fact that in nature xylose, rather than arabinose, is commonly associated with glucose, suggests that xylose is produced from glucose by the oxidation of the primary alcohol group,* the aldehyde group being protected from attack owing to the form of combination in the complex molecule concerned ; the same explanation may account for the frequent association of cellulose with xylans. Spoehr f has isolated the lactone of glucuronic acid from cactus gum, and suggests that glucuronic acid is broken up under the influence of sunlight into carbon dioxide and xylose. THE CHARACTERIZATION OF SUGARS. In order to characterize a sugar, the following procedure may be followed : — I. Ascertain whether the substance is a reducing or non- reducing sugar by adding a little of the neutral aqueous solution to a little Fehling's solution previously diluted with three times its volume of water and boiled to see that it is not changed by boiling alone. Boil the mixture for about one minute. If at the end of this time no red or brown precipitate of cuprous oxide is formed the sugar is non-reducing. * De Chalmot : " Am. Chem. J.," 1893, 16, 610. t Spoehr : " The Carbohydrate Economy of Cacti," Carnegie Inst. Pub., Washington, 381, 42. 75. CHARACTERIZATION 87 All monosaccharides reduce Fehling's solution, but some disaccharides, such as sucrose and trehalose, are so constituted that the reducing aldehydic or ketonic group is masked, and is only set tree after hydrolysis. If the sugar is non-reducing, boil a fresh portion for a short time with a little dilute hydrochloric acid ; neutralize and test once more with Fehling's solution as above. The solution should now reduce owing to the hydrolysis of the di- or tri- saccharide to monosaccharides. It must be borne in mind that other substances besides sugars reduce Fehling's solution, and consequently due pre- caution must be taken to exclude the presence of these before applying the test. 2. Ascertain whether the substance is a pentose (for tests see p. 90) or a hexose. 3. If a pentose is not found, distinguish between aldo- hexose and ketohexose (for tests see p. 96). 4. If the substance is a reducing sugar, whether pentose or hexose, its further identification usually depends upon the production of a crystalline derivative by means of phenyl- hydrazine or a similar compound. Phenylhydrazine reacts with sugars containing either an aldehyde or ketone group to form, in the first place, phenylhydrazones, which in many cases are characteristic crystalline solids, but are usually soluble in water ; this reaction may be illustrated thus : — CH20H(CHOH)4CHO + H2NNHC6H5 = CH20H(CHOH)4CH : NNHCsHs + H^O Dextrose or Glucose. Glucose Phenylhydrazone. If, however, an excess of phenylhydrazine be employed, a second hydrazine complex is introduced into the compound, and the resulting substance is termed an osazone. Both glucose, fructose, and mannose yield the same osazone : — CH20H(CHOH)3— C— CH : NNHCgHg N . NHQHs which is called glucosazone. * This is due to the fact that these three sugars differ only in the con- figuration of their two terminal carbon atoms, a difference which is elimin- ated when they are converted into their osazones (cf. p. 95). 88 THE CARBOHYDRATES The osazones being, for the most part, insoluble in water, serve as a valuable means of isolating a sugar from a dilute solution ; their identity can then be readily established by means of their crystalline form, melting-point, solubility, and optical activity. Other special tests employed for the identification of individual sugars will be given under the various sugars in the following pages. The identification of the constituents of a mixture of a number of different sugars may require special methods depending on the use of specific hydrolytic enzymes or of special yeasts which may fermicnt away certain hexoses, or all hexoses, leaving only the non-fermentable pentoses. The following list, taken from a paper by Chapman,* shows the behaviour of certain species of Saccharomyces towards several of the more commonly occurring sugars : — Species. Dex- trose- Fruc- tose. Man- nose. Galac- tose. Maltose. Sucrose Lactose. S. CerevisicB . + + + + + + o S. ,, Carlsherg + + + + + + o S. Pastorianus + + + + + + o S. ellipsoideus + + + + + + o S. Marxianus . + + + + o + o S. Exiguus + + o + o + o S. Ludivigii + + + o o + o S. anomalus . + + + o o + o S. fragilis + + + + o + + Kefir + + + o o + + (The sign -f indicates that the yeast in question is capable, and the sign o that it is incapable, of bringing about fermentation.) An individual description of the various naturally occurring sugars will now be given. MONOSACCHARIDES. A. PENTOSES. The pentoses, which are sugars containing five carbon atoms, have the general formula C5H10O5, would not appear to be common in the free state ; their presence has been * Chapman : " J. Chem. Soc," 1917, III, 216. MONOSACCHARIDES 89 recorded in the leaves of carrot, mangold, potato, sunflower, TropcEolum and turnip,* and also in Opuntia phcsacantha.'\ Pentosanes, however, which may be regarded as polymerized anhydrides of pentose are very widely distributed in the vegetable kingdom, forming cell wall constituents and entering into the composition of various gums, mucilages, and pectins. With regard to their physiological significance, it is im- possible to say whether they are direct products of photo- synthesis ; if, as Spoehr points out, the formation of sugar in a green leaf is a series of additions of molecules of formalde- hyde, the presence of pentose is to be expected. There is, however, no evidence that this occurs in the green plant. On the other hand, they may have their origin in the oxidation of hexose. The facts that in the germination of seeds, the amount of total pentoses falls as development proceeds and that in some instances the amount is high at certain phases, thus in Parthenium argentatum a high percen- tage of pentose coincides with the period of growth during which the production of rubber is at its highest, suggest that pentoses are definite stages in the elaboration of other substances. As a food material the value of pentoses is variable ; whilst xylose has a high nutritive value for Aspergillus, it, together with other pentoses, is not utilized by Saccharomyces. In the higher plants, Spoehr % has shown that the respiration of Cactacece is not depressed when the hexoses are insignificant in amount, § and that the formation of pentosanes is bound up with certain conditions, especially water content and tempera- ture. Thus in the Cactaceae, a low water content coupled with a high temperature results in a decrease in the amount of monosaccharides and an increase in polysaccharides and pen- tosanes. On the other hand, a high water content and a low temperature are associated with an increase of mono- saccharides and a decrease of pentosanes and polysaccharides. * Davis and Sawyer : " J. Agr. Sci.," 1914. 6, 406. t Spoehr : " Carnegie Inst. Pub.," 1919, No. 287 ; " Plant World." 1917, 20, 365. X spoehr : loc. cit. § It is not uncommonly assumed that in the respiratory activity of higher plants, hexoses are the significant fuel. 90 THE CARBOHYDRATES Only four pentoses have so far been recorded as occurring in the combined state and entering into the composition of plant materials ; these are arabinose, xylose, ribose, and apiose. The structural formulae of these substances is given below, in order that their relationship to the hexoses and to each other may be appreciated : — OH CHO Hi. HOC.H HOC.H H . C— OH i Arabinose CHO HCOH HOC . H HCOH I H . C . OH I H Xylose CHO HCOH ■i HCOH HCOH I HC— OH H Ribose CHO H . C . OH C.OH CH,OH CHoOH Apiose GENERAL PROPERTIES OF PENTOSES. A number of colour reactions are available for the charac- terization of pentoses. 1. Thomas's Reaction.-\ — A freshly prepared 0*3 per cent solution of ^-naphthol in concentrated sulphuric acid is care- fully poured down the side of a test-tube containing a few cubic centimetres of the solution to be tested ; if a pentose is present a deep blue ultramarine ring is formed at the junction of the two liquids on gently shaking ; the colour gradually changes to green-brown. 2. BiaVs Reaction. — To a few cubic centimetres of the reagent raised to boiling-point in a test-tube, add a few drops of the pentose or pentosan solution and raise again to boiling- point. A green colour indicates a pentose, a methyl pentose, or glucuronic acid. The colour is soluble in amyl alcohol, and shows an absorption band between the C and D lines. 3. Add a small quantity of powdered gum-arabic to a few cubic centimetres of 18 per cent of hydrochloric acid together with a few crystals of phloroglucinol ; place in a water bath and gradually raise to boiling-point ; remove from time to * It will be seen that apiose represents an abnormal type of sugar possessing a branched chain. t Thomas : " Bull. Soc. Chim. Biol.," 1925, 7, 102. PENTOSES 91 time and watch for the appearance of a reddish-violet colour ; when this appears, remove from the water bath, cool, and shake up with amyl alcohol ; the solution in the alcohol has an absorption band between the D and E lines. 4. When boiled with 12 per cent of hydrochloric acid or sulphuric acid, pentoses give rise to furfural which is carried off by the escaping steam ; if this is allowed to impinge upon a filter paper moistened with a drop of aniline acetate a bright pink colour is formed. CHOH— CHOH CH— CH II I! II CHj CHOH . CHO — 3H2O = CH C . CHO \ \/ OH O Methyl pentoses under these conditions give methyl fur- fural, while hexoses give small quantities of hydroxy-methyl- furfural. CHOH— CHOH CH— CH 11 " I' CH2OH— CHOH CHOH CHO — 3H2O = CH^OH C C . CHO O All furfural derivatives give similar colour reactions to fur- fural both with aniline acetate and with phloroglucinol and orcinol (see below) ; were it not for the fact that hydroxy- methylfurfural is itself readily decomposed further into levu- linic acid and formic acid, CeHgOa + aH^O = HCOOH -f CH3 COCH^ CH, COOH neither of which give the above colour reactions, the test described would not be specific for pentoses. In carrying out the above test it must therefore be borne in mind that a very faint positive reaction should not be taken as evidence of the presence of pentose without further evidence. This reaction has also been made the basis of a method for the quantitative estimation of pentoses (see p. 137). 5. Pentoses reduce Fehling's solution and yield osazones but are not fermentable. 92 THE CARBOHYDRATES PROPERTIES OF INDIVIDUAL PENTOSES. Arabinose. Arabinose is best obtained by the hydrolysis of cherry gum with 4 per cent sulphuric acid ; it can also be obtained by the hydrolysis of gum-arabic and of peach gum and mesquite gum * (Prosopis jutiflora). Arabinose has a very sweet taste, is dextro-rotatory, ac in lO per cent solution =+ 105°, crystal- lizes in prisms, and melts at 160° ; it reduces Fehling's solution, and yields with diphenyl hydrazine a characteristic diphenyl hydrazone, melting at 204-205°. f Xylose. Xylose may be obtained by the hydrolysis of xylane or wood gum, and also from brewers' grains, maize, fruits, straw, and various forms of cellulose. It is a very sweet substance and shows an optical activity of aj) =+ 19° i^i a 10 per cent solution, it crystallizes in prisms, melting at 144-145°, and gives a phenylosazone of melting-point, 161°. When oxidized with bromine and boiled with cadmium carbonate it yields cadmium xylonate, which with the cadmium bromide in solution forms a sparingly soluble crystalline double salt (C5H906)2Cd . CdBrj . 2H2O.J Xylose may be conveniently obtained, in about a 12 per cent yield, by boiling i kg. of corn cobs § (previously soaked and washed in 2 per cent ammonia solution) for two hours under a reflux condenser with 8 litres of 7 per cent sulphuric acid. The solution is filtered on a Buchner funnel through cloth, and is then carefully neutralized with precipitated chalk. After filtering, the solution is treated with lead acetate, filtered, freed from lead by hydrogen sulphide, again filtered and * Anderson and Sands : " Ind. Eng. Cliem.," 1925, 17, 1257. t Neuberg : " Ber. deut. chem. Gesells.," 1900, 33, 2243 ; ToUens and Maurenbrecher : id., 1905, 38, 500. X Widstoe and ToUens : id., 1900, 33, 136. § Hudson and Harding : " J. Amer. Chem. Soc," 1917, 39, 1038 ; 1918,40,1601. Ling and Nanji : " J. Chem. Soc," 1923, 123, 620. Irvine, loc. cit. PENTOSES 93 decolorized with animal charcoal. The filtrate is evaporated under reduced pressure and the calcium sulphate precipitated by the addition of alcohol ; the filtered solution is then evaporated to a viscous syrup and crystallized from alcohol or from glacial acetic acid. Ribose. This pentose has been obtained as a product of the hydrolysis of yeast nucleic acid. According to Robinson * it is probably not pre-existent in this substance but is pro- duced by optical inversion during hydrolysis from the xylose contained in the nucleic acid. Apiose. This is a rare pentose obtained by the hydrolysis of the glucoside apiin contained in parsley ; it yields a bromo- phenylosazone, m.p. 211-212°. Owing to its abnormal struc- ture (see formula, p. 90) it does not yield furfural when heated with hydrochloric acid and gives no colour with phloroglucinol and hydrochloric acid. Methyl Pentoses. There is no evidence that methyl pentoses occur free in the plant ; they are, however, associated with the pentoses as cell wall constituents, and also occur as glucosides. Their constitution is represented by the formula CH3CHOH(CHOH)3CHO When heated with 10 c.c. of concentrated hydrochloric acid and 2 c.c. of acetone, the methyl pentoses give a violet colour which is permanent, in contradistinction to pentoses, which also yield a violet colour which, however, fades within one hour. Heated with concentrated hydrochloric acid, methyl pentoses give off methyl furfural which with aniline acetate gives a yellow colour ; whereas furfural which would be * Robinson : " Nature," 1927, 120, 44, 656. 94 THE CARBOHYDRATES obtained from pentoses under the same conditions gives a pink colour. [a] Rhamnose has been obtained by the hydrolysis of a number of glucosides, e.g. quercitrin, hesperidin, and xantho- rhamnin, and also saponins. The substance forms glistening crystals, m.p. 93° ; a^ =+ 8-07°, and gives a phenylosazone melting at 180°, and a naphthyl hydrazone melting at 192°. [h) Fucose, which is isomeric with rhamnose, may be obtained by the hydrolysis of sea-weeds by means of dilute sulphuric acid ; it crystallizes in microscopic needles, and yields a hydrazone, m.p. 172-173°. (c) Quinovose, another methyl pentose isomeric with rham- nose, is produced by the hydrolysis of quinovite, a substance formed by boiling quinovin contained in the bark of Cascarilla hexandra with alcohol and hydrochloric acid. [d) Isorhamnose and Rhodeose are two methyl pentoses obtained by the hydrolysis of the glucoside convolvulin. B. HEXOSES. Theory accounts for the existence of no less than thirty- two sugars of the molecular formula CgHiaOe having a straight six carbon atom chain. Of these sixteen are aldoses of the type CHgOH . CHOH . CHOH CHOH CHOH . CHO Aldohexose and the remaining sixteen are ketoses containing the ketonic group attached either to the second or third carbon atom of the chain — CH2OH CHOH . CHOH CHOH . CO . CHjOH or CH2OH CHOH CHOH CO . CHOH CH^OH 2 Ketohexose 3 Ketohexose The various possible isomeric aldoses and ketoses differ only in the spatial relationships of the OH and H groups. Although most of the aldoses and a few of the ketoses have been synthesized, only three aldoses, glucose, mannose, and galactose, and two ketoses, fructose and sorbose, have so far been identified in nature. HEXOSES 95 The following formulae illustrate the relationship between the five naturally occurring hexoses :■ — CHO HCOH I HOCH HCOH HCOH I HCOH H Glucose CHO in HO' I HOCH HCOH HCOH I HCOH H Mannose CHO HCOH I HOCH I HOCH HCOH I HCOH H Galactose H HCOH io I HOCH HCOH HCOH I HCOH H Fructose H HCOH io I HCOH HOCH I HCOH I HCOH H Sorbose From these formulae it will be seen that the spatial arrange- ment of the third, fourth, fifth, and sixth carbon atoms in glucose, mannose, and fructose is identical ; for this reason they all give the same osazone when once the first and second carbons have been condensed with phenylhydrazine ; on the other hand, they all give different hydrazones in which only the terminal carbon atom i is involved, leaving the rest of the chain from carbon atoms 2-6 different in each case. Further evidence for the close relationship existing between the three sugars, glucose, fructose, and mannose is furnished by the fact that if a 5 per cent solution of any one of these three sugars is treated with one-tenth of its volume of 10 per cent caustic potash and left in an incubator for twelve to twenty-four hours at 37° C, the solution will be found to contain all three sugars. This may be accounted for by assuming that the two terminal links in the six carbon chain of all three sugars can undergo molecular rearrangement to the so-called enolic form as under — ■ CHO H— C— OH Glucose CHO HOCH 1 Mannose CH2OH io Fructose CHOH II COH I Enolic modi- fication in which form they all become identical, and the change back from the enol form may give rise to any or all of the three. The significance of this lies in the explanation it offers for the possible interconversion of these sugars in the plant. 96 THE CARBOHYDRATES DISTINCTION BETWEEN ALDOSES AND KETOSES. To distinguish an aldose from a ketose use is made of the fact that on heating with concentrated hydrochloric or hydro- bromic acid a ketose is more readily converted into chloro- or bromo-methylfurfural than is an aldose, as may be seen from the formulae : — CHOH-CHOH CHOH-CHOH CHpHCHOH— CH CHOH CHjOH— CH COH CHjOH O O Aldose Ketose CH CH li II CHXIC C . CHO O Chloro-methj'lfurfural The production of the furfural derivative from the ketose involves much less rearrangement than from the aldose. On this fact depends the two reactions of Seliwanoff and of Fenton.* Seliwanoff Reaction. — Warmed on a water bath with an equal volume of concentrated hydrochloric acid and a crystal of resorcin, a ketose solution turns rapidly red while a hexose develops a colour much more slowly. There are no convenient general reactions for distinguish- ing hexoses as a class from any other group of sugars, but each of the hexoses occurring in nature is readily identified by characteristic reactions. GLUCOSE OR DEXTROSE. Occurrence. The substance which is commonly known as grape sugar occurs, together with levulose or fruit sugar, in a number of sweet fruits, in honey, and in the seeds, leaves, roots, and blossoms of a great many of the higher plants. Glucose is formed by the hydrolysis of cane sugar, of glucosides, and of many polysaccharides, such as starch, cellulose, etc. * Fenton and Gostling : " J. Chem. Soc," 1901, 79, 361, 807. GLUCOSE 97 Preparation. The most convenient source for the preparation of glucose on a small scale is cane sugar. One hundred and twenty c.c. of 90 per cent alcohol mixed with 5 c.c. of fuming hydro- chloric acid are heated at 45-50° ; 40 grams of powdered cane sugar are now added, the mixture being kept thoroughly stirred. After two hours the solution is allowed to cool, and a little anhydrous glucose is added to induce crystallization. In the course of a few days the resulting crop of crystals is filtered off and washed with a little dilute alcohol ; it is recrys- tallized by dissolving in half its weight of warm water and adding twice as much 90-95 per cent alcohol, filtering warm and setting aside to cool. On a commercial scale glucose is best prepared by heating freshly prepared potato or maize starch freed from nitro- genous material with dilute sulphuric * acid in sealed copper vessels under 3 atmospheres pressure for half an hour. When the hydrolysis is complete, the acid is removed as calcium sulphate by the addition of powdered chalk, and the filtered solution, after being decolorized by means of animal charcoal is evaporated in a vacuum ; a little anhydrous glucose is then introduced, and the syrup is allowed to crystallize, the crystals being separated from the mother liquor by means of the centrifuge. Prepared in this way the glucose forms a rather soft cake of small crystals of the hydrate CgHigOe . HgO ; it is liable to contain small quantities of maltose, isomaltose (p. 117), and dextrin from which it may be further purified by crystallization from alcohol. Commercial dextrose is employed as a substitute for cane sugar for the sweetening of cheap jams, etc., but its sweetness is only about two-thirds that of cane sugar. In the United States it is used largely in the manufacture of ice cream, chewing gum, etc., and owing to its high osmotic pressure and low sweetening power it is recommended for use in condensed milk. * More recently the use of hydrochloric acid has been recommended ; this involves a modification of the technique for the removal of the acid. 7 98 THE CARBOHYDRATES Properties. Glucose separates from alcoholic solution or from concen- trated aqueous solutions at 30-35° in needle-shaped crystals, which are anhydrous ; from cold aqueous solutions, however, it crystallizes with one molecule of water (CgHiaOe . HgO) in the form of plates. It is readily soluble in water, but only very slightly soluble in absolute alcohol. It is readily fer- mented by yeast. Glucose is dextro-rotatory, a^ = 52-3° ; it is sometimes known as dextrose to distinguish it from the laevo-rotatory sugar levulose with which it is frequently found associated in ripe fruits. Reactions. 1. In the presence of ammonia, glucose can reduce silver from its salts. A little glucose is added to a solution of silver nitrate to which have been added a few drops of caustic potash and just sufficient ammonia to redissolve the brown precipitate. On warming the mixture the silver is deposited on the sides of the test tube, forming a mirror. 2. Nylander's Test. — When boiled with a solution of glucose Nylander's reagent turns brown and finally black owing to the precipitation of bismuth oxide and metallic bismuth. The reagent is prepared by dissolving 2 grams of bismuth oxynitrate and 4 grams of Rochelle salt in 100 grams of 10 per cent caustic soda solution. 3. Add to the solution basic lead acetate and ammonia. If glucose be present, a white precipitate comes down, which turns red. This reaction is not given by cane sugar. 4. Add to the solution a little copper sulphate solution and an excess of caustic potash. On warming, a yellow to red precipitate is formed. This reaction also is given by levulose, maltose and other reducing sugars. 5. On warming with Fehling's solution, a red precipitate is given by dextrose, levulose, maltose, and other reducing sugars. 6. Add a little Barfoed's reagent and warm. A red GLUCOSE 99 precipitate floating as a thin film on the surface of the liquid indicates dextrose. This reaction is also given by levulose and other hexoses but not by cane sugar or maltose. The reagent, which should be freshly made up, is prepared by dissolving 5 grams of copper acetate, and 5 grams of sodium acetate, in 100 c.c. of water containing i c.c. of glacial acetic acid. 7. The addition to the solution of picric acid and caustic soda results in the formation of a blood-red coloration, due to picramic acid. This reaction is also given by other reducing sugars. 8. On boiling the solution of glucose with an equal volume of caustic potash, a yellow-brown colour results ; on acidifying with dilute nitric acid the colour lightens and a smell of burnt sugar is produced. 9. Glucose reacts with phenylhydrazine to give an osazone. To 5 c.c. of an approximately 5 per cent solution of glucose, add 4 or 5 drops of phenylhydrazine and about the same amount of glacial acetic acid. (If phenylhydrazine hydro- chloride is used, add about enough solid to cover a threepenny piece and an equal .quantity of sodium acetate.) Place the mixture in a boiling water bath for about half an hour and then remove ; a golden yellow crystalline precipitate will have been formed. On examination under the microscope the needle-shaped crystals will be seen to be gathered together in clusters resembling wheat sheaves. Glucosazone melts at 204-205° with decomposition ; it is insoluble in water but soluble in alcohol, the solution being laevo-rotatory in contra- distinction to that of maltose which is dextro-rotatory. The constitutional formula of glucose is given on p. 84. Microchemical Tests. For microchemical tests for sugars, the reduction of copper salts in the presence of excess of alkali is generally employed, but these are not altogether satisfactory, owing to the amount of diffusion which takes place, and also because sucrose, if its presence in a tissue be suspected, must first be hydrolysed by boiling with acid before the reduction will take place. loo THE CARBOHYDRATES Mangham * and others have obtained excellent results by the use of the osazone test for microscopic work ; if properly performed, it is much more satisfactory than any other, and has the advantage of being a very delicate test for some sugars. For example, a -015 per cent solution of glucose will give a definite reaction. The main disadvantage of the method is in its comparative slowness. Two solutions are required : — {a) I gram of phenylhydrazine hydrochloride dissolved in 10 grams of glycerol. {b) I gram of sodium acetate dissolved in 10 grams of glycerol. If necessary the solution of these substances may be hastened by means of heat, and before use the solutions should be filtered. Glycerol is used because its penetrative power is greater than that of water, and also because it will not evaporate and deposit crystals of the substances used. For use, one drop of each fluid is placed on a glass slip and mixed thoroughly. The section, which must be more than one cell in thickness, is laid in the mixture and covered with a cover glass. The preparation is heated on a hot water oven for about half an hour, and is then allowed to cool ; the osazone crystals will form in varying degrees of rapidity. In order that familiarity with the method may be gained, the reagents may be mixed on the slip with drops of sugar solution of different concentrations heated for varying periods and examined periodically after cooling. Maltose gives an osazone characterized by dense rosettes of lemon-yellow crystals, which are broader and larger than those obtained with dextrose and levulose. Dextrose and levulose may be distinguished by the fact that methylphenylhydrazine gives a crystalline osazone with levulose and not with dextrose. * Mangham: "New PhytoL," 1911, lO, 160; "Ann. Bot.," 1915. 29, 360. HEXOSES ror FRUCTOSE OR LEVULOSE. Occurrence. Fructose occurs in most sweet fruits and in honey, together with both cane sugar and dextrose, but usually in exoess of the latter two. It is formed in equal quantity with dextrose by the hydrolysis of cane sugar, and the resulting mixture, known as invert sugar, may occur in sucrose-producing plants, such as sugar beet and sugar cane, if kept for some time after gathering. Much discussion has centred around the origin of levulose in the actively assimilating leaf. It is often considered to be chiefly employed in building up new tissue whilst the glucose is consumed in respiration (see Vol. II.). It may be more abundant than glucose as in Galanthus nivalis * and in oat straw, a subject which is considered on page no. Preparation. The separation of pure levulose from invert sugar on a small scale is not easy to carry out, but the operation is per- formed on a large scale by making use of the fact that on treating invert sugar with milk of lime the levulose is converted into an insoluble calcium compound, which may be filtered off and purified, while the glucose remains in solution. The easiest means of preparing levulose in the laboratory is to hydrolyse inulin by boiling i part of this substance with 5 parts of -5 per cent sulphuric acid f for one hour ; the acid is then removed by means of barium carbonate, and the solution, after being treated with animal charcoal and filtered, is eva- porated at a low temperature to a thin syrup. The latter is then crystallized from alcohol after sowing with a crystal of pure levulose. A modification of this method is employed for the manufacture of pure levulose. | * Parkin : " Biochem. Journ.," 1912, 6, i. t Diill (" Chem. Zeit.," 1895, 19, 216) recommends the use of oxalic acid ; see also Wiechmann : " Z. d. Vereins Deut. Zuckerind.," 1891, 41, 331- + Cf. Stein : " Proc. Internat. Confer. Sugar lud.," April, 1908. 102 THE CARBOHYDRATES Properties. Levulose separates from alcohol in hard rhombic crystals, which have the composition CgHiaOg ; from concentrated aqueous solutions, however, it crystallizes in needles with water of crystallization 2C6H12O6 . H2O. It is fairly soluble in hot absolute alcohol and ether, and may thus be separated from other sugars which are insoluble in these solvents. Levulose is strongly leevo-rotatory and exhibits slight muta-rotation ; its rotatory power is very dependent on temperature, a^^" = — 93° in a 10 per cent solution. Reactions. 1. To a solution of levulose mixed with an equal volume of concentrated hydrochloric acid a few grains of resorcin are added. On warming, a deep red coloration results, and finally a brown-red precipitate. The precipitate is soluble in amyl alcohol, giving a deep red solution. This reaction is given by all keto-hexoses and by carbo- hydrates such as cane sugar and raflfinose which give rise to them on hydrolysis. 2. Levulose gives the same reactions as dextrose with salts of copper and picric acid. 3. Levulose with milk of lime forms an insoluble com- pound ; dextrose does not. 4. Levulose gives with phenylhydrazine the same osazone as glucose, namely glucosazone. 5. With methylphenylhydrazine it gives, in alcoholic solution, an osazone * crystallizing in needles ; m.p. 158-160°. (Distinction from glucose and mannose.) Constitutioji. Fructose is a 2 keto-hexose whose constitution may be represented by either of the two formulae : — * Neuberg : " Ber. deut. chem. Gesells.," 1902, 35, 961. HEXOSES 103 -O- CH2— (CHOH)3— C— OH— CHoOH I Normal crystalline fructose CHjOH— CH— (CHOH).,— C— OH . CH.pH II y-fructose Haworth and his fellow-workers * from their considerable experimental work conclude that the normal form of hexoses, both aldehydic and ketonic and of pentoses, is the amylene oxide form, and accordingly the ordinary form of fructose is represented by the formula I. It has, however, been shown by Irvine and Steele f that fructose, as it occurs in sucrose and inulin, is present in a so-called y-form which is more active than in its normal state. The y-form, which has the butylene oxide configuration shown in formula II., differs from ordinary, or normal, fructose in the fact that it reduces potas- sium permanganate readily ; it may be produced by leaving ordinary fructose in contact with acid for an hour and then neutralizing ; the solution has thus acquired the power of decolorizing permanganate. SORBOSE. Sorbose is a 2 keto-hexose, isomeric with fructose, of the formula— OH H OH i- I I I H OH H It does not occur naturally, but is produced by the oxidative action of Bacterium xylinum upon the alcohol sorbitol ; it was, in fact, first isolated from the juice of Pyrus aucuparia which had been kept for some months exposed to the air. It has since been shown that the fresh juice contains no sorbose but only the corresponding alcohol sorbitol. Bertrand :j: * Haworth and Hirst : " J. Chem. Soc," 1926, 1858. Avery, Haworth, and Hirst: id., 1927, 2308. Goodyear and Haworth : id., 1927, 3140. t Irvine and Steele : id., 1920, 117, 1474. X Bertrand : " Ann. Chim. Phys.," 1904 [8], 3, 200. CH,OH . C— C ^C— CO . CH2OH 104 THE CARBOHYDRATES subsequently found that Bacterium xylinum had the peculiar power of oxidizing a — CHOH group to CO provided the hydroxyl was adjacent to another hydroxyl group on the same side of the molecule ; thus it could oxidize mannitol or sorbitol which contain the grouping — H H — C C CH^OH OH OH but not dulcitol which contains the grouping — H OH — C C— CHjOH OH H Sorbose is not fermentable by yeast. GALACTOSE. OccMrrefice. This sugar has rarely been recorded as occurring free in nature. Von Lippmann * claims to have found it on ivy berries after a sudden frost, which is analogous with the in- crease of the rafhnose content of sugar beet under like con- ditions. In its polysaccharide form of galactan, galactose forms a constituent of mucilages, such as agar and carrageen obtained from sea weeds ; it also occurs in the gums of the peach and plum, and is a constituent of the pectic substances of carrot, turnip, and many fruits such as apple and pear. In all these instances galactose is accompanied by other sugars, which may be either hexoses or pentoses. Further, galactose is a constituent of the glucoside digitalin, of the anthocyan idaein, and forms the carbohydrate constituent of the group of lipins known as cerebrosides and galactolipins. In all cases it may be set free by hydrolysis with mineral acid. Preparation. A convenient material for the preparation of galactose is agar, which on hydrolysis yields a mixture of sugars amongst * Von Lippmann : " Ber. deut. chem. Gesells," 1910, 43, 361 1. GALACTOSE 105 which galactose predominates. Pure galactose is, however, more easily prepared from lactose ; for this purpose i kg. of lactose is boiled for two hours with 2*5 litres of water containing 50 grams of sulphuric acid ; the solution is neutralized with barium carbonate, filtered, and concentrated ; the galactose is crystallized by the addition of a mixture of I part of ethyl with 2 parts of methyl alcohol.* The estimation of galactose depends upon its oxidation by nitric acid, under specified conditions, to mucic acid and weighing the latter ; the results obtained vary in the hands of different workers, and considerable practice is required to obtain consistent values. Properties. Galactose crystallizes in minute hexagonal crystals, which melt at 164°. It is strongly dextro-rotatory, a^ = 8i'5°, and exhibits muta-rotation. Galactose after association with ordinary yeast for some time is fermentable, but it is not acted upon by 5. Ludimgii and S. anonialus. Detection. 1. The hexagonal form of the crystals is characteristic of galactose. 2. It gives a methylphenylhydrazone (m.p. 190-191°). 3. It reduces Fehling's solution somewhat more slowly than glucose ; 10 c.c. Fehling's solution =0*5 1 gram galactose. 4. On oxidation with nitric acid it yields mucic acid. Five grams of substance are heated in a beaker with 6 c.c. of nitric acid (sp. gr. I'I5) until two-thirds of the liquid have been evaporated off. After twelve hours the mucic acid formed will have separated, and may be washed with lO c.c. of water. If other insoluble substances are present, place the filter paper with the solid in a dilute solution of ammonium carbonate to extract the mucic acid as ammonium salt. Filter once more, and evaporate the filtrate almost to dryness, and acidify with nitric acid ; the precipitate is pure mucic acid. * Dept. of Commerce, Bur. of Standards, Washington, No. 416. io6 THE CARBOHYDRATES MANNOSE. Occurrence. There is no record of the free occurrence of mannose in plants ; in its polymerized or polysaccharide form, however, it is widely distributed as a constituent of the so-called hemicelluloses contained in the cell walls of the seeds of peas, coffee, date, etc. It is also a constituent of salep mucilage [Orchis Morio). Preparation. Mannose may be prepared by the hydrolysis of the hemi- cellulose contained in the endosperm of ivory nuts, Phytelcphas macrocarpa, which are extensively used in the manufacture of vegetable ivory buttons. The turnings are added to ten times their weight of boiling i per cent caustic soda and allowed to stand for half an hour with occasional stirring. The liquor is then decanted off and the residue washed with water and dried ; 500 grams of this material are mixed with an equal weight of 75 per cent sulphuric acid and allowed to stand for twenty-four hours. The resulting substance is dis- solved in water, diluted to 5-5 litres, and then boiled for two and a half hours. The solution is neutralized with barium carbonate paste and filtered through a thin layer of animal charcoal. The last traces of barium are removed by the careful addition of dilute sulphuric acid and filtering. The filtrate is concentrated over a boiling water bath until it contains 87-88 per cent of total solids ; it is then mixed with an equal volume of glacial acetic acid, seeded with a few crystals of mannose and then frozen. On allowing the mass to thaw slowly in a refrigerator, the mannose will crystallize out.* Properties. Mannose has a sweet taste ; when dry, it is a hard crumbling substance, which, however, deliquesces and is readily soluble in water ; it is only slightly soluble in hot alcohol and is * Clark : "J. Biol. Chem.," 1922, 52, i. Patterson: "J. Chem. Soc," 1923, 123, 1139. DISACCHARIDES 107 insoluble in ether. It is dextro-rotatory, [a]u^" =+ 14-36° in 10 per cent solution, but when freshly prepared it is laevo- rotatory. Mannose is readily fermentable by yeast. Detection. 1. Mannose is most readily detected and estimated by means of its phenylhydrazone, which is almost insoluble in water, and forms almost at once on adding phenylhydrazine acetate to an aqueous solution of the sugar ; the phenylhydra- zone is soluble in a very large volume of boiling water, and separates in fine prisms from the solution on cooling. These crystals melt at 195-200°. An excess of phenylhydrazine converts mannose into glucosazone, which is identical with the substance obtained under similar conditions from both glucose and fructose. 2. Mannose reduces Fehling's solution, 10 c.c. = -04307 gram mannose. C. HEPTOSES. A number of heptoses of the formula C7H14O7 have been synthesized, but only two are known to occur naturally. One of these, mannoketoheptose, occurs in the avocado pear, Persea gratissima, and the other, sedoheptose, in the stonecrop, Sedum spectabile.* Both are ketoheptoses and are not fer- mented by yeast. DISACCHARIDES. The disaccharides, as is implied by their name, give rise on hydrolysis to two molecules of monosaccharide which may both be hexoses, or one may be a hexose while the other is a pentose ; the latter type of pentosehexose disaccharide, which is comparatively rare, is dealt with on page 121. The true hexose disaccharides of the general formula C12H22O11 may be divided into two classes : — ■ (a) Those giving rise on hydrolysis to two molecules of the same hexose, such as maltose, isomaltose, cellobiose, iso- cellobiose, gentiobiose, trehalose, and isotrehalose. * La Forge : " J. Biol. Chem.," 1920, 42, 367. io8 THE CARBOHYDRATES (b) Those giving rise on hydrolysis to two different hexoses such as sucrose, turanose, lactose, and melibiose. The isomerism between the various members of the first group may be due to a different mode of attachment of the two hexoses, involving in some cases the reducing groups so that, as in the case of trehalose, sucrose, and turanose, the resulting disaccharide has no reducing properties. On the other hand, two structurally identical sugars may differ in stereochemical formula, i.e. in the spatial arrangement of the two constituent sugars with the resultant production of two isomeric a- and j8-disaccharides corresponding to the a- and j8-glucoses ; such a relationship is found to exist between maltose and isomaltose, the former of which is hydrolysed by maltase while the latter is only attacked by emulsin. Similarly, the disaccharides gentiobiose and cellobiose appear to belong to the jS-glucosides, since they are not attacked by maltase but are acted upon by emulsin. In addition to the above considerations, an exact knowledge of the nature of the anhydride ring of the constituent mono- saccharides is requisite for a complete understanding of the constitution of given disaccharide ; this may be seen by the alternative formulae given for glucose on page 83. Action of Enzymes on Disaccharides. {a) Hydrolytic Enzymes. — The hydrolysis of disaccharides is effected by enzymes such as maltase and emulsin, which act on more than one substrate, and in some cases the hydrolysis can only be effected by a specific enzyme such as invertase (sucrase), which acts only upon sucrose. Attempts to utilize enzymes for the synthesis of disac- charides as well as for their hydrolysis were initiated by Croft- Hill who, by acting upon a solution of glucose with a yeast extract of maltase, was able to synthesize a disaccharide to which he gave the name of revertose, but which was subse- quently identified as isomaltose, the jS-glucosidic isomer of maltose. Since then, largely as the result of the work of Bourquelot * and his co-workers, gentiobiose, cellobiose, and * Bourquelot : " Ann. d. Chim.," 1920, (9), 13, 5. DISACCHARIDES 109 a number of similar disaccharides and glucosides have been synthesized. The two sugars, sucrose and maltose, have, however, so far resisted all attempts at their synthesis by enzymes, although both have been synthesized by chemical means.* {b) Fermenting Enzymes. — Contrary to the assertion of Fischer that disaccharides are not attacked by yeasts until they have been hydrolysed by the appropriate enzyme con- tained in the yeast, Willstatter f concluded that both maltose and lactose are directly fermentable, since he was able to effect fermentation of these by distillery yeasts which contained only very little maltase and were entirely free from lactase. The fact that Saccharomyces Marxianus, which is known to be free from maltase, is unable to ferment maltose, he attributes to its not possessing a maltozymase rather than to any deficiency in maltase. CANE SUGAR, SUCROSE OR SACCHAROSE. Occurrence. Cane sugar is one of the most widely distributed substances to be found in the vegetable kingdom. Besides forming about 20 per cent of the juice of the sugar cane, Saccharum officinaruni, and about 10-20 per cent of that of the beetroot, it is found in varying quantities in the wood of m.aple and birch, and in Sorghum saccharatum ; it occurs, moreover, in wheat, maize, barley, in carrots, and in madder root. In most sweet fruits it is found together with a greater or lesser quantity of dextrose and levulose, which may possibly have been formed from it by hydrolysis. It also is found in the leaves of many plants associated with glucose and maltose. The following table, compiled by Kulisch, gives the relative proportions of cane sugar and hexoses found in various fruits. Pineapple . Strawberry . Apricot Ripe banana Apple * Pictet : " Bull. Soc. Chim.," 1920 "Compt. rend.," 1928, 186, 727. t Willstatter : " Zeit. physiol. Chem., Cane Sugar. Hexoses. 11-33 1-98 6-33 4-98 604 2-74 500 1000 1-5-40 7-13-00 [4]. 27, 650. Pictet and Vogel : ." 1925. 150, 287. I 10 THE CARBOHYDRATES In honey practically only invert sugar is found, although the sugar found in the flowers by the bees is commonly cane sugar. The hydrolytic agent in this case is most probably the formic acid secreted by the bees. Cane sugar also has been recorded as occurring in Sphagnum, Hypnum, and Pellia* The relative proportions of the three sugars sucrose, levulose, and dextrose in certain plants have been studied by Collins & Gill.f Thus in the case of Helianthus tuherosus, they found that during the period August-December, the period of formation of the tubers and thus of translocation, the total sugar of the stalks reaches a maximum and then falls to a low value in December. The amount of sucrose and levulose follows a similar course, but the dextrose, which is in greatest abund- ance in August, shows a sudden drop in September and then increases, so that in December it is the chief sugar present, being more than twice as abundant as either sucrose or levulose. The accompanying table gives the actual figures calculated to percentages of the living plant : — Aug. Sept. 4. Oct. 2. Oct. 30. Dec. 13. Sucrose Dextrose Levulose Total sugar 0-I5 0-37 0-29 0-36 Oil i-i8 I-I5 0-27 0-90 035 0-55 1-37 0-24 0-58 0-24 o-8i 1-65 2-32 2-27 I -06 In the instance of oat straw, the preponderating sugar at the end of vegetative activity is levulose not dextrose, which suggests that the nature of the reserve material determines the variety of the residual sugar. In the artichoke, the for- mation of inulin means the fixation of levulose, wherefore there will be a surplus of dextrose. In the oat, on the other hand, dextrose is converted into starch so that there is a residuum of levulose. In this argument Collins and Gill con- clude that the hexoses have their origin in sucrose. In the * Goris and Vischniac : " Bull. Sci. Pharm.," 1913, 20, 390. t Collins and Gill : "J. Soc. Chem. Ind.," 1926, 45, 63 T. SUCROSE III development of the tuber, the following table gives the analysis of samples expressed in percentages of dry matter : — Oct. 2. Oct. 30. Dec. 13. Total sugar, including inulin Free-reducing sugar Levulose .... 46-76 7-03 6-72 52-87 5-20 4-82 5.5-27 8-24 8-54 In this connection brief allusion may be made to the work of Miller * on sorghum and maize in the leaves of which the maximum of sugars was reached between noon and 5 p.m. after which there was a gradual decrease until dawn. The water-insoluble carbohydrates reached a maximum later than the sugars and a decrease did not begin till about midnight, the minimum being at about dawn. It was also observed that the reducing sugars varied far less than the non-reducing sugars over the twenty-four hours. The conclusion drawn by many that sucrose is the first sugar of photosynthesis is a matter of dispute, an aspect of the subject which is considered in the second volume of the present work. Preparation. The two chief sources for the preparation of cane sugar on a manufacturing scale are the sugar cane and the beet. The processes used in both cases are more or less similar, and con- sist in obtaining, purifying, concentrating and, lastly, crystal- lizing the juice. The juice is generally obtained from the cane by crushing, as much as 85-95 per cent of the juice being expressed in this way ; in some cases it is extracted by diffusion, which consists in immersing the cane in water, when the sugar diffuses out of the cells into the surrounding water while the indiffusible colloids remain behind. The crude juice is then boiled with milk of lime, in order to neutralize any acid present and to precipitate coagulable proteins, and is subse- quently treated with sulphur dioxide. After filtering, the solution is concentrated in a vacuum and allowed to crystallize, * Miller : "J, Agric. Res./' 1924, 27, 785. 112 THE CARBOHYDRATES the mother liquor being separated by centrifugalizing ; the crystals may be used at once as brown sugar, or may be refined. When the beet is used, the roots are first cut into slices and subjected to diffusion, the same quantity of water circu- lating through a series of vessels in such a manner that the fresh water first passes over material from which most of the sugar has already been extracted, and as the solution becomes more concentrated, it comes into contact with material which is increasingly richer in sugar. In this way the aqueous extract attains a concentration of from 12-15 per cent.* This solution is then boiled with lime and saturated with carbon dioxide to decompose any calcium saccharosate which may have been formed ; it is then filtered and again saturated with carbon dioxide or a mixture of this gas and sulphur dioxide to pre- cipitate the last traces of calcium, and also to decolorize it ; the older process of filtration through animal charcoal is thereby rendered unnecessary ; the solution is then boiled and filtered and the clear filtrate is concentrated in a vacuum and allowed to crystallize. The uncrystallizable residue which remains is known as molasses ; a further yield of sugar may be obtained from this residue by the addition of lime to the cold solution or of strontia to the boiling solution whereby the cane sugar in the molasses is converted into the insoluble cal- cium or strontium saccharosate, which may be filtered off and decomposed by a current of carbon dioxide into cane sugar and calcium or strontium carbonate. The molasses are sometimes fermented for the manufacture of rum or may be used for cattle food ; they are also used in the manufacture of boot blacking. By suitable methods of cultivation, seed selection and use of nitrogenous and potash fertilizers the amount of sugar con- tained in the beet has been raised from 10'6 per cent in the period 1880-90 to about 15 per cent in the period 1900-IO, and the beetroot is gradually displacing the sugar cane as a source of sucrose. * The residue remaining after the extraction of the sugar is employed for cattle food. SUCROSE 113 Constitution. Whilst it has long been known that sucrose on hydrolysis yields molecular proportions of glucose and fructose, it was first shown by Irvine and Steele * that the fructose occurred in the y-form and not in its normal form in combination with glucose. The constitutional formula f for cane sugar, based on the amylene oxide formula for the glucose constituent and the butylene oxide formula for the y-fructose, is as follows : — XH o- CH^OH I O (CHOH)3 (CHOH)., O I CH CH 1 I I CHjOH CH2OH Glucose y-Fructose Repeated attempts to synthesize cane sugar from glucose and fructose failed owing to the fact that the fructose requires to be combined with the glucose in its active or y-form. Appreciating this fact, Pictet and Vogel | prepared the acetyl derivative of y-fructose, and uniting this with the acetyl derivative of glucose, by shaking the two in chloroform solution with phosphorus pentoxide, they obtained octacetyl sucrose which on hydrolysis yielded a compound showing all the characteristics of the natural sucrose. Properties. Cane sugar crystallizes from water in monoclinic crystals which do not contain water of crystallization ; it is readily soluble in water and only slightly soluble in alcohol ; it is dextro-rotatory, its specific rotation being a^ =+ 66-5. When heated to 160° it melts to a glassy mass known as barley sugar, which gradually becomes crystalline again ; if heated to 190-200° it is converted into an uncrystallizable * Irvine and Steele : " J. Chem. Soc," 1920, 117, 1474. t Haworth and Hirst : id., 1926, 1858. Avery, Haworth, and Hirst : id., igz'j, 2308. t Pictet and Vogel : " Compt. rend.," 1928, 186, 727. 8 114 THE CARBOHYDRATES brown substance known as caramel, which is used for colouring beer and wine. Reactions. 1. Solutions of cane sugar heated with concentrated hydrochloric acid turn reddish-pink. 2. If warmed with concentrated hydrochloric acid and a few crystals of resorcin a deep red colour is produced owing to the liberation of levulose. 3. Cane sugar does not react with phenylhydrazine. 4. Cane sugar does not reduce Nylander's reagent. 5. Solutions of cane sugar do not reduce Fehling's solution until they have been inverted by boiling for a short time with a few drops of dilute sulphuric acid ; if then made alkaline and boiled with Fehling's solution reduction ensues. If a solution in water is boiled with a few drops of mineral acid, the sign of the optical activity of the solution changes from -f to — . This change, which is known as inversion, is due to the fact that the mineral acid hydrolyses the cane sugar, converting it into equal molecular proportions of the two sugars dextrose and levulose, and since the optical activity of levulose is greater than that of dextrose the resulting invert sugar is laevo-rotatory. Aqueous solutions of cane sugar, if kept for some time, gradually become inverted, the change being somewhat accelerated by prolonged boiling. Extremely small quantities of acid suffice to effect the change in a boiling solution; thus 80 parts of cane sugar dissolved in 20 parts of water are completely hydrolysed by heating in boiling water for one hour with an amount of hydrochloric acid corresponding to 0-005 per cent of the weight of the sugar ; within certain limits, however, the action is accelerated by increasing the concentration of the acid. If, however, the acid is too strong and the heating be continued too long, the solution is liable to darken and decompose. Moreover, prolonged action, even at temperatures of 10-15°, of DISACCHARIDES 1 1 5 concentrated acids was found by Wohl * and by Fischer f to produce exactly the opposite phenomenon, known as reversion, by which the simple molecules, more especially those of levu- lose, are made to condense together to form complex dextrin- like substances, as well as a disaccharide iso-maltose. 6. Sucrose is fermentable by ordinary yeast, but this has been attributed to the fact that such yeast is possessed of invertase which hydrolyses the sucrose previous to its fer- mentation. TURANOSE. CiaHa^Oii. This is a disaccharide formed by the partial hydrolysis of the trisaccharide melecitose (see p. 124) ; it reduces Fehling's solution, and on hydrolysis yields glucose and levulose ; it is therefore isomeric with sucrose. MALTOSE. Ci^H^jjOii. Maltose does not appear to have so wide a distribution in the plant as has sucrose. The hydrolytic action of diastase on starch yields maltose — Starch Maltose Dextrin From this it might be expected that where starch is stored and subsequently digested, maltose would appear. But not infrequently maltase also is present by the action of which the maltose is converted into hexose sugars, so that if the preparation of the material is such as to destroy or to preserve maltase, maltose will or will not appear in the subsequent analysis. It is, possibly, for this reason that discrepant results have been obtained. Maltose has been described as occurring in the leaves of Tropceolum, Pyrola, Populus, and Linncea, whilst, on the other hand, its presence has been denied in the leaves of the snowdrop, potato, and mangold.J Gillot § describes the occurrence of maltose in the rhizomes and roots of Mercurialis perennis and, from the variations in amount, * Wohl : " Ber. deut. chem. Gesells.," 1890, 23, 2092. t Fischer : id., 1890, 23, 3687. X See Vol. II., chapter on " Photosynthesis." § Gillot : " Recherches Chimique et Biologiques sur le Genre Mer- curialis," Nancy, 1925. 8 • ii6 THE CARBOHYDRATES •25-2 per cent of dry weight, which obtain in the different phases of the life-history of the plant, he concludes that maltose, in this instance, is not a transitional sugar but a true reserve material comparable to starch and sucrose. In the germination of the barley maltose is produced, but it does not accumulate owing to the action of maltase which, as has already been stated, converts it into hexose. Maltose is also formed by the action of diastase and other enzymes on glycogen. In preparing maltose from starch, the diastase which is employed is usually introduced in the form of malt, which is barley that has been allowed to sprout and is then killed by suddenly heating to a temperature sufficient to stop the further growth of the barley without destroying the diastase. The malt is then stirred up with starch and water, and kept at a temperature of 60-62° for about half an hour ; by the end of this time about 80 per cent of the starch has been converted into maltose and 20 per cent into dextrin. Dextrin itself is also converted into maltose by diastase, but the reaction is very slow, and in practice sufficient time is not allowed to effect this change. Properties and Reactions. Maltose is readily soluble in water, and crystallizes from this solvent in slender white needles, having the composition C]2H220ii, H2O ; its aqueous solution is strongly dextro- rotatory : ai3=+ 137°; freshly made solutions exhibit a higher rotation than older ones, owing to a negative muta- rotation. 1. Maltose reduces Ny lander's reagent, but not Barfoed's reagent. 2. Maltose reduces Fehling's solution without previous hydrolysis, and can therefore be estimated directly by this means. 3. When treated with phenylhydrazine, as described under glucose, it gives an osazone (m.p. 206°), which is soluble in 75 parts of boiling water, and can be crystallized from this solvent in rosettes of plates or broad needles ISOMALTOSE 117 resembling sword blades ; alcoholic solutions of maltosazone are dextro-rotatory. (Distinction from glucosazone.) 4. On hydrolysis, by boiling with dilute mineral acid, maltose breaks up into two molecules of glucose — C12H22O,, + H,0 - aQHiPe the rotatory power of the solution being thereby diminished. 5. Maltose is fermentable by ordinary yeast, but not by S. Marxianus * and S. Ludwigii. As yeast ordinarily contains maltase, it was generally thought that hydrolysis by this enzyme was a preliminary to fermentation by zymase. Ac- cording to Willstatter,t however, a distillery yeast free from maltase is able to ferment sucrose at V^ 4-6, which is a degree of acidity at which maltase is unable to act. The constitutional formula assigned by Haworth and Peat % to maltose is — I ° 1 CHOH— (CHOH)2— CH— CH— CH2OH . O . O— CH— (CH0H)3— CH— CHjOH from which it appears that the union between the two glucose molecules is through the fourth carbon atom of one and the aldehydic carbon atom of the other ; it can therefore be de- scribed as a-glucosido-4-glucose. ISO-MALTOSE. CiaH^^Oi,. Not a little confusion exists with regard to the use of the term iso-maltose ; the name was first given to a sugar obtained by Fischer § by the action of concentrated hydrochloric acid upon glucose, and this same substance has since been shown to be formed also by the action of dilute hydrochloric acid upon strong solutions of glucose. Subsequent workers, || * Croft Hill : " Proc. Chem. Soc," 1901, 17, 45. t Willstatter : " Zeit. physiol. Chem.," 150, 287. X Haworth and Peat : " J. Chem. Soc," 1927, 844. § Fischer : " Ber. deut. chem. Gesells.," 1890, 23, 36S7 ; 1895, 28, 3024. II Georg and Pictet : " Helv. chira. Acta.," 1926, 9, 444. Berlin : " J. Amer. Chem. Soc," 1926, 48, 1107. ii8 THE CARBOHYDRATES however, claim that the action of acid on glucose yields a mixture containing gentiobiose in addition to iso-maltose. In a study of the reversible nature of enzyme action, Croft Hill,* in attempting to synthesize maltose by the action of maltase upon glucose, obtained some maltose and in addi- tion an unfermentable sugar which he termed revertose, dehberately avoiding the name isomaltose " because this designation has been applied to several differing substances and revertose is different from any of these." Later Arm- strong showed it to be a j8-glucoside and considered it to be identical with Fischer's iso-maltose. According to Lintner and Dullf malt diastase acting upon starch produces, in addition to maltose and dextrin, some unfermentable sugar, iso-maltose; this observation was subse- quently confirmed by Ling, but in the opinion of the latter author, Fischer's iso-maltose produced by the action of acid upon starch is not identical with that produced by diastase.|| A method for preparing iso-maltose, due to Ling and Nanji,$ consists in allowing a solution of precipitated malt diastase to act upon crude amylopectin, or upon a^-hexa- amylose prepared from it, at 50° until the rotatory power remains constant, and then fermenting away any maltose or glucose ; the mixture is then filtered, evaporated, and extracted with alcohol. Thus prepared, iso-maltose is a white, amorphous, hygro- scopic power having aD=+l40°; it forms an osazone, m.p. 150°, which is soluble in hot water or in absolute alcohol. Iso-maltose is not attacked by maltase but is hydrolysed by emulsin and is therefore a j8-glucoside ; it is not fermented by yeast. It should, however, be noted that Haworth,§ who ex- amined a sample of isomaltose prepared by Ling and Nanji, was unable to observe any structural difference between this sample and maltose itself. * Croft Hill : " Ber. deut. chem. Gesells.," 1901, 34. 1384- " J- Chem. Soc," 1903. 83, 580. t Lintner and Diill : " Z. angew. Chem.," 1892, 5, 268. + Ling and Nanji : " J. Chem. Soc," 1923. 123, 2681. § Haworth : " J. Soc. Chem. Ind.," 1927, 46, 300 T. . DISACCHARIDES 119 CELLOBIOSE. Ci,.H,.Pii. This is a disaccharide obtained from cellulose by the action of glacial acetic acid and acetic anhydride in the presence of concentrated sulphuric acid. The resulting acetyl deriva- tive, on treatment with alcoholic potash, yields cellobiose. It reduces Fehling's solution and gives an osazone melting at 198°. On hydrolysis, it yields two molecules of glucose and is thus isomeric with maltose, but unlike this sugar it is not hydrolysed by maltase but is attacked by emulsin. From these facts Haworth and Peat * conclude that cellobiose and maltose are structurally identical, differing only in the stereo- chemical configuration of their glucose residues. Thus cello- biose is represented by the same formula, as maltose (see p. 117) only is a j8-glucosido-4-glucose, whereas maltose is the cor- responding a-compound. Iso-cellobiose. An isomeric sugar, isocellobiose, was obtained in the form of its acetyl derivative together with cellobiose acetate on acetolysis of cellulose ; on hydrolysis of the acetyl derivative with baryta, iso-cellobiose f was set free. GENTIOBIOSE. Ci.Ha.On. This disaccharide % is obtained by the partial hydrolysis of the trisaccharide gentianose (see p. 125) ; by the action of emulsin it is converted into two molecules of glucose, from which it follows that gentiobiose is a j3-glucoside. It has been synthesized by the action of emulsin on glucose, § a method || which provides a more convenient source for its preparation, and also by the action of concentrated hydro- chloric acid on glucose. *[| Gentiobiose is the biose of * Haworth and Peat : " J. Chem. Soc," 1926, 3094. t Ost and Prosiegel : " Zeit. angew. Chem.," 1920, 33, 100. Ost : id., 19-26, 39, 1 1 17. X Zemplen : " Bar. deut. chem. Gesells.," 1915, 48, 233. § Herissey, Bourquelot, and Coivre : " J. Pharm. Chim.," 1913- 7> 44i- II Georg and Pictet : " Helv. chem. Ace," 1926, 9, 444. Berhn : " J. Amer. Chem. Soc," 1926, 48, 1107. M For this method of preparation, see Harding : " Sugar," 1922, 240. I20 THE CARBOHYDRATES amygdalin *; as the result of its synthesis f and from other considerations its constitution may be represented by the formula — i ° 1 i ° 1 CHOH . (CHOH)3 . CH . CH^ . O . CH . (CHOH)3 . CH . CHjOH from which it appears to be a j8-glucosido-6-glucose. TREHALOSE. C^^K.^O^i. Trehalose is a disaccharide very widely distributed among the fungi, J including ergot and myxomycetes,§ in moulds such as Aspergillus niger, in Selaginella lepidophylla \\ and in various Florideae.^ It does not reduce Fehling's solution and is strongly dextro-rotatory, ap = + 199°. When boiled with 5 per cent sulphuric acid for six hours, it is converted into two molecules of glucose.** It is also hydrolysed by the enzyme trehalase contained in many fungi. LACTOSE OR MILK SUGAR. Cy^R-^fi^i. This disaccharide, though of considerable importance in the animal kingdom, is never found in plants. It reduces Fehling's solution and on hydrolysis, by the enzyme lactase or by dilute mineral acids, it yields molecular proportions of glucose and galactose. MELIBIOSE. CiaH^jjOu. This disaccharide ff is not a naturally occurring sugar, but is produced by the partial hydrolysis of the trisaccharide raffinose ; it is dextro-rotatory, a^ = + 143°. It yields on hydrolysis molecular proportions of glucose and galactose. Owing to the fact that this sugar is hydrolysed by bottom fermentation yeasts but not by top fermentation yeasts, it * Haworth and Wylam : " J. Chem. Soc." 1923, 33, 3120. t Iwanoff : " Biochem. Zeit.," 1925, 162, 455. JBourquelot : " Bull. soc. mycol. France," 1905. 21, 50. § Helferich, Bauer, and Weigand : " Annalen," 1926, 447, 27. II Anselmino and Gilg : " Pharm. Zeit.," 1913, 58, 563 ; Lippmann : " Ber. deut. chem. Gesells.," 1912, 45, 343i- ^ Kylin : "Zeit. physiol. Chem.," 1915, 94, 337. ** Winterstein : id., 1894, 19, 70. tt Scheibler and Mittelmeier : " Ber. deut. chem. Gesells.," 1890, 23, 1438. DISACCHARIDES 121 may be used to distinguish between these varieties of Sac- charomyces. The constitution * of meHbiose is represented by the formula — CHOH . (CHOH)3 . CH— CHj— O— CII . (CHOH)3 . CH . CH^OH ! o ■ ' o Glucose Galactose DISACCHARIDES PRODUCED BY THE UNION OF A HEXOSE WITH A PENTOSE. Several disaccharides have been discovered which on hydrolysis yield one molecule each of a hexose and a pentose ; some of the more important of these are the following : — PRIMEVEROSE. C^^H^^Oi^. Primeverose is prepared from the glucosides primeverin and primulaverin occurring in Primula officinalis.'f This sugar has ai)— 379° and melts at 210°. It has a free aldehyde group and would therefore appear to have the constitution — CHO . [CH0H]4 . CH, . O . CH . CH . [CHOH], . CH„OH O This disaccharide has also been found to occur in the glucosides gentiacaulin and monotropitin, the latter of which occurs in Monotropa hypopitys, in the bark of Betula lenta, and in the fresh roots of Spircea Ulmaria, S. Filipendula, and S. gigantea. The fact that this carbohydrate has thus been found to occur in five families, namely, Betulaceae, Monotropeae, Primulaceae, Gentianaceae, and Rosacese, would indicate that it has a much wider distribution than was formerly suspected.^ VICIANOSE. C11H20O10. This disaccharide is obtained by the hydrolysis of the glucoside vicianin occurring in Vicia angustifolia, and gein * Charlton, Haworth, and Hickinbottom : " J. Chem. Soc," 1927, 1527. Haworth, Leach, and Long : id., 1927, 3146. t Goris and Vischniac : " Compt. rend.," 1919, 169. 871, 975. I Bridel : id., 1924, 179, 991. 122 THE CARBOHYDRATES obtained from Geum urbanum, and is found to be composed of one molecule of glucose and one of arabinose.* STROPHANTHOBIOSE. Ci^H.^Oio. This disacchaiide likewise occurs in a glucoside, stro- phanthin. On hydrolysis it yields mannose and rhamnose (CeHi^OJ.t TRISACCHARIDES. RAFFINOSE. C,oH,,0 18^-^32^ia- This sugar occurs in cotton seeds, barley, eucalyptus, lotus, $ and also in the beetroot ; the juice of this latter con- tains on an average about 15 per cent of cane sugar but only 0-02 per cent § of raffinose. The molasses from beet sugar re- fineries, however, contain from 2-3 per cent of raffmose (hence the name) and form the chief commercial source of this sugar. As the concentration of the raffinose increases it tends to crystallize out together with the cane sugar in the form of mixed crystals having a peculiar and characteristic pointed appearance quite different from ordinary cane sugar. Numerous methods || have been described for preparing pure raffi.nose from molasses, but they are mostly rather tedious and a more convenient source for its preparation is cotton-seed meal ^ Raffinose crystallizes with five molecules of water in clusters of slender glistening needles or prisms whose composition is expressed by the formula CigHggOie . 5H2O. It dissolves in water and in methyl alcohol, in which latter solvent cane sugar is only sparingly soluble, but is hardly soluble in ethyl alcohol, whereas cane sugar is appreciably soluble. * Bertrand and Weisweiller: " Compt. rend.," 1908, 146, 1413. Herissey and Cheymol : id., 1925, 180, 384 ; and 181, 565. f Feist : " Ber. deut. chem. Gesells.," 1900, 33, 2091. X Hemmi : " J. Coll. Agr. Imp. Univ. Sapporo," 1921, 9, 249. § Strohmer : " Oest. Ung. Z. f. Zuckerind u. Landw.," 1910, 39, 649. II V. Lippmann : " Die Chemie d. Zuckerarten," 3rd ed., Braunschweig, Vol. II., p. 1628. ^ Englis, Decker, and Adams : " J. Amer. Chem. Soc," 1925, 47, 2724. Harding : " Sugar," 1922, 240. TRISACCH ARIDES 1 2 3 It is strongly dextro-rotatory, a^ — + 104-4°, in 10 per cent solution, and consequently cane sugar in which raffinose occurs as an impurity appears to contain more than 100 per cent of sucrose when estimated polarimetrically ; hence raffinose is sometimes known as " plus sugar." It does not reduce Fehling's solution, nor does it react with phenylhydrazine. On careful hydrolysis raffinose breaks up at first into levu- lose and a disaccharide — melibiose. ^18^32016 + HgO = CgHj^Og -{- CijHjg^ii Raffinose Levulose Melibiose On heating further the melibiose itself is broken up as follows : — CjjHgjOn + HjO = CjHijOg + CgHijOg Melibiose Dextrose Galactose If boiled with mineral acid, therefore, raffinose gives rise to a mixture of dextrose, levulose, and galactose. According to Neuberg,* raffinose is hydrolysed by emulsin into cane sugar and galactose. (See below.) Raffinose, unlike cane sugar, is completely fermented by bottom 'fermentation yeast to alcohol and carbon dioxide, whereas top fermentation yeast is only able to ferment it parti- ally, converting the levulose complex into carbon dioxide and alcohol and leaving melibiose unattacked. These facts have been made use of by Bau f for detecting and for estimating raffinose. From its behaviour on hydrolysis the constitution may be represented by the formula J : — . o . . o- HOCH2 .CH . {CHOH)2— "C O— CH— (CHOH)2— CHOH— CH— CHj— O— CH— (CHOH)3— CH— CHtOH CH3OH • Fructose. Glucose. Galactose. * Neuberg : " Biochem. Zeit.," 1907, 3, 519. t Bau : " Chem. Zeit.," 1894, 18, 1797 ; 1897, 21, 185 ; 1902, 26, 69. t Haworth, Hirst, and Ruell : " J. Chem. Soc," 1923, 123, 312.5. 124 THE CARBOHYDRATES Detection. There are no rapidly performed characteristic tests for rafRnose. The only really rehable method of identifying it is to isolate the substance by precipitating the strontium compound in alcoholic solution, filtering off the precipitate and decom- posing it by a current of carbon dioxide. The resulting solution is then evaporated and the residue extracted with alcohol to remove sucrose and other sugars which are more soluble in alcohol than raffinose. The pure substance should be identified by its crystalline form and optical properties. Another way of identifying raffinose * is to add to the solution a little decoction of fresh yeast, to act as nutriment, and then to sterilize the solution ; a pure culture of top fer- mentation yeast is then added to the solution and the fermen- tation is allowed to proceed in a thermostat at 31° ; when it is completed, the solution is boiled with animal charcoal, filtered, and evaporated to a syrup ; the latter is then, while still hot, poured into hot alcohol and on cooling it is filtered ; the filtrate is then precipitated by mixing with l| vols, of ether. After twenty-four hours the supernatant liquid is poured off and the residual syrup, which consists of melibiose, is converted into its osazone which is characterized by its crystalline form and melting-point, 1 78-1 79°. f Finally, Neuberg % has proposed making use of emulsin for the identification of raffinose. MELECITOSE. C^^Yi^^O^^, 2H,0. This is a sugar which occurs in the sap of Larix europcea, in Persian manna, and especially in the manna exuded from the twigs and needles of Pseudotsuga Douglasii ; it crystallizes with two molecules of water in rhombic prisms, and is dextro- rotatory (au =+ 83°). It does not reduce Fehling's solution, and on hydrolysis yields first a molecule of glucose and a disac- charide — turanose, C12H22O11 — which subsequently itself breaks * Bau : loc. cit., 1897, 21, 185. f I 7- ToUens : " Zeit. physiol. Chem.," 1902, 36, 239. JThe amount chosen should be sufficient to produce from -03 to 0-3 gram of phloroglucide. § This is best prepared, according to ToUens, by shaking up equal volumes of aniline and water in a test tube and adding glacial acetic acid drop by drop until the turbid solution suddenly becomes clear. 138 THE CARBOHYDRATES red colour appears when the two hquids come in contact with each other, the solution is free from furfural, and the distillation can be discontinued. The furfural contained in the united distillates is then precipitated from solution by means of phloroglucinol which reacts according to the equation — - CsH.Oj + CeH^Oa = CnHgOa + 2H,0 90 126 To this end about the amount of phloroglucinol * likely to be required by the furfural obtained is dissolved in hydro- chloric acid (sp. gr. i-o6), and added to the furfural solution, and the total volume is then made up to 400 c.c. with more of the same acid. The solution at once turns yellow, then becomes turbid, and, on the next day, the greenish-black pre- cipitate of the phloroglucide is filtered off on to a tared Gooch crucible; the precipitate is washed with 150 c.c. of water, dried for four hours at 97°, then cooled in a desiccator and weighed in a weighing bottle. f From the weight {a) of the precipitate, which under ordinary conditions should lie between 0-03 and 0-3 gram, the weight of furfural, pentose, or pentosane may be calculated by substituting the value of (a) in one of the following formulae : — a lies between 0-03 and 0-3 gram. Furfural = (a -(- -0052) X -5185 Pentose — (a + -0052) x 1-0075 Pentosane = {a + -0052) x -8866 in which -0052 is the weight of phloroglucide, which remains in solution under the conditions of the experiment as given above. If the precipitate weighs less than 0-03 gram or more than 0-3 gram, one of the following formulae must be employed : — Weight of precipitate-<-o3 gram. Weight of precipitate>-o-3 gram. Furfural = (a + 0-0052) X 0-517 Furfural = (a + 0-0052) X 0-518 Pentose = (a + 0-0052) X 1-017 Pentose = (a + 00052) X 1-0026 Pentosane = (a + 0-0052) x 0-8949 Pentosane = {a + 00052) x 0-8824 * The phloroglucinol employed must be pure. To ascertain this, test as follows : Dissolve a small quantity in a few drops of acetic anhydride, heat almost to boiling, and add a few drops of concentrated sulphuric acid ; a violet colour indicates the presence of diresorcinol ; if more than a faint coloration appears, the sample should be rejected. t This is necessary to prevent the phloroglucide, which is hygroscopic, from absorbing moisture. ESTIMATION 139 According to Boddener and Tollens,* a considerable saving in time may be effected by precipitating the phloroglucide in hot solution, i.e. between 80 and 85°. The reaction then takes place according to the equation — • QH.O^ + QH0O3 = C„H,0. + sHp so that the precipitate actually weighs less than the one pro- duced in the cold ; the precipitation is, however, complete in from one and a half to two hours. The weight of furfural corresponding to the precipitate so obtained may be calculated by adding -ooi (to allow for the phloroglucide remaining in solution) and multiplying the resulting figure by 0-571. The number so obtained if multiplied by i -935 gives the correspond- ing amount of pentose or if multiplied by 1703 gives the amount of pentosane. The method is, however, not suitable if it is desired to estimate the methyl-pentosans as distinct from the pentosans, in which case Krober's method as modified by Ellett f and Mayer J should be employed. Reducing Sugars Other than Pentose. Various gravimetric methods of estimating reducing sugars have been suggested ; the outstanding feature of all these methods is that they yield reliable results only if carried out under strictly controlled conditions. One of the most reliable methods is that of Brown, Morris, and Millar ; § the pre- cipitated cuprous oxide is washed, dried, and weighed after conversion into cupric oxide by ignition ; 1| from the weight of cupric oxide obtained, the equivalent weight of either dextrose, levulose, invert sugar, or maltose may be determined by reference to tables which will be found in the original paper. Compared with Brown, Morris, and Millar's method, that of Allihn,^ once extensively used, is more cumbersome. The * Boddener and Tollens : " J. Landw.," 1910, 58, 232. t Ellett : id., 1905, 53, 13. X Mayer : id., 1907, 55, 261. § Brown, Morris, and Millar : " J. Chem. Soc," 1897, 71, 94. II Alternatively, the cuprous oxide may be reduced in a current of hydrogen and weighed as copper. T[ Allihn : " J. prak. Chem.," 1880, [2], 22, 63. 140 THE CARBOHYDRATES official method of the American Association of Official Agricul- tural Chemists is that devised by Munson and Walker.* Estimation of Glucose as Osazone. The following method of estimating glucose as osazone in the products of the action of malt upon starch is recommended by Davis and Ling : f 20 c.c. of solution containing 2-3 grams of starch products per 100 c.c. are mixed with I c.c. of phenyl- hydrazine and 1-5 c.c. of 50 per cent acetic acid. After heating for an hour % over a water bath, the liquid, which has by this time evaporated to a small bulk, is filtered through a tared Gooch crucible, and the crystalline osazone is washed with 20-30 c.c. of boiling water, so that the total filtrate does not exceed 50 c.c. ; the precipitate is then dried in a steam oven and weighed ; under these conditions, o-i gram of glucose gives 0-0505 gram of glucosazone. Estimation of Natural Mixtures of Sugars. Several methods § have been devised for the estimation of the constituents of sugar mixtures, such as occur in plant extracts and in fermentation liquors, use being made of yeasts to ferment away sugars and of enzymes, such as invertase, to hydrolyse disaccharides, etc.]] The following method is that described by Davis, Daish, and Sawyer : ^ The freshly plucked leaf material is dropped into boiling 95 per cent alcohol to which a little 0-88 ammonia is added, to destroy the enzymes. This leaf material is placed in a Soxhlet and is extracted with the same alcohol for eighteen to twenty hours. The extract, after evaporation to a small bulk under reduced pressure, is made up to a known volume with water. A portion is evaporated to dryness for the determination of the dry weight, and the remainder is precipitated with basic lead acetate, filtered, and made up to * Munson and Walker : " J. Amer. Chem. Soc," 1907, 39, 541. t Davis and Ling : " Journ. Chem. Soc, Lond.," 1904, 85, 24. t The heating should not be continued for more than one hour. § See also " Note." p. 514. II Davis : " J. Soc. Chem. Ind.," 1916, 35, 201. Nanji and Beazeley : id., 1926, 45, 220. ^ Davis and Daish : " J. Agric. Sci.." 1913, 5, 437. Davis, Daish, and Sawyer : id., 1916, 7, 255. ESTIMATION 141 a known volume ; this is solution A. A portion of this solution is freed from lead by sodium carbonate and made up to a known volume ; this is solution B. Solution B is divided into portions : (i) For the direct determination of the reducing power due to dextrose, levulose, maltose, and pentose, and also the combined rotation. (2) For determining sucrose by inverting with invertase,* and with 10 per cent citric acid ; each of these values should agree closely. (3) For the deter- mination of pentoses by the Krober method ; and (4) For the estimation of maltose. To do this 50 c.c. of the solution are made slightly acid with hydrochloric acid, and hydrogen sul- phide is bubbled through in order to remove the last traces of lead. Any precipitate is filtered off, and a current of air is passed through the solution to remove the sulphuretted hydro- gen. The resulting solution should be absolutely free from lead, else the yeast will not grow in it, and faintly acid to litmus. To it are added 5 c.c. of yeast water and the mixture sterilized by twenty minutes heating at 115-120° C; when cool it is inoculated with a little yeast and incubated at 25° C. for three or four weeks. f On the completion of fermentation, 5 c.c. of alumina cream are added to clarify the solution, and the whole is well boiled ; it is then filtered and the precipitate washed until the filtrate and washings measure 100 c.c. An aliquot portion is used for determining the reducing power. The yeast must be a pure strain free from maltase, thus all sugars except maltose are fermented away. C. POLARI METRIC METHODS. The presence of an asymmetric carbon atom confers upon a compound the property of optical activity, by which is meant the power of the substance to rotate to the right or to the * The invertase required for this purpose is prepared by washing fresh- pressed beer yeast, to remove adherent wort, packing it into a large wide- mouthed bottle and adding 30-50 c.c. of toluene, which percolates through the mass. The bottle, covered with a sheet of paper, is left in a warm place at a temperature of 25°-30° C. At the end of a fortnight, nearly the whole is liquefied ; it is then filtered on a Buchner funnel. The filtrate yields a highly active preparation of invertase, free from maltase and zymase. f 0-2 to 0-5 gram of cane sugar are completely fermented in about three weeks in these conditions. 142 THE CARBOHYDRATES left the plane of a beam of circularly polarized light passing through it. The polarimeter is much used in ascertaining the strength of sugar solutions, but before describing the mode of using it, it is desirable to consider briefly the principles which are involved. When a ray of light enters a crystal of any system other than the cubical, it is broken up into two rays, the ordinary and the extra-ordinary, provided the beam of light is not coincident with the optical axis of the crystal. This pheno- menon is known as double refraction. These two rays, the ordinary and the extra-ordinary, do not behave similarly ; the former conforms to the ordinary laws of refraction, but the latter does not ; further, the two rays are polarized in directions at right angles to one another. In order to make use of these facts, it is necessary to be able to examine the extra-ordinary ray alone ; that is, the two rays must be separated one from the other. This is effected by a Nicol's prism, which consists of two plates of Iceland spar fixed together by means of Canada balsam. A ray of light enters one side of the prism, and is broken up into the ordinary and the extra-ordinary ray ; on reaching the layer of balsam, the former is totally reflected, whilst the latter passes on through the other plate and emerges at the side opposite to its entry. If a second Nicol be placed in the path of this ray, the latter will pass through in different amounts according to the angle which the second prism makes with the first. If the interposed Nicol be parallel to the first Nicol, the ray will pass through entirely ; if the second Nicol be rotated, the light passing through will be less and less in amount until, when the two prisms are at right angles to each other, no light passes at all. If the rotation be continued, the light will again pass through in gradually increasing quantities until the prism has been rotated through an angle of i8o° from its original position, when the whole light will again pass through freely. Many liquids and solutions of solids possess what is known as optical activity, which means that they can rotate the plane POLARIMETER 143 of vibration of a ray of polarized light passing through them ; so that, on emergence from the liquid, the new plane is inclined either to the right or to the left of the original plane. This is known as the rotation of the plane of polarized light. Laurent's Half-Shadow Polarimeter. — ^This apparatus con- sists of a tube containing two Nicol's prisms, of which one is fixed and is known as the polarizer, while the other can be rotated and is called the analyser. A quartz plate which covers half the field of vision is fixed just behind the polarizer. The liquid or solution to be examined is contained within a glass tube with polished ends, and is placed in position between the quartz plate and the analyser. The analyser is fixed in a tube which can be rotated, the degree of rotation being read from a divided circle. Leaving out of consideration the quartz plate, the beam of polarized light passes through the liquid and so becomes rotated ; it follows, therefore, that the vibration plane of the analyser will no longer be at right- angles to the plane of polarization of the light striking it, therefore light will enter the analyser, and in order to bring about complete extinction, the analyser must be rotated either to the right or to the left. This angle of rotation is a measure of the optical activity of the substance under observation, and according to the direction of rotation, the substance is termed dextro- or laevo-rotatory. In Laurent's polarimeter the illu- mination is obtained from a sodium flame, and this light before entering the tube containing the liquid must pass through the plate of quartz. When the instrument is set in the zero position, the whole field is equally illuminated, but on in- troducing the liquid, one-half of the field becomes the darker ; equal illumination can be obtained by rotating the analyser. If this position be passed, the field is once more unequally illuminated, but in a reverse manner, that is to say, the half which was originally dark is now light, and vice versa. As the exact position of equal illumination is somewhat difficult to determine, several readings should be made and the mean of these taken as the correct value. 144 THE CARBOHYDRATES The specific rotation of a substance is defined as the angular rotation produced by a column of liquid i dm. in length, which contains i gram of the active substance in each cubic centimetre. This quantity is expressed by the symbol a^^, the numeral indicating the temperature at which the measure- ments were made, and the letter d standing for the yellow line of the sodium flame which is used as the source of illumination. The use of this quantity a^ for determining the number of grams of active substance in a given solution will be rendered apparent from the following considerations. Supposing we have a solution containing an unknown number of grams, m, of active substance per c.c, and we fill a tube of length / dm.* with this solution and observe its angular rotation to be a. ^, , , , ... rof substance in I c.c. of "\ If a layer i dm. long containing i gram|jjq^j^p^^^^^^^^^^^^^.^^ j o^, Then „ / ,, „ „ i „ „ ,, la D / „ ,. ,. m „ „ „ mla And this would be the observed angle of rotation (a). .-. a = w X / X ttjj a D / X aj,- The angle of rotation is determined as follows : — 1. Find the zero reading when no solution is between the polarizer and analyser. For this purpose the mean of at least three readings, differing by only two or three minutes, should be taken. 2. Fill the tube with the liquid, taking care to avoid the introduction of air-bubbles. 3. Insert the tube and determine the new reading at which the illumination of both halves of the field is equal. The mean of three readings should again be taken. The difference between the initial and the final readings is the angle of rotation. The following experiment performed on a solution known to contain glucose may be quoted in illustration of the method : — * The length of the tube must be expressed in decimetres. POLYSACCHARIDES 145 Initial reading of polariscope, without any solution = o" 30' Final ,, ,, ,, with glucose ,, = 3° 45' Difference (a) = 3° 15' or 3-25° Length of tube containing the solution (/) = 2 dms. Specific rotation of glucose (a^) = 52-5° From which m = — - — - — = -0300 2 X 52-5 ^ ^ .•. the strength of the solution is 309 per cent. It is of course obvious that correct values can only be obtained by this method on the assumption that the liquid contains only a single optically active substance. Plant extracts should be treated with lead acetate in the manner described above. Some substances, e.g. glucose, exhibit the phenomenon of muta-rotation, that is to say, the rotation of their solutions varies according to the length of time that they have been made up ; the maximum rotation is given by a freshly-made solution, but the rotatory power gradually decreases until it finally becomes steady. The attainment of the final condition is greatly accelerated by warming the solution in the presence of a little alkali, but the solution must of course be cooled before a reading is taken. FURTHER REFERENCES. Armstrong : " The Simpler Carbohydrates and Glucosides," London, 1924. Mackenzie : " The Sugars and their Simpler Derivatives," London, 1913- Cramer : " Les sucres et leurs Derives," Paris, 1927. POLYSACCHARIDES. The second great group of carbohydrates, namely the non- sugars or polysaccharides, are substances of high molecular weight, mostly amorphous and insoluble in water. Like the di- and tri-saccharides, the polysaccharides on hydrolysis break up into sugars containing five or six carbon atoms, and they may therefore be looked upon as anhydrides of these substances. In the absence of any exact knowledge regarding their molecular weights, their formulae are written (CgHioOs)!! or 10 146 THE CARBOHYDRATES (C5H804)n according as they give rise to hexoses or pentoses on hydrolysis. The value of " w " has not been determined as yet for any particular case, but there is reason to believe that it is fairly high. The various methods adopted for the eluci- dation of this point have led to such widely different results that a description of them here would not serve any useful purpose. HEXOSANS. The general formula for all substances belonging to this group is (CfiHioOajn, which indicates that on hydrolysis they yield hexoses ; for this reason they may be termed hexosans. GLUCOSANS. Starch or Amyluni. Starch is one of the most widely distributed substances in the vegetable kingdom ; it may be found in green leaves as a temporary reserve of the photosynthetic products ; as a more or less permanent reserve food-material it occurs in seeds and fruits, where it is not infrequently accompanied by other reserves, for instance proteins ; in the vegetative parts, such as tubers, the living cells of the pith, medullary rays, and cortex of roots and stems ; and also in the latex of certain plants, e.g. Euphorbia. When especially stored, the amount of starch may be considerable ; thus in cereals it may form from 50 to 70 per cent of the dry weight of the grains, and in potatoes from 15 to 30 per cent of the dry weight of the tubers. As is well known, starch grains from different sources show much variety in size and shape, and occur in association with plastids, in which, as Schimper demonstrated, they have their origin. Not only are the microscopic charac- ters of starch grains of diagnostic value, but the different varieties of starch can be grouped into generic, specific, and varietal classes which correspond with the classification of plants based on the ordinary morphological features.* Brief mention may be made of the ideas held regarding the physical nature of starch grains. As is well known, the gran- * Reichert : " Amer. Journ. Bot.," 1916, 3, 91. GLUCOSANS - 147 ules not infrequently exhibit a more or less well-marked stratification which years ago was thought to correspond to the alternation of day and night. The " apposition " theory held that new layers were added to those already formed, each layer being separated from the next by a thin film of air. Nageli, on the other hand, came to the conclusion that the lamellation was due to the differences in the water-content of the several layers, and that the grain was made up of minute particles, the so-called micellae. He held the view that the growth of the grain took place not by apposition but by a process of intussusception, that is to say, new material was intercalated between the micellae, and either gave rise to new micellae, or was used up in increasing the size of the old ones. Schimper expressed the idea that the grains were really of a sphsero-crystalline nature, which view was modified by Meyer, who says that the grain is made up of two kinds of needle-shaped crystals composed respectively of a- and ^-amylose ; he also states that in those grains which are coloured red with iodine, for example, those found in the cells of the root-cap of Allium Cepa, in the seed- coats of Chelidonium and in Oryza sativa, var. glutinosa, dextrin and amylo-dextrin occur. On the other hand, the ordinary grains which are coloured blue with iodine, are made up almost entirely of sphaero-crystals of amylose arranged in layers. According to Kraemer,* the starch grains of the potato are composed of colloid and crystalloid substances arranged in lamellae which are distinct and separate one from the other. At the point of origin of growth, the hilum, and in the alter- nate lamellae, the colloid preponderates and is associated with the crystalloid cellulose ; in the other lamellae the crystalloid granulose is in the greater proportion. He also states that the peripheral layer is elastic and porous, and may be an- hydride of cellulose. Dennison also has expressed the view that the outer layer of the grain is different from the more internal parts, and may be a carbohydrate not fully poly- merized to starch. * Kraemer : " Bot. Gaz.." 1902, 34, 341. TO* 148 THE CARBOHYDRATES The amount of starch present in the leaf varies with the specific physiology of the plant and with the climatic con- ditions. Thus, in Japan, the starch content of evergreen leaves begins to diminish in November. In January, the coldest month, a minimum is reached, in fact, starch may be entirely absent, and at the end of February an increase begins. Miyake,* the maker of these observations, does not comment on the fat content, wherefore a comparison between his re- sults and those of other workers is not possible (see p. 3). Many monocotyledonous plants are characterized by the absence of starch, for example Scilla nutans, Phleum pratense, Allium, etc., but in some of these cases starch granules may occur in the guard-cells of the stomates, in the bundle sheath of the leaves, and also in the bulbs at the base of the growing shoots ; further, in certain plants which normally form sugar, e.g. Musa, H enter ocallis, and Muscari, starch will appear when much sugar accumulates. On the other hand, many members of this same class of plants are fairly constant starch producers, e.g. Lilium tigrinum, Pontederia cordata, Ananas saliva, Canna indica, Tradescantia virginica, Juncus communis, and Alisma Plantago. There are many peculiarities in this occurrence of starch in the Monocotyledons ; for instance, in Scilla nutans it is absent, whilst in Scilla siberica it is quite abundant ; further, the former plant, if fed with cane sugar in a solution of suitable strength, does not form it, while, on the other hand, starch-free plants of Scilla siberica under the same treatment do form starch, the experiment being carried out in the absence of light. In the Mycetozoa, in which starch is normally absent, starch formation may be in- duced under the influence of acid and a supply of sugars.f In the plant starch occurs, as is well known, in the form of variously shaped microscopic bodies composed of concentric layers ; the granules have an organized structure and possess the power of double refraction. • Miyake : " Bot. Gaz.," 1902. 33, 321. t Boas : " Biochem. Zeit.," 191 7, 78, 308. STARCH 149 Preparation of Starch. The method of preparation varies according to the source employed. From wheat flour it may be obtained by stirring up this material thoroughly with water, and allowing the mix- ture to stand until the gluten contained in the flour undergoes fermentation, when it dissolves and may be removed by wash- ing. On a small scale the separation is most conveniently effected by kneading some flour in a muslin bag which is held under a stream of water. The starch granules are hereby washed through the muslin, while the gluten remains behind in the bag as a sticky grey mass. Starch may also be obtained from potatoes by macerating them with water and separating the non-starchy material from the starch by filtration. The starch is then allowed to settle at the bottom of the water, when it is collected and dried. Purification. Malfitano and Moschkoff * give the following method for the purification of starch : A I per cent colloidal solution of starch is frozen and then allowed to melt. When melted, most of the starch is deposited in a floccular precipitate, whilst the clear liquid contains some starch and the greater part of the mineral impurities. On repeating the operation four or five times, the purified product yields less than -02 per cent of ash. Even the purest starch yields on incineration a small amount of ash constituents chiefly of phosphates which were in organic combination with the material (see Amylopectin, p. 152). In addition to phosphates, varying quantities of silica are found in the ash, the amount depending on the source from which the starch was prepared. Silica is not a true constituent of the starch proper, but is associated with another substance, known as amylohemicellulose,t which occurs in greater quantities in some starches than in others, notably in the starches of barley, wheat, rice, tapioca, maize, and sago,| * Malfitano and Moschkoff : " Compt. rend.," 1910, 151, 817. t Ling and Nanji : " J. Chem. Soc," 1923, 123, 2672 ; 1925, 127, 652. X Clayson and Schryver : " Biochem. Journ.," 1923, 17, 493 ; Schryver and Thomas : id., 497. 150 THE CARBOHYDRATES while potato and arrowroot starch contain hardly any (cf. Amylohemicellulose, p. 153). Properties. Air-dried starch contains a considerable quantity of water, as much as 20 per cent being not uncommon ; it can be made to part with this water by carefully heating to 100°. If heated to about 200° it is converted into a sticky soluble substance, which is probably a mixture of isomeric substances of the empirical formula CgHioOg, known as British gum or dextrin (q.v.). Starch is insoluble in cold water, but if dry starch is finely ground for some time in an agate mortar and then stirred up with cold water and filtered through a gravimetric filter paper, such as will retain the finest suspended solids, the filtrate may be shown to have taken up some of the starch in colloidal solution since on addition of a solution of iodine a deep blue coloration results. If a suspension of starch in water is heated, the particles gradually swell and finally burst, forming an opalescent solution, known as starch paste, which is more or less mucilaginous according to the amount of starch employed ; the optimum temperature for bringing about this change varies with the starch as may be seen from the following figures : — Rye . 55° C. Wheat . . 62° C. Maize . 68° C. Rice . 72° C. Potato . . 72° c. Too high a temperature tends to the formation of lumps, and it is generally best not to boil the solution but to add a fine suspension of starch in cold water to the requisite amount of warm water heated over a boiling water bath. A solution of starch so prepared is not to be regarded as a true molecular disperse solution, but as a colloidal solution of soluble amylose thickened by a suspension of the insoluble gelatinizing material amylopectin. A solution of starch paste undergoes a change on keeping, known as retrogradation, and deposits a white fiocculent pre- cipitate which, microscopically, resembles starch. For this STARCH 151 reason, the precipitate has been described as artificial starch. The change is retarded by keeping the paste at 60°. According to Fernbach and Wolff * green malt contains an enzyme " amylocoagulase " which can accelerate the change described. The precipitate is insoluble in hot water and its formation is due to a dehydration and aggregation of the coUoidally sus- pended particles of the original solution ; certain it is that the change is influenced by the presence of electrolytes.^ In order to facilitate the preparation of starch solution for indicator purposes, a number of so-called " soluble starches " have been prepared. These are in reality starch which has been treated in a variety of ways by chemicals whereby it is rendered more soluble, without having suffered a sufficiently profound change to influence its ability to give a blue colour with iodine. Lintner's soluble starch is prepared by exposing starch to the action of 7-5 per cent hydrochloric acid for a week and then washing with cold water until free from acid. The Composition of the Starch Grain. Nageli % was the first to suggest that the starch grain was made up of two distinct constituents, but some years elapsed before his views were supported by reliable chemical evidence. In view of the fact that the terminology employed by the earlier investigators was irregular, a brief historical resume is desirable before considering the present state of our know- ledge of the subject. The researches of Nageli have shown that when starch is treated with dilute hydrochloric acid, malt extract, or saliva, a considerable portion goes into solution, leaving a transparent skeleton undissolved. The soluble portion, which gives a blue colour with iodine, Nageli regarded as the true starch constituent of the granule, and named it granulose ; on the other hand, the undissolved skeleton, which he described as not turning blue with iodine (see below), he considered to be of a cellulose nature, and called it starch cellulose or amylo- cellulose. * Fernbach and Wolff : " Ann. Inst. Pasteur," 1904, 18, 165. t Samec : " Kolloidchem. Beihefte.." 1912, 3, 123. 4: Nageli : " Die Starkekorner," Zurich. 1858. 152 THE CARBOHYDRATES On the other hand, Meyer * was of opinion that starch granules consisted essentially of two substances known respec- tively as a- and j3-amylose. The former, which was insoluble, he regarded as an anhydride which could be converted into the soluble P variety by the action of superheated steam. He also thought that when starch is acted upon by hydro- chloric acid it is converted into amylo-dextrin, and considered that amylo-cellulose, which Nageli regarded as an original constituent of the starch granule, was in reality identical with amylo-dextrin, and therefore a secondary product of the action of acid on the amylose. It is to the French workers Maquenne and Roux,t and Fernbach and Wolff $ that we owe the first definite ideas concerning the existence of two distinct substances. They stated that starch granules consist of two substances, amylo- cellulose or amylose. and amylopectin ; the term amylocellu- lose was not equivalent to Nageli's starch cellulose but to his granulose ; we thus get the following equivalents : — Nageli. Meyer. Maquenne and Roux. Outer layer or starch a- Amylose. Amylopectin. cellulose. Inner layer or granulose. /3-Amylose. Amylocellulose or amylose. Maquenne first stated that amylose formed 80 per cent and amylopectin 20 per cent by weight of the starch granule ; the former substance was described as being soluble in water and giving a blue colour with iodine, while the latter would only swell in water without dissolving, and was erroneously stated to give no colour with iodine. The isolation of amylopectin was first effected by Gatin Gruzewska,§ who treated starch with i per cent caustic soda whereby the granules burst and the amylose constituent entered into solution leaving the swollen outer shells of amylo- pectin ; on neutralizing with acetic acid, the amylopectin shrivels and can be filtered off and washed and dried. Ling * Meyer : " Unters. ii. d. Starkekorner," Jena, 1895. t Maquenne and Roux : " Compt. rend.," 1903. 137, 88 ; 1905, 140, 1303- I Fernbach and Wolff : id., 1904, 138, 819. § Gatin Gruzewska : " Compt. rend. Soc. biol.," 1908, 64, 178. STARCH 153 and Nanji have furnished further methods for distinguishing between amylose and amylopectin ; they find that when starch paste, warmed to 50° C, is treated with precipitated diastase, prepared from ungerminated barley, and dried by means of absolute alcohol, the amylose constituent is converted com- pletely into maltose whilst the amylopectin is hardly acted upon ; the maltose may be removed by dialysis leaving the amylopectin. If, on the other hand, precipitated barley dia- stase is used, which has not been dried by alcohol, amylo- pectin also is attacked yielding a product to which they give the name a^-hexa-amylose. A further distinction between amylose and amylopectin was observed by Samec and von Haefft * who showed that amylopectin contains, on an average, 0-175 per cent of P2O5 in organic combination as a carbohydrate ester of phosphoric acid ; later Samec and Mayer f were able to demonstrate that after the removal of the phosphoric acid from amylo- pectin, the phosphorus free carbohydrate, to which they gave the name of erythroamylose, could be re-esterified with phosphoric acid to a viscous jelly which, however, contained 2-19 per cent P2O5. The same authors also showed that amylose has a^ = 189°, while amylopectin has aj, = 195-196°. With regard to the relative amounts of the two constituents. Ling and Nanji J claim to have established an almost constant ratio of 66 per cent, amylose to 33 per cent of amylopectin, while, on the other hand, Samec and Hofft claim that the pro- portions in potato starch are 17 per cent of amylose to 83 per cent of amylopectin. In addition to amylose and amylopectin. Ling and Nanji § have also found a substance described as amylohemicellulose to be associated with cereal starch grains, but the quantity varies considerably in different starches. To summarize our knowledge with regard to the two constituents of the starch grain : — * Samec and von Haefft : " Kolloidchem. Beihef.," 1913, 5, 141: 1914. 6, 23. t Samec and Mayer : " Compt. rend.," 1921, 193, 321. X Ling and Nanji : " J. Chem. Soc," 1923, 123, 2666. § Ibid., 1925, 127, 630. 154 THE CARBOHYDRATES Amylase forms 66 per cent of the granule ; it is a poly- merized a-hexa-amylose ; is soluble in water and gives a clear bright blue colour with iodine ; it is converted by barley or malt diastase completely into maltose at 50° C. Amylopectin forms 33 per cent of the granule ; it is a polymerized phosphoric acid ester of aj3-hexa-amylose ; when made into a paste with hot water, it gives a bluish-black coloration and precipitate with iodine (amylopectin extracted with alkali gives a violet coloration with iodine) ; it is de- phosphated and depolymerized by barley diastase at 50°, yielding a/3-hexa-amylose. Amylohemicellulose is the name given by Ling and Nanji to a substance associated with and, in the case of the cereal starches, apparently forming an integral part of the granule. Starches of the potato and arrowroot, on the other hand, contain hardly any of this material, although the tuber of the potato actually contains a considerable quantity which, in- stead of forming part of the starch granule, remains attached to the cell wall. Amylohemicellulose contains from I-2-I-3 per cent of ash and is regarded by Ling and Nanji as a calcium, mag- nesium, and iron salt of a silicic and phosphoric ester of a- amylose ; it is converted by malt diastase quantitatively into maltose but, unlike amylose, it is unacted upon by barley diastase. On the other hand, like amylose it gives a blue colour with iodine, and being associated in some cases with the cell wall it is liable, when occurring in wood, to be mistaken there for starch.* Action of Acids on Starch. The action of acids on starch varies according to the strength of the acid, the duration of the action, and the tem- perature of the experiment. To complicate matters, there are considerable divergences in regard to the interpretation of the results obtained by the different workers. As an illustration of the very different effects which may be produced under different conditions, the following experiments may be quoted. * Ling and Nanji : " J. Chem. Soc," 1925, 127, 652. STARCH 155 By acting on starch at the ordinary temperature with 12 per cent commercial hydrochloric acid for twenty-four hours, Brown and Morris found that granules, while retaining their external features, had acquired the power of dissolving in hot water without the formation of paste. The addition of alcohol to such a solution caused the immediate precipitation of a white substance known as soluble starch, which is turned blue by iodine, is strongly dextro-rotatory, [a],, = 202°, and does not reduce Fehling's solution. On the other hand, if starch is boiled for some time with dilute hydrochloric acid, it is converted into glucose, a fact which is made use of in estimating starch. That maltose is also produced as an intermediate product of the acid hydrolysis of starch has been shown by Fernbach and Schoen,* and also by Weber and Macpherson,t who have proved it to be present in commercial glucose (see p. 97). Accompanying the conversion of starch into glucose there is, however, the formation of varying quantities of gummy sub- stances known as dextrins (q.v.) ; it is, however, not known for certain whether these dextrins are formed directly by the action of the acid on the starch, or whether they are produced by the condensing action of the acid on the glucose already formed ; there is, moreover, great difference of opinion with regard to the nature and number of these substances which are formed. According to Nanji and Beazeley J some iso-maltose is always formed during acid hydrolysis of starch. Action of Malt Diastase on Starch. The action of an extract of malt § on starch paste is complex in that it involves liquefaction and saccharification.|| These two changes are effected by different enzymes as is in- dicated by keeping a mixture of starch paste and malt extract at 70° C. After some minutes the paste becomes less viscous * Fernbach and Schoen : " Bull. Soc. Chim.," 1912, [iv], li, 303. t Weber and Macpherson : "J. Amer. Chem. Soc," 1895, 17, 312. I Nanji and Beazeley : " J. Soc. Chem. Ind.," 1926, 45, 215 T. § Barley malt has been shown to contain a great many different enzymes capable of acting upon lichenin, mannan, cellobiose andmaltase, etc. ; it is the action upon starch only which is here being considered. II Oblsson : " Zeit. physiol. Chem.," 1922, 119, i. 156 THE CARBOHYDRATES and develops but little reducing power : if now the mixture be cooled to 50° and a fresh amount of malt be added, the reducing power of the solution rapidly develops owing to the saccharifying action of the second enzyme which was all but inactivated at the higher temperature.* The process of saccharification is essentially hydrolytic, whereby the starch molecule is successively broken down to a number of substances of lower molecular weight such as dextrins and sugars ; this change may be conventionally represented as follows : — (QHioOs)!! + H,0-> (CeHioO,)x + CioH^Pn Starch Dextrin Maltose though actually other sugars such as iso-maltose or glucose may be formed according to the conditions. The production of glucose for example was demonstrated by Ling and Baker f by acting upon starch with malt diastase at 70° ; its pro- duction has been attributed to the further hydrolysis of the maltose by maltase contained in the diastase, but as maltase is not active above 55° this is not possible, and, as Ling and Nanji have shown, the glucose is actually produced from the hydrolysis of jS-glucosido-maltose (see below). A detailed study of the action of diastase upon the two constituents of the starch grain, namely amylose and amylo- pectin, was undertaken by Ling and Nanji ; J these authors find that the action of barley diastase at 50° upon amylose is to convert it quantitatively into maltose without the pro- duction of any intermediate substances. On the other hand, amylopectin is first converted into aj8-hexa-amylose and the further hydrolysis of this substance by malt diastase results in the production of a series of maltodextrins, a trisaccharide j8-glucosido-maltose, iso-maltose, maltose, and glucose depend- ing on the conditions of the experiment. Thus malt diastase acting at 70° upon aj8-hexa-amylose converts it into malto- dextrin-a and thence into 2 molecules of a hexatriose ^- * Both saccharifying and Uquefying enzymes are destroyed at 80°. t Ling and Baker : " J. Chem. Soc," 1895, 67, 702, 739. Ling and Davis : " J. Fed. Inst. Brewing," 1902, 8, 475 ; " J. Chem. Soc." 1904. 85, 16. X Ling and Nanji : id., 1925. ^27, 639. STARCH 157 glucosido-maltose which contains both an a- and a jS-Hnking. These facts are best explained by the following formulae : — 1/3 a\ in a\ 10 aj3-Hexa-amylose Maltodextrin-a j3-Glucosido-maltose From which it appears that ajS-hexa-amylose is composed of six hexose residues united together by four ^- and two a- linkages ; the conversion of this into maltodextrin-a results from the fission of one jS-linkage ; the further fission of yet another )3-linkage yields 2 molecules of the trisaccharide jS-glucosido-maltose * which must have the constitution — o ^ I I CH, . CH . CH(CHOH),-C . HOH iso-maltose. P O J CH— (CHOH),— CH . CHOH— CHg > Oo CHjOH CHOH . CH(CHOH) ' o— ,— CH maltose. j3-glucosido-maltose since it is broken up by emulsion into glucose and maltose and by maltase into glucose and iso-maltose, showing it to have both an a- and jS-linking. On the other hand, the amylose constituent of the starch grain would in Ling and Nanji's opinion appear to be an a-hexa-amylose f of the formula — • / \ |a a e\ b K / in which the six sides, a, h, c, d, e, and /, represent glucose anhydrides, CgHioOg, united together through oxygen atoms at * j3-Glucosido-maltose yields an osazone, m.p. 122°. Ling and Nanji : loc. cit., p. 2679. t Ling and Nanji : " J. Chem. Soc." 1923, 123, 2684. 158 THE CARBOHYDRATES the six corners of the hexagon ; such a molecule would yield on hydrolysis 3 molecules of maltose, ab, cd, and ef, which is in agreement with the observed fact that amylose yields only maltose. Action of Bacteria on Starch. In 1903 Schardinger * isolated from retting flax a bacillus to which he gave the name Bacillus macerans ; this organism when grown on 5 per cent starch paste, liquefies the starch and sets up an active fermentation with the evolution of carbon dioxide and hydrogen, and the production of acetone f and butyl alcohol. In the course of a few days these products give way to the formation of acids, and after about a week a liquid remains from which Schardinger isolated two crystal- line substances which he described as a- and ^-dextrin. The former of these gives with iodine a dark green compound crystallizing in needles, while the latter gives dark reddish- brown prisms. Schardinger ascribed to these compoimds the formulae (C6Hio05)4 and (C6Hio05)6 and named them tetra- and hexa-amylose respectively. Pringsheim and his colla- borators X subsequently investigated these compounds more fully and found that on treating them with acetic anhydride and zinc chloride they were acetylated and at the same time depolymerized ; the product obtained from the a-dextrin (C6Hio05)4 was shown to be an acetylated diamylose (C6Hio05)2, while that obtained from the ^-dextrin (C6Hio05)6 was a triamylose (C6Hio05)3. For these reasons he regarded Schar- dinger's a- and jS-dextrin as polymerized di- and triamyloses respectively as shown by the formulae — a-Dextrin — Tetra-amylose [(CeHio05)2]2 Diamylose {C^Yiiffd^)^ j3-Dextrin = Hexa-araylose [(CsHio05)3]2 Triamylose (CgH,oOB)3 * Schardinger : " Zeit. Nahr. Genussm.," 1903, 6, 874 ; " Zentr. Bakt. Parasitenk.," 1905, [ii], 14, 772 ; 191 1, 29, 188. t During the Great War this type of fermentation was first employed on a large scale for the production of acetone. X Pringsheim and Langhans : " Ber. deut. chem. Ges.," 1912, 45, 2533 ; Pringsheim and Eissler : id., 191 3, 46, 2959. STARCH 159 in which the basic molecule is enclosed in a round bracket, while the degree of polymerization is indicated by the square brackets. According to Pringsheim,* the a and j3 series of dextrins are derived from amylose and amylopectin respectively, and diamylose and triamylose he regards as the basal nuclei of amylose and amylopectin to which substances he assigns the following structure :— — CH o CH (CHOH). L O CH -0 CHOH I -CH O Choh — CH., (CHOH)^ I -CH Amylose [(C6Hi„05)2]„ O- -CH (CHOH), O CH O i CHOH -CH, O CH,--CHOH— CH— (CHOH). -CH . (CHOH),- -CH I O 1h- -CHOH— CHo O Amylopectin [(CgHio05)3],, Moreover, the fact that by the action of cold concentrated acid upon glycogen, he has also obtained triamylose leads him to believe that the amylopectin of starch is identical with glycogen. In Ling and Nanji's f opinion, however, the basal unit of the polymerized amylose and amylopectin of the starch granule are a-hexa-amylose and a^-hexa-amylose respectively (for formulae, see p. 157). Reactions. I. The appearance of the grains under the microscope and their action on polarized light in the presence of water are well known. * Pringsheim : " Ber. deut. chem. Gesells.," 1924, 57, 1581. t Ling and Nanji : "J. Chem. Soc," 1923, 123, 2683. i6o THE CARBOHYDRATES 2. The most characteristic reaction of starch is the blue colour produced with iodine. The composition of this blue substance varies ; it contains, on an average, about i8 per cent iodine, and cannot be formed unless a small quantity of hydri- odic acid, which is always present in small amounts in ordinary solutions of iodine, be present. The blue colour is discharged on heating the solution, but reappears on cooling. The dried substance may, however, be heated to ioo° without under- going alteration. If the starch grains are very small, or relatively so few in number that they might easily be overlooked, Meyer's pro- cedure for their detection may be followed. A section of the material to be examined is cut, and is first treated with a fairly dilute solution of iodine in potassium iodide, the excess of the reagent is then removed, and the section is irrigated with a concentrated aqueous solution of chloral hydrate. This causes the starch grains to swell, and at the same time the other cell contents are dissolved, as are also the starch grains in time. The fact that iodine sometimes gives a blue colour with a soluble cell constituent led to the assumption of the presence of a so-called soluble starch. There is, however, no need for such an interpretation, since the blue colour ob- served in the epidermal cells of Saponaria officinalis, for example, is attributable to the action of iodine on the glucoside saponarin, CaiHaiOia, which Barger * has shown to be present and to give this reaction. The blue colour given by starch with iodine was originally regarded by Mylius f as a definite chemical compound, and the same view is taken by Murray $ but by others it is con- sidered to be a physical adsorption of colloidally dispersed iodine by the starch acting as a protective colloid ; § the particular shade of colour produced probably depends upon the degree of dispersion of the iodine (cf. dextrin, glycogen, etc.). •Barger: " Ber. deut. chem. Gesells.," 1902, 35, 1296. t Mylius : id.. 1887, 20, 688. I Murray : " J. Chem. Soc," 1925, 127, 128S. § Barger and Field : id., 1912, loi, 1394- STARCH i6i 3. Starch grains are insoluble in cold water, but in hot water they swell up and form an opalescent solution which, if strong enough, will on cooling eventually form a paste. 4. Starch is precipitated from its aqueous solution by alcohol or by basic lead acetate (cf. Inulin and Dextrin). 5. Boil a little starch paste solution with a few drops of dilute sulphuric acid in a test tube, and from time to time remove a little of the solution, cool it and test with iodine solution ; when the starch has been converted into dextrin the blue colour at first formed will give way to a plum colour. If boiled too long only dextrose will remain which gives no colour with iodine. The solution will, however, after making alkaline, reduce Fehlings' solution. 6. Cautiously heat a little starch on a porcelain basin until it has acquired a light fawn colour. Cool and extract with cold water, and filter ; the dextrin produced being soluble in cold water is thus separated from the starch. On adding iodine to the solution a plum colour is produced. Estimation of Starch. The chief difficulty in estimating starch by determining the amount of reducing sugar formed after appropriate hydrolysis lies in the error caused by the presence of pentosans. Lintner overcame this difficulty by estimating the pentosans by the phloroglucinol method (see p. 137) and deducting a proportionate amount from the reducing power after hydrolysis, on the assumption that xylose and arabinose have approxi- mately the same reducing power as glucose. A method for the determination of starch in barley or wheat due to Ling, Nanji, and Harper * makes use of the fact that when a paste of any of the starches, or materials containing starch, is treated with barley diastase at 50°, the amylose is converted into maltose and the amylopectin into aj8-hexa- amylose leaving the amylohemicellulose of the cereal starches as an insoluble residue. Cereal starches, owing to the presence of amylohemi- cellulose, do not give the same percentage of maltose as other * Ling, Nanji, and Harper : " J. Inst. Brewing," 1924, 30, 838. II 1 62 THE CARBOHYDRATES starches which do not contain this substance. The ratio amylose : amylopectin though approximately 2 : i in most cases is not quite constant, and for this reason, in addition to the variation in the activity of the barley diastase, a control is carried out upon pure potato starch, and from the deter- mination of the maltose as a percentage of dry starch the amylose : maltose ratio can be deduced. If this ratio has been estabhshed for one set of conditions, and the same conditions are applied to a cereal starch, it is possible to determine the amylose : maltose ratio for that cereal. The method, for the details of which the original paper should be consulted, gives the true starch content exclusive of hemi- cellulose, and the results are sHghtly lower than those given by malt diastase. The following method depending on the hydrolysis of starch by hydrochloric acid and the subsequent estimation of the glucose produced, is only rehable if there are no pentosanes or other substances present which on hydrolysis would yield reducing sugars. About 3 grams of the substance in as fine a state of division as possible are covered with 50 c.c. of cold water and shaken at frequent intervals ; after an hour the insol- uble portion is filtered off and washed with water until the total filtrate measures 250 c.c. ; the addition of a little alumina shaken up with water will frequently facilitate clear filtration. The soluble carbohydrates contained in the fil- trate may if desired be determined both before and after inversion. The residue remaining on the filter paper is then transferred to a flask with a 250 c.c. graduation mark and heated for two and a half hours under a reflux condenser with 200 c.c. of water and 20 c.c. of hydrochloric acid (sp. gr. 1-125). After cooling, the solution is neutralized with caustic soda and made up to 250 c.c, whereupon it is filtered and the amount of glucose contained in an aliquot portion of the filtrate is estimated by Fehling's or Benedict's solution. The amount of glucose found when multiplied by O-p gives the weight of starch. DEXTRINS 163 The following method for the estimation of starch in barley is due to Horace T. Brown * : — Five grams of the powdered or crushed grain are extracted for three hours in a Soxhlet extractor with alcohol (sp. gr. 0-90) ; the residue is then thoroughly boiled with lOO c.c. of water, and, after cooling to 57°, 10 c.c. of active malt extract are added and the mixture is set aside for one hour ; it is thereupon boiled and filtered into a flask with a 200 c.c. graduation mark ; the residue is thoroughly washed with water, and, after cooling, the filtrate and washings are made up to 200 c.c. The cupric reduction of 20 c.c. of the solution is determined under the conditions laid down by Brown, Morris, and Millar,t the maltose being calculated according to Table XI in that paper [loc. cit., p. 100), after correction for the re- duction due to the malt extract. The starch equivalent to this maltose is then ascertained by assuming that 84-4 parts of maltose correspond to lOO parts of starch. The malt extract is prepared by digesting 10 grams of fresh finely-ground malt for two to three hours with 200 c.c. of water and filtering. A method of starch estimation due to von Fellenberg f depends on the solution of the starch in a hot solution of calcium chloride, its precipitation by iodine and the decom- position of the iodine precipitate by alcohol. DEXTRINS. The term dextrin is applied to substances which are formed from starch by the action of heat alone or of diastase or mineral acids. Occurrence. In the plant dextrins may occur as transitory substances whenever starch is being acted upon by diastase ; further, certain dextrins may occur in a more permanent form. Thus the sap of the epidermal cells of Arum italicum turn reddish- violet on the application of iodine. The aqueous extract of * Horace T. Brown : " Trans. Guiness Research Lab.," 1903, i, 89. t Brown, Morris, and Millar : " J. Chem. Soc, Lond.," 1897, 71, 94- t V. Fellenberg: " Mitt. Lebensm. Hyg.," 1916, 7, 369. II* 1 64 THE CARBOHYDRATES such cells gives on evaporation a transparent sticky substance. This also gives with iodine a violet coloration ; after boiling, the colour reaction with iodine is red, and after digestion with diastase a reducing sugar is found. Formation from Starch. The question of the formation of dextrins from starch by the action of diastase has been the subject of a great many researches, and has, at different times, resulted in the postu- lation of the existence of a large variety of dextrins and intermediate products, such as amylo-, achroo-, erythro-, and malto-dextrin, amylases, amyloins, glycoamylins, etc., many of which did not survive for long. The chief facts observed during the action of malt extract on starch may be very briefly summarized as follows : If, say, a 10 per cent starch paste is left in contact with malt extract at 50°, the mass rapidly liquefies and the solution acquires a sweet taste owing to the conversion of starch into maltose ; if the latter substance be estimated from time to time, it will be found that the reducing power of the mixture increases rapidly at first until, after about two hours, the amount of maltose present corresponds to about 80 per cent of the starch em- ployed, when practically no further change takes place. The change in the starch paste can also be demonstrated by peri- odic testing with iodine solution ; the blue-black coloration gradually becomes less and less marked until various shades of red are obtained, finally the iodine gives no distinctive coloration. A corresponding fall in the optical activity of the solution can also be observed, but as the activity is still greater than what it should be for maltose alone, it must be concluded that some other substance is formed at the same time as the maltose, and that its reducing power is less but its activity is greater than that of maltose. The amount of this " non- maltose " product of diastatic activity varies directly with the temperature, and increases considerably at the expense of the maltose if the temperature be kept at or above 60° ; if to such a product, rich in non-maltose, a fresh quantity of malt extract be added, the non-maltose will be attacked and converted into DEXTRINS 165 maltose until the amount present again attains the value 80 per cent, which is the normal maximum ; this experiment, which is due to Brown and Morris,* shows that the non-maltose is composed of different constituents, some of which are con- verted into maltose by diastase more readily than others ; moreover, experiments have shown that these substances behave differently towards yeast, some being more readily fermentable than others. This non-maltose constituent represents a mix- ture of the various dextrins mentioned above as having been described by several authors. General Properties of Dextrins. From what has been said above, it will be seen that the term dextrin comprises a number of substances some of which are not at all well defined. The following may, however, be regarded as approximately representing the characteristics of all substances included in this group : — ■ 1. They are amorphous substances which are readily soluble in water to form gummy solutions, which are used as a sub- stitute for natural gum ; they are precipitated from aqueous solutions by the addition of alcohol. 2. Dextrins in strong solution give a precipitate with basic lead acetate. 3. As their name implies, they are strongly dextro-rotatory, in which respect they resemble starch. 4. They give either a red colour or no colour at all with iodine, 5. They are not fermentable by yeast alone, but are fer- mented by a mixture of yeast and diastase acting together, which is no doubt due to their slow hydrolysis in the first place by the diastase and the subsequent fermentation of the maltose so produced. 6. They do not reduce Fehling's solution when pure. 7. They are converted into glucose on hydrolysis with mineral acids. As has already been mentioned, starch when suddenly heated to about 200° is converted into a substance commercially * Brown and Morris : " J. Chem. See, Lond.," 1SS5, 47, 527. 1 66 THE CARBOHYDRATES known as dextrin. The use of starch for stiffening hnen depends on some such similar change produced in the starch by the heat of the iron. Although a great many different dextrins have from time to time been described, comparatively few of them are sufficiently well defined to warrant any description here. Amylo-dextrin. — This substance is obtained by the action of ungerminated barley diastase at 50° C. and precipitated by alcohol. It is a white powder slightly soluble in cold water, but readily in hot. It is strongly dextro-rotatory (^j, = + 196) does not reduce Fehling's solution, and gives a blue colour with iodine. Erythro- dextrin. — This is a solid which dissolves readily in water, has a rotatory power of ^j, = + 196°, and with iodine produces a red-brown colour. The existence of erythro-dextrin as a chemical entity is, however, disputed by Ost, who says that it is a mixture of achroo-dextrin with starch ; an artificial mixture of achroo- dextrin with | per cent of starch also produces a red colour with iodine. Achroo-dextrin. — This substance is optically active, has the value a^ — -]- 192°, gives no colour with iodine, and has a sweetish taste. M alto-dextrin. — In addition to the above dextrins, Brown and his collaborators, and Ling and Nanji * have described the following malto-dextrins which are non-crystalline inter- mediate products of the action of diastase on starch and pos- sessing cupric-reducing power : malto-dextrin-cc CaeHgaOgi (ajj=i8o°), and malto-dextrin-^, C24H4.202it (au — I73"5°), and stable dextrin. X According to Ling and Nanji, malto-dextrin-a is an inter- mediate product in the degradation of aj3-hexa-amylose to j3-glucosidomaltose (see p. 156), and they regard the stable dextrin of Brown as a malto-dextrin of the highest type. * Ling and Nanji : "J. Chem. Soc, Lond." 1925, 127, 636. t Ling and Baker : id., 1895, 67, 703 ; 1897. 71, 517. + Brown and Millar : id., 1899, 75, 286. GLYCOGEN 167 COMMERCIAL DEXTRIN. Commercial dextrin is prepared by heating starch to about 230-260° ; it is a yellowish-brown powder, while that prepared by acid hydrolysis of starch is an almost colourless solid with a choncoidal fracture, or else a white powder resembling starch. It is composed chiefly of achroo-dextrin mixed with varying quantities of erythro-dextrin and glucose. It dissolves in an equal volume of water to give a neutral sticky solution with a faint sweet taste ; the solution is strongly dextro- rotatory. Dextrin is insoluble in alcohol and ether. GLYCOGEN. This substance, although one of the most important and widely distributed reserve foods in the animal kingdom, has a restricted distribution in plants. It occurs abundantly in certain Fungi, especially in Saccharomyces cerevisece, where it may sometimes form as much as 30 per cent of the dry weight. It has also been described as forming part of the cell-contents in Myxomycetes, Flagellates, and in certain Algae including the Cyanophyceae. In the yeast plant the glycogen varies in amount according to the physiological phase of the organism, and, it appears, accumulates and disappears often with great rapidity. The glycogen appears in the cells of Saccharomyces during the early stages of fermentation as minute refractive granules scattered through the protoplasm ; after a few hours these granules give place to small vacuoles, which in turn are re- placed by one large vacuole, which may occupy the greater space in the cell.* Wager and Peniston,t have shown that the amount of glycogen present is correlated with the periodical fluctuations in the fermentative activity. When yeast is placed in a nutrient fluid, e.g. Pasteur's solution, fermentation may start at once, in which case it was found that the cells float and contain very little glycogen, while the cells which contain much glycogen sink to the * Harden and Rowland : " J. Chem. Soc, Lond.," 1901. 79, 1234. t Wager and Peniston : " Ann. Bot.," 1910, 24, 45. 1 68 THE CARBOHYDRATES bottom. After an hour or two the cells begin to rise, and they become distributed throughout the medium after the lapse of four or five hours. The fermentation is now much more active, and the amount of glycogen in the cells less. The next five to fifteen hours is the period of maximum vegetative activity, during which the glycogen disappears ; then it slowly reappears, and later on much more rapidly, at which phase there is a marked decrease in budding. At the height of fermentation, or immediately after, the glycogen increases rapidly, and a large number of cells sink to the bottom of the fluid. If the medium be not exhausted, the process may be repeated two or three times. These facts suggest that the yeast, although surrounded by a medium rich in soluble carbohydrate, uses its glycogen reserve in the first instance and, moreover, is not able to utilize the free sugar without first elaborating it to glycogen and mannan. Elias and Weiss * find that the reaction of the nutrient medium has a bearing upon the amount of glycogen produced, there being a marked increase in the presence of alkali. Although glycogen and mannan may be looked upon as a temporary reserve food.f for yeast-cells rich in glycogen retain their vitality much longer than those in which there is little or none, the fact that in the spores of species of Mucor and in sclerotia glycogen does not appear until growth has com- menced, points to the conclusion that in these plants, at any rate, it is not primarily a storage product. Kohl considers that since it is more abundant in Saccharomyces during active gemmation, it is not exclusively a reserve substance, but an intermediate product in the formation of alcohol from the sugar. In the animal kingdom, according to Hoppe-Seyler, glyco- gen is an invariable constituent of almost all developing cells ; it is found also in the muscles and blood, and chiefly in the liver, where it is stored in larger quantities. It may be remarked that there is little doubt that the glycogen obtained frdm'animal and plant sources are identical. * Ellas and Weiss : " Biochem. Zeit.," 1922, 127, i. t See Warkany : id., 1924, 150, 271. GLYCOGEN 169 Preparation. The following method of obtaining glycogen was devised by Pflijger.* Fresh finely-cut liver is stirred up with water and 60 per cent caustic potash, and heated for two hours ; the filtered solution, containing 15 per cent of potash, is then mixed with an equal volume of 96 per cent alcohol, and the precipitated glycogen is collected and washed with a mixture of I part of 15 per cent potash with 2 parts of 96 per cent alcohol ; if necessary, the substance may be redissolved and purified in the same way. Glycogen may also be prepared from yeast, but not in a particularly pure state, in the following manner : A quantity of baker's yeast, which has been previously well washed with water, is mixed with fine well-cleaned sand and ground very thoroughly in order to rupture the cells. The mixture is then placed in a vessel with about thrice its volume of water and heated for some time, being constantly stirred. The liquid is then filtered off, cooled, and strong alcohol added to the filtrate in order to precipitate the glycogen, which is filtered off. The glycogen so obtained may be purified by redissolving it in water, adding a little acetic acid, and boiling in order to remove any proteins which may not have been removed by the initial heating, filtering, and precipitating with alcohol. An elaborate method has been described by Harden and Young,f which has been modified by Ling, Nanji, and Paton.| Dried yeast, a commercial by-product of many breweries, is boiled for two hours with 2 per cent caustic soda, and after removal of the insoluble cell wall residue, the crude glycogen is precipitated by alcohol and freed from protein and nucleo- protein by heating with 60 per cent caustic potash. From this solution the glycogen is once more precipitated by alcohol, and is further purified from mannan (yeast gum) by precipi- tating the latter from a warm alkaline solution by means of Fehling's solution. The filtrate containing the glycogen is * Pfluger : " Pfliiger's Archiv f. Phys.," 1902, 91, 119. and 1903, 93, 163. t Harden and Young : " J. Chem. Soc," 1912, loi, 1928. I Ling, Nanji, and Paton : " J. Inst. Brewing." 1925, 31, 316. 170 THE CARBOHYDRATES then acidified with acetic acid and dialysed, the last traces of copper being removed electrolytically. The further purifica- tion of the glycogen is effected by repeated alternate pre- cipitation by alcohol and solution in water. According to these same authors, some of the glycogen of the yeast cell occurs in the plasma and is readily extracted by water, while another portion, which is less readily extracted by water, is associated with the cell wall. The former modifica- tion, which resembles the amylose constituent of the starch granule, produces in water a faintly opalescent solution which gives a pure red colour with iodine but no precipitate. The other modification, associated with the cell wall, appears to be a phosphoric ester of the form associated with the plasma and is comparable with amylohemicellulose ; its solution in water is opalescent and gives with iodine a reddish-brown precipitate. Properties. Pure glycogen is a snow-white amorphous solid. It is readily soluble in hot water, forming an opalescent solution, from which it may be precipitated again by alcohol, provided small quantities of dissolved salts are present ; lOO c.c. of a I per cent solution when mixed with 200 c.c. of absolute alcohol remain clear, but on adding 003-005 gram of sodium chloride, an immediate precipitate is formed. Glycogen is strongly dextro-rotatory, a^ = + 198-9°, and is coloured red to brown by iodine ; it does not reduce Fehling's solution, but is broken down by diastase into dextrin and maltose, and by acids into glucose. The fact that cold concentrated hydrochloric acid con- verts glycogen into triamylose is considered by Pringsheim to establish the identity of glycogen with amylopectin of starch. Identification. 1. The opalescent appearance of its aqueous solution is characteristic ; it is strongly dextro-rotatory. 2. A brown coloration is given with iodine solution. 3. A white precipitate is given with basic lead acetate in strong solutions only. GLYCOGEN 171 4. It does not reduce Fehling's solution. 5. On boiling with mineral acids, it is converted into dextrose. Estimation. This is best effected by heating the aqueous solution for three hours in a boiling water bath with about 2-2 per cent HCl, and then neutralizing and estimating the resulting glucose by means of Fehling's solution ; the amount multi- plied by 0-9 gives the weight of glycogen. According to Ling, results so obtained are vitiated by the presence of mannose which is produced by the partial hydrolysis of the mannan. He recommends hydrolysis by boiling for three hours with 8 per cent sulphuric acid and estimating iodometrically * the glucose and mannose produced before and after removal of the mannan. LICHENIN AND ISO-LICHENIN. Lichenin is the name given to a water-soluble polysaccharide extracted from " Iceland moss " — Cetraria islandica — and other lichens. When an aqueous extract of Iceland moss is concen- trated, a gelatinous precipitate of lichenin is formed while the solution contains a substance known as iso-lichenin. The latter substance, also known as lichen starch, is dextro-rotatory and gives a blue colour with iodine, and is said by Pringsheim and others f to be identical with amylose of the starch grain. Lichenin, on the other hand, is optically inactive and gives no blue colour with iodine ; according to Pringsheim it owes its gelatinizing properties to the fact that it is a carbohydrate ester of silicic acid. J On the other hand, Hess§ claims to have prepared highly purified ash-free samples of lichenin possessing unimpaired gelatinizing properties as compared with the less purified material, from which he concludes that the gelatinizing power * See Baker and Hulton : " Biochem. Journ.," 1920, 14, 754. t Pringsheim and Kusenack : " Zeit. physiol. Chem.," 1924, 137, 265 ; 1925, 144, 241. Pringsheim and others : " Ber. deut. chem. Gesells.," 1924. 57. 1581- J Cf. p. 154, under Amylo-hemicellulose. § Hess : " Zeit. angew. Chem.," 1924, 37, 993, 172 THE CARBOHYDRATES is not dependent upon the existence of an ethereal siHcate as stated by Pringsheim.* That there is a close relationship between lichenin and cellulose is shown by the work of Karrcr and Joos,t and also by the fact that lichenin J can be converted by the action of a lichenase contained in barley diastase into cellobiose, the disaccharide obtainable from cellulose (see p. 119), and that on acetolysis it yields, like cellulose, octacetycellobiose. When lichenin is heated in glycerol at 240° it is converted into a substance lichosan, an anhydride of glucose of the formula — , O , CH— CHOH . CHOH . CH . CH . CH.,OH ! o 1 Since cellulose may likewise be converted into a glucose anhydride cellosan, hchenin and cellulose are both regarded as products of associated glucose anhydrides. By a comparison of the optical rotation in cuprammonia solution of cellulose and lichenin, however, Hess § has shown that these two substances are not structurally identical. The same conclusion is reached by Herzog and Gonell H ; they were, however, able to establish the identity of plant cellulose with that of animal origin (Tunicin). Lichenin is widely distributed in nature and has been found by Karrer and Staub ^ in Evernia vulpina, Usnea harhata, Parmelia furfuracea, and in barley, oats, maize, spinach, beans, hyacinth bulbs, and other plants. The same authors have isolated from the ahmentary canal of the snail. Helix pomatia, the enzyme lichenase which also occurs in barley and other plants and can hydrolyse lichenin to glucose in a few hours ; the enzyme is comparable with cytase which attacks reserve cellulose, and for this reason they are inclined to look upon lichenin as a reserve cellulose. The fact that a * Pringsheim and Kusenack : " Zeit. physiol. Chem.," 1924, 137, 265. t Karrer and Joos : " Biochem. Zeit.," 1923. 136, 537 ; " Helv. Chim. Acta," 1924, 7, 144. t Pringsheim, Knoll, and Kasten ; " Ber. dent. chem. Gesells.," 1925. 58, 2135. Pringsheim : " Biochem. Zeit.," 1926, 172, 411. §Hess: "Zeit. angew. Chem.," 1924, 37. 993- \\ Herzog and Gonell : " Zeit. physiol. Chem.," 1924, M'? 63. \\ Karrer and Staub : " Helv. Chim. Acta," 1924, 7, 159. FRUCTOSANS 173 lichenase should occur in the snail is significant in view of the wide distribution of lichenin in the plant world. PARA-DEXTRANE AND PARA-ISODEXTRANE. These substances have been isolated from Boletus ediilis and Polyporus hetulinus respectively. The former gives a yellow colour with chlorzinc iodide, and the latter a blue when treated with iodine and sulphuric acid. Both give glucose on hydrolysis. FRUCTOSANS. INULIN. This substance is commonly found as a reserve food-stuff, of the same nature as starch, existing in a state of solution in the cell sap of a number of plants belonging to the natural order Compositge, e.g. in the tubers of the dahlia and artichoke [Helianthus tuberosus), and in the fleshy roots of the chicory [Cichorium Intihus). It has also been described as occurring in the following natural orders : Violaceas, Malpighiacege, Drose- raceae, Candolleaceae, Goodeniaceae, Campanulaceae, Lobelia- ceae, Myoporineae, Liliaceae, and Amaryllidaceae ; also in some Algae, e.g. Neomeris. Inulin, or closely allied substances, are not infrequently found in company with starch, especially in some Monocoty- ledons, and exhibits the same peculiarity in its occurrence, as has already been remarked upon in connection with starch in monocotyledonous plants (p. 148). Thus in Iris pseudacoriis starch is present but not abundant, in Iris Xiphium both starch and inulin are present in quan- tity ; Scilla nutans has inulin but no starch, while Scilla sibirica, and also Hyacinthus and Muscari botryoides have both starch and inulin. According to Grafe and Vouk * and Melchior,t inulin is found in the leaves of Cichorium intibus and Marcgravia spp., and is considered to be a direct assimilatory product. On the other hand, Colin J finds that the leaves of Helianthus tuberosus * Grafe and Vouk : " Biochem. Zeit.," 1912, 43, 424 ; 1913, 56, 249. t Melchior : " Ber. deut. bot. Gesells.," 1924. 42, 198. I Colin : " Compt. rend.," 1918, 166, 224, 305 ; " Bull. Assoc. Chim. Sue," 1919. 37> 121 ; " Bull. Soc. chim. biol.," 1925, 7, 173. 174 THE CARBOHYDRATES never contain inulin, but do contain dextro-rotatory sugars and starch. The formation of inuhn begins in the stem and is completed in the tubers. Thus the inuhn must be formed from dextro-rotatory sugars synthesized in the leaves. Preparation. Inulin may be obtained from dahlia tubers, of which it forms from 10-12 per cent, by crushing them and pressing out the liquid and filtering ; the residue is then boiled up with a little water and some precipitated chalk and filtered again. The two filtrates are then united and once more boiled with chalk in order to neutralize any acids, and while still warm treated with lead acetate until no further precipitate is formed. The filtered solution is then saturated with hydrogen sul- phide, filtered, neutralized with ammonia, evaporated to small bulk, and mixed with an equal volume of alcohol. After one or two days, crude inulin may be filtered off ; it may be further purified by warming in aqueous solution with animal charcoal, filtering, and adding alcohol ; the precipitated inulin is then washed with alcohol and ether, and dried over sulphuric acid. According to Kiliani,* it may also be prepared by boiling crushed dahlia tubers with water and a Httle chalk, filtering and freezing the filtrate. When the water cools, the precipi- tate is filtered off, re-dissolved in hot water and frozen out once more. After repeating this process several times, the inulin is washed with methyl alcohol, ethyl alcohol, and finally ether. Characters. Pure inulin forms a white starchy tasteless powder of a sphaero-crystalline nature ; it swells up and is readily dissolved in hot water, alkalis, etc., and may be recovered from the aqueous solution by the addition of alcohol, in which it is practically insoluble, or by freezing. Highly purified inulin should give less than 0-2 per cent of ash, but the removal of the last traces of inorganic substances is so difficult as to suggest that they form a definite part of the molecular com- * Kiliani : " Annalen," 18S0, 205, 147. FRUCTOSANS 175 plex.* Iriulin is Icevo-rotatory, aj,=— 35°, and is non- reducing. Unlike starch it does not give a paste with water, nor does it give a blue colour when treated with iodine. Diastase has no effect upon it ; it may, however, be hydrolysed by the ferment inulase, or by mineral acids, by which reagents it is converted into fructose. Whilst the final product ob- tained is ordinary fructose, the initial product is, presumably, y-fructose, since inulin has been shown to be a polymerized form of this active sugar. The low osmotic pressure which solutions of inulin exert suggests a large molecule, but its molecular structure appears to be less complex than that of starch. Identification. In many plants the presence of inulin is indicated by the well-known sphaero-crystals which are obtained on steeping the fresh tissues for some time in strong alcohol ; this deposi- tion is not, however, always so characteristic ; thus in Mono- cotyledons the inulin is frequently found, after treatment with alcohol, in amorphous masses. The sphaero-crystals and the amorphous concretions of inulin are readily soluble in warm water, and thus may be distinguished from calcium phosphate which may occur in cells in shapes similar to those of inulin. These two substances may be further recognized by the fact that sulphuric acid completely dissolves inulin, whereas it forms with calcium phosphate insoluble calcium sulphate. The following tests also may be performed : — 1. Green's Test. — Sections of the material, which have been soaked for some time in absolute alcohol, are treated with a saturated solution of orcin in strong alcohol, and then boiled in hydrochloric acid. The masses of inulin disappear and a red colour results. If phloroglucin be substituted for the orcin, the resulting coloration will be reddish-brown. 2. Molisch's Test. — The sections are treated with a 10 per cent alcoholic solution of a-naphthol, then a few drops of strong sulphuric acid are added and the preparation warmed. A deep violet coloration ensues, and the inulin is dissolved. * Irvine and Steele : " J. Chera. Soc," 1920, 117, 1474. 176 THE CARBOHYDRATES These colour reactions are indicative of the formation of sugar by the hydrolysis of the inulin by the acids employed in the tests ; it is therefore important, before employing these reactions, to make sure that no free sugars are present in the material to be examined, and to wash the preparations thoroughly with alcohol in order to remove them. Since inulin does not reduce Fehling's solution, this re- agent may be employed to ascertain whether any reducing sugars are present in the material before employing the above tests for inulin. The following reactions may be carried out with a solution of inulin. 3. Basic lead acetate gives no precipitate with inulin. 4. Inulin is precipitated from solution by alcohol. 5. Hydrolyse with mineral acid and test for levulose. Physiological Significance. It is of interest to find that the nature of the reserve carbo- hydrates may often be correlated to the habitat of the plant. Parkin * points out that these reserve substances of aquatic plants and of plants inhabiting wet situations take the form of starch, e.g. Sparganium, Alisma, Listera, Orchis, and Schizo- stylis ; whereas, on the other hand, inulin, generally associated with sugar, is the characteristic carbohydrate reserve in those Monocotyledons inhabiting dry situations, e.g. Allium, Aspho- delus, Anthericum, Yucca, Tritona, Iris Xiphium, etc. In this connection f reference must be made to the work of Lidforss, who showed that plants inhabiting wet situations fall into two distinct categories ; those like Elodea, Chara, and Stratiotes, which hibernate at the bottom of the pond or stream, contain starch but no sugar ; while those which live on the banks where their rhizomes, or other organs of storage, pass the winter out of the water, e.g. Myosotis and Menyanthes, contain sugar during the winter months. In the former case a temperature of —2° C. to —4° C. is fatal, while in the latter case the death point is about —7° C. * Parkin : " Phil. Trans. Roy. Soc, Lond.," B, 1899. 191, 169. t See Blackman : " New Phyt.," 1909. 8, 354. FRUCTOSAXS 17; This peculiarity also obtains for many arctic plants ; Miyake, Wulff, and others have shown that cold, which means physiological dryness, is conducive to sugar production, so that arctic plants frequently exhibit but a small amount of starch, and relatively large quantities of sugar. Stahl has shown that the leaves of mycotrophic plants, which generally show a feeble transpiration, seldom contain starch, its place being taken by glucose. Lidforss also has shown that the winter green vege- tation of Sweden is characterized by the absence of starch from the leaves, the mesophyll, in its place, containing relatively large quantities of sugar, and sometimes oil during the winter months. In summer the leaves of these plants contain starch, which, on the advent of winter, is converted into sugar, from which starch is formed on the rise of temperature in the spring.* Then, again, it is not uncommon to find sugar stored in the periderm of trees and in the leaves of evergreen plants during the winter ; starch, however, may be found in the leaves of evergreen trees during the cold season, its presence being due to feeble photosynthesis. Reference may be made here to the well-known fact that potatoes turn sweet on exposure to cold. This conversion of starch into sugar is most active at 0° C, and the action de- creases with the rise in temperature, so that above 7° C. no sugar is thus produced. Also if the tubers are suddenly sub- jected to a temperature of —1° C, no sugar will be produced. The amount of sugar formed is not great, its maximum being about 3 per cent of the wet weight ; the limit of the process depends on the concentration of sugar, and, as Czapek has shown, the transformation of the starch may be prevented, on a lowering of the temperature, if the concentration of sugar be sufficient. If these sweet potatoes be exposed to a higher temperature, all the sugar that remains — some has been used up in respiration — is reconverted into starch. CEcologically these characters are of value to the plant ; for if the water of the cell sap be frozen, the salts held in solution become concentrated and will eventually precipitate the soluble • See also Maximow : " Bar. deut. bot. Gesells.," 1912, 30, 52. 12 178 THE CARBOHYDRATES proteins. Parkin points out that the presence of inuUn * in the cell sap of the parenchymatous tissues would retard the evaporation of water. It is a well-known fact that water in the presence of oil may be much over-cooled before ice-forma- tion takes place, and the freezing-point of water in which other substances, e.g. sugar, are dissolved is depressed, and thus the danger arising from the salting out of the proteins is mini- mized. But, notwithstanding these facts, plants are frequently subjected to temperatures sufficiently low to cause ice to be formed, and as the water is thus withdrawn, the sugar becomes more concentrated until it will also crystallize out. Both these processes generate heat, which may be sufhcient in amount to enable the protoplasm to live. And this is, accord- ing to Mez and Lidforss, the explanation of the presence of sugar in winter leaves. At the same time we must be careful not to push such explanations too far, for there are many exceptional cases ; thus Ewart has pointed out that Dicraniim which contains much oil is less resistant to cold that Bryum, and other mosses, in which such substances are absent. The beetroot also is very susceptible to cold, notwithstanding the fact that it contains much sugar ; similarly the seeds of the hemp and willow, which contain much oil, are easily killed by desiccation, whereas the oil-containing seeds of the linseed are highly resistant. Such divergent phenomena must depend on the constitution of the protoplasm. Again, oil is a convenient form of reserve food, especially in small organisms and in reproductive bodies, where space is limited and lightness is all-important and it is desirable to store a maximum of potential energy in the minimum of bulk. INULIN-LIKE SUBSTANCES. A number of ill-defined substances similar to inulin have been described as occurring in various plants. The chief of these are : — Graminin in Agrostis, Fesluca, Triticum. Arrhenatherum, and other grasses. * See also Grafe and Vouk : " Biochem. Zeit.," 1913, 56, 249. HEMICELLULOSES 179 Irisin in Iris pseudacoriis. Phlein in Phleiim pratense and Phalaris arundmacea. Sinistrin in Scilla maritima. Triticin in Triticum repois, Draccena australis and Dra- ccBna rubra. Of these fructosans, graminin, and triticin are not precipi- tated from neutral or acid solutions by heavy metal salts. With barium hydroxide they give insoluble compounds, but the corresponding calcium and strontium compounds are. soluble. They do not reduce Fehling's solution and yield only fructose on hydrolysis. All these compounds possess the same characteristics ; they are laevo-rotatory, yield fructose on hydrolysis, and are fairly soluble in cold water. The majority are difficult to crystallize, and their solutions yield a gum-like substance on evaporation. It is possible that some, at any rate, of these substances may bear the same relation to inulin as dextrin does to starch. HEMICELLULOSES. Whilst it is possible on a physiological basis to distinguish between food reserve polysaccharides such as starch, inulin, and glycogen, on the one hand, and the typical structural polysaccharide cellulose on the other, there occur in the plant a number of related compounds whose physiological role is less sharply defined. These substances are associated with the structural elements of the plant and form part of the cell wall but they may, on occasion, be attacked by appropriate enzymes, secreted in the plant, and be utili-'.ed as food. Owing to their dual function, and to emphasize their relation- ship to cellulose, they are commonly termed reserve celluloses. Whilst they resemble cellulose in many of their physical properties, they differ from cellulose in their chemical pro- perties. Included in the hemicelluloses are mannan, galactan, and pentosan, which have been isolated from wheat and rye bran, from beans and pea pods, and from lichens, and wood gums which have been isolated from wood. T2 i8o THE CARBOHYDRATES Properties. Schulze * first proposed for this group of substances the term hemicellulose, characterized by their insolubiUty in water, solubiUty in alkaH, and precipitation from their alkaUne solution by acid or by alcohol. On hydrolysis by means of dilute acid they give origin to one or more monosaccharides which may be either hexoses or pentoses, whilst cellulose, which is more difficultly hydrolysed, yields glucose only. Although hemicelluloses are normally insoluble in water, and are not extracted from wood by hot water, they become soluble in water after extraction by means of alkali ; the galactan of coniferous wood is, however, an exception, being, according to Schorger, completely extractable from the wood by hot water. From alkaline solutions some, but not all hemicelluloses are precipitated as copper compounds on the addition of boiling Fehling's solution ; among those not precipitated are the arabans of the beet and cherry gum. f Constitution. The earlier workers looked upon hemicelluloses as poly- merized anhydrides of pentoses or hexoses or of mixed sugars as is indicated by the names xylan, araban, mannan, galactan, galactoaraban, galactoxylan, etc. It has, however, been shown by O'DwyerJ for the two hemicelluloses of beech wood that they are not true polysaccharides in that they contain acid groups, one in the form of a galacturonic acid and the other a glycuronic acid residue, from which facts these compounds would appear to be more closely related to the pectins (see below) than to cellulose. By extraction of various starches with normal sodium hydroxide, after a preliminary digestion with taka-diastase, Schryver and his co-workers have obtained solutions from which they were able to precipitate by acetic acid a hemi- * Schulze : " Zeit. physiol. Chem.," 1892, 16, 391. t Salkowski : id., 1901, 34, 171. X O'Dwyer : " Biochem. Journ.." 1926, 20, 656. HEMICELLULOSES i8i cellulose to which they assign the formula sCgHioOs + 2H2O (see also p. 154). MANNAN. Mannan occurs in salep mucilage, and has been extracted by Ritthausen * and Effront f and others from wheat and barley. Mannans are also found in Penicillium glaiiciim, ergot, in the roots of several plants such as asparagus, chicory Helianthus and Taraxacum ; also in the wood and leaves of many trees, such as lime, chestnut, apple, mulberry, certain Oleaceae and conifers ; the so-called reserve celluloses and hemicelluloses contained in seeds of Palmaceae, LiHacese, elder, cedar, and larch, and many other plants, are also very rich in mannans. Evidence for the occurrence of a manno- galactan in the American white oak has been furnished by O'Dwyer.J The mannan of the vegetable ivory, the endosperm of the seeds of Phytelephas macrocarpa, may be prepared in 8-10 per cent yield by treating the ground ivory meal with five times its weight of 10 per cent caustic soda for half an hour ; § the black alkaline liquid is filtered through copper gauze, and the residue after washing with water is boiled for half an hour with five times its weight of 20 per cent caustic soda ; the solution is then precipitated by the addition of one-third of its volume of rectified alcohol spirits and the precipitate after washing with the same precipitant is dissolved in hot water ; after adding sufficient acetic acid to render just acid, the solution is boiled for a few minutes when the mannan is precipitated as a white powder. The mannan of vegetable ivory was shown by Baker and Pope II to be contaminated with laevulo-mannan and galacto- mannan. It was shown by Pringsheim ]f that ivory nut shavings were * Ritthausen : " J. prakt. Chem.," 1867, 102, 321 ; and " Chem. Zeit.," 1897, 21, 717. t Effront : " Compt. rend.," 1897, 125, 38, 116. I O'Dwyer : " Biochem. Journ.," 1923, 17, 501. § Patterson : " J. Chem. Soc," 1923, 123, 1139 ; Schmidt and Grau- mann : " Ber. deut. chem. Gesells.," 1921, 521, 1867. II Baker and Pope : " J. Chem. Soc," 1900, 77, 696. t Pringsheim : " Zeit. physiol. Chem.." 191 2, 80, 376. 1 82 THE CARBOHYDRATES hydrolysed by certain bacteria to mannose and a trisaccharide mannotriose, and Paton, Nanji, and Ling * have found that the nuts themselves contain an enzyme capable of hydrolysing the mannan to mannose with probably the intermediate formation of mannotriose. A substance known as yeast gum, which occurs in consider- able quantities in yeasts of weak fermenting power.f is also a mannan ; the amount of this substance present in yeast is in inverse proportion to the amount of glycogen (cf. p. i68), but it is not regarded as a reserve substance ; its solution, which has a strong foaming power in water, does not reduce Fehling's solution. PARAMANNAN. Paramannan is a variety of mannan which is characterized by being much more resistant to hydrolysis ; this substance, which is contained in coffee beans, is only slightly acted on by hot dilute mineral acids, potassium chlorate, and hydrochloric acid, but dissolves in a concentrated hydrochloric acid solution of zinc chloride. CARUBIN OR SECALANE. Carubin J is the name given to a substance occurring in the seeds of Ceratonia siliqua, and in various cereals such as rye and barley. In its characters it closely resembles mannan, and by some authors is regarded as identical with it ; when dry, it is a spongy friable substance which swells upon the addition of water. It is soluble in cold water and is optically inactive. Its sugar is fermentable and non-crystalline. XYLAN. This substance may be obtained by extracting sawdust from the wood of deciduous trees with dilute caustic soda after preliminary extraction of the sawdust successively with organic solvents, water, ammonia, and finally washing with water. The yield of xylan obtainable from birch wood is, according to Schorger, 197 per cent, but the amount obtained * Paton, Nanji, and Ling : " Biochem. Journ.," 1924, 18, 451. t Hashitani : " J. Inst. Brew.," 1927, 33. 347- jEffront : " Compt. rend.." 1897, 124, 200, and 125, 116 and 309. HEMICELLULOSES 183 from coniferous wood is much less. Xylan also occurs in corn cobs and in straw, from which latter source it may be conveniently prepared in a state of purity * by extraction with caustic soda and precipitation by means of an alkaline solution of copper sulphate. Xylan was formerly thought to be a polymerized anhydride of xylose, since it gives rise only to this sugar on hydrolysis ; the observations of O'Dwyer on the hemicelluloses of American white oak render this view no longer tenable (see p. 181). Xylan, precipitated from alkaline solution by the addition of acid, is soluble in hot water ; but water will not extract it from wood in the first instance. ARABAN. This substance, which on hydrolysis gives rise to arabinose, is associated with xylan in wood ; it is not clear whether it occurs as a distinct individual, or whether it is combined with other material ; according to O'Dwyer beech wood contains two hemicelluloses, one of which contains xylose in combination with glycuronic acid, while the other contains arabinose combined with galacturonic acid. Another substance yielding arabinose on hydrolysis and which has been regarded as a true carbohydrate or poly- saccharide form of arabinose is cherry gum ; this substance is exuded from the bark of the cherry and yields on hydrolysis chiefly arabinose with a small quantity of xylose, whereas the wood of the cherry extracted with alkali yields a product which produces chiefly xylose. Gum-arabic likewise contains an araban since it yields chiefly arabinose on hydrolysis. An araban has also been described by Ehrlich as arising from the hydrolysis of protopectin (see p. 202). WOOD GUM. This term is applied to the hemicelluloses extractable from wood by caustic soda. A true gum should at least swell up if not dissolve in water, but many of the so-called wood gums * Salkowski : " Zeit. physiol. Chem.," 1902, 34, 162. 1 84 THE CARBOHYDRATES are not directly extractable by boiling with water, and occur in the wood in such combination that they are only soluble in water after precipitation from the alkaline solution with which they were extracted from the wood. There is a good deal of variation in the composition and properties of the various wood gums ; thus while hard woods may contain up to 20 per cent of xylan, the wood of gymnosperms con- tains only about i per cent of this substance but contains, on the other hand, galactans * ; amounts of galactan varying from 8-17 per cent have been found in Larix occidentalis.'\ According to Schorger and Smith % this substance is characteristically associated with coniferous wood, though it has also been reported in the wood of angiosperms such as aspen, white oak, and apple ; it is not certain whether all these woods contain the same galactan. The so-called e-galactan which occurs in the wood of Larix orientalis, to the extent of about 8-17 per cent, is a white powder which dissolves readily in cold water, and in this respect bears no true resemblance to a gum. GALACTAN. The term galactan is apphed to any non-reducing substance which on hydrolysis gives rise to galactose ; while the plant world supplies a great many substances which yield galactose on hydrolysis, the number of such substances which yield this sugar only, unaccompanied by other sugars, is small. A number of other galactans have from time to time been described as occurring in the seeds of lucerne, lupin, and in beets ; these are ill-defined substances which in the past have been distinguished by prefixing letters of the Greek alphabet to the term galactan, but the present state of our knowledge concerning them does not justify a fuller description. * Schorger and Smith : " J. Ind. Eng. Chem.," 1916, 8, 494. t This fact has been commercially exploited in America for the manu- facture of raucic acid by the oxidation with nitric acid of hydrolysed sawdust ; the galactan has also been recommended as a source of alcohol. I Schorger and Smith : " J. Ind. Eng. Chem.," 1916, 8, 494. HEMICELLULOSES 185 MIXED GALACTANS. Other sources of galactan which, however, do not yield exclusively galactose on hydrolysis are of common occurrence. Such substances have been variously described as galacto- arabans, galacto-xylan, galacto-mannan, etc., according to the sugars to which they give rise ; these occur notably in the mucilaginous extracts of seaweeds and form the agar and carragheen extracts of commerce (see below, p. 191). Under this heading should be included galacto-araban, sometimes wrongly described as para-galactan, which occurs in the cell walls of the cotyledons of many plants, e.g. Lupinus luteus and other species, Phoenix dactylifera, Cocos nucifera, and other palms, Soja hispanica and Coffea arabica, where it forms a reserve food-material which is digested on germination. Galacto-araban is a white solid which is insoluble in water and cuprammonia ; it dissolves in hot potash. On heating with nitric acid it is oxidized to mucic acid. Microchemically it may be identified by its insolubility in the reagents men- tioned, and also by the fact that with phloroglucin and hydro- chloric acid it gives a red coloration on warming. No colour is given in the cold. Its association with cellulose prevents the latter exhibiting some of its reactions ; thus the cellulose is unacted upon by cuprammonia unless the galacto-araban be removed ; this may be done by boiling in dilute hydrochloric acid. Other substances giving rise to galactose are the pectins (p. 192). AMYLOID. Amyloid is the name given to a substance occurring in the seeds of paeonies and certain cresses,* which yields on hydrolysis with dilute sulphuric acid a mixture of galactose, glucose, and xylose. It is a colourless substance, and is in- soluble in cold water, but swells up into a slimy mass in hot water ; it is soluble in cuprammonia solution. Amyloid does not reduce Fehling's solution, but is oxidized by nitric acid to mucic and trihydroxy-glutaric acids. It gives a blue colour with iodine. * Winterstein : " Zeit. physiol. Chem.," 1893, 17, 353. 186 THE CARBOHYDRATES GUMS. The natural gums were formerly thought to be carbo- hydrates of the general formula (C6Hio05)n ; the researches of O'Sullivan, however, have shown that they are not simple carbohydrates, since on hydrolysis they give rise to sugars mixed with complex acids of high molecular weight. The gums themselves retain the character of acids, and it would appear that the molecule of a gum is composed of a number of sugar residues grouped around a nucleus acid in such a way as to leave the acid group exposed. The gums are translucent amorphous substances, some of which dissolve in water completely, giving a sticky solution, while others merely swell up in water and form a sort of jelly ; they are all insoluble in alcohol. The natural gums must be distinguished from starch gum or dextrin, which is an artificial product obtained from starch, by the following characteristics : — 1. Solutions of natural gums arc Igevo-rotatory, whereas those of dextrin are dextro-rotatory. 2. Basic lead acetate precipitates natural gums from solu- tion, but has no action on dextrin in weak solutions. 3. Natural gums on hydrolysis yield chiefly galactose and pentoses such as arabinose or xylose, whereas dextrin yields glucose only. The hydrolysis of gums takes a long time to complete — from eighteen to twenty-four hours — whereas dextrin is easily hydrolysed. 4. On oxidation with nitric acid, natural gums yield chiefly mucic acid (CgHioOg) together with some saccharic (CeHioOg) and oxalic (C2H2O4) acids, whereas dextrin yields chiefly oxalic acid together with a small quantity of saccharic and tartaric (C4H6O6) acids. As they occur in nature, the true gums are mostly com- bined with potassium, calcium, or magnesium in the form of salts, from which the free acid can be isolated by the action of a stronger acid. GUMS 187 The classification of gums is, for want of more accurate knowledge, based chiefly on their solubility in water : — (a) Gums, such as arabin, which are completely soluble. (b) Gums which are partially soluble, such as cerasin and bassorin. {c) Mucilages and pectic bodies which swell up with water and dissolve, and in concentrated solution form a jelly. The classification, however, is by no means rigid, many natural gums being composed of mixtures of several kinds of gums. In the separation of gums from the tissues of the plant advantage is taken of their solubility in water ; it is found in practice, however, that in many cases mere maceration in water does not remove all the gum present. Microchemical Reactions. Microchemically, gum and mucilage may be recognized by their solubility and swelling respectively in water. Both are insoluble in alcohol and ether. With other reagents the results differ in different examples. Thus with iodine either a blue or a yellow colour may result, while in other cases the blue coloration is only obtained after treatment with chlor- zinc iodide or sulphuric acid and iodine, indicating a close association with cellulose ; this type of mixed gum, e.g. gum tragacanth, is not stained by such dyes as ruthenium red (an ammoniacal solution of ruthenium sesquichloride), whereas true gums, such as those of apricot, cherry, peach, etc., are stained red. They show different degrees of solubility in cuprammonia. Many of these substances stain well with corallin soda, and they also, especially the mucilages, show a great avidity for stains such as aniline blue and aniline violet. GUM-ARABIC. This substance is a mixture of calcium, magnesium, and potassium salts of a weak acid of unknown constitution, to which earlier writers gave the name of arable acid or arabin. O'SuUivan,* however, applied the term arable acid to a *0'Sullivan: "J. Chem. Soc," 1884, 45, 41 ; 1891, 59, 1029. 1 88 THE CARBOHYDRATES substance of the formula C23H38O22, which he regarded as the nucleus acid around which a number of sugar residues are grouped ; by hydrolysis under varying conditions, it is possible to split off successive sugar residues with the formation of acids of gradually decreasing molecular weight, until finally the nucleus acid free from all carbohydrate residues remains, and it is this acid that he calls arable acid ; the natural gum itself would, according to him, be a diarabinan-tetragalactan- arabic acid of the formula 2C10H10O8, 4C12H20O11, C23H30O18, which is combined with the calcium, magnesium, and potas- sium. The arable acid of the earher authors, which is the acid set free from the natural gum by the removal of the calcium, magnesium, and potassium, may be prepared by acidifying a concentrated aqueous solution of gum-arabic with hydro- chloric acid, and adding alcohol. The pure substance is a white amorphous glassy mass which dissolves in water to give a Isevo-rotatory solution. Ten per cent sulphuric acid converts this arable acid into metarabic acid, which swells up in water, but does not dissolve. Reactions. Solutions in water (10 per cent) of arable acid and other varieties of gum-arabic give, according to Masing,* certain more or less definite reactions. 1. They are not precipitated by {a) a cold saturated solu- tion of copper acetate ; [h) 10 per cent solution of lead acetate ; [c) solution of ferric chloride (sp. gr. 1-2). 2. A 5 per cent solution of silicate of potash produces a cloudiness or a precipitate which is partially or wholly soluble on adding an excess. Arabic acid either does not respond to this reagent, or merely gives a slight turbidity, and the same applies to the gums obtained from certain species of Cactus, Albizzia, Acacia catechu, Acacia leucophlcea, and other plants. 3. Stannate of potash gives similar reactions, and in the case of arable acid produces a precipitate which is soluble in excess. * Masing : " Archiv d. Pharm.," 1879, [3], 15, 216 ; 1880, 17, 34, 41 ; " Year Book of Pharmacy," 1881, 191, GUMS 189 4. A solution of neutral sulphate of aluminium (10 per cent) generally gives a precipitate which is, in many cases, soluble in potash. 5. Basic lead acetate yields a precipitate which is entirely or partially soluble in excess. GUM TRAGACANTH. This gum occurs in species of Astragalus, and consists of about 8-10 per cent of soluble calcium, magnesium, and potassium salts, together with about 60-70 per cent of in- soluble salts, which only swell up in water to a jelly. The water-soluble portion is said to contain a substance, poly- aribanan-trigalactan-geddic acid, which on hydrolysis breaks up into arabinose, galactose, and geddic acid, an isomer of arable acid. The part soluble in water, when treated with baryta water, gives two isomeric tragacanthan-xylan-bassoric acids, which on hydrolysis yield a pentose sugar tragacanthose, xylose, and bassoric acid C14H20O13. Von Fellenberg * has shown that the water-insoluble constituent of tragacanth and bassorin gums is a methoxylated compound ; it dissolves in alkali undergoing hydrolysis with the liberation of methyl alcohol. The de-esterified compound is named bassoric acid and yields on hydrolysis large quantities of galacturonic acid showing it to be allied to the pectic acid derived from pectin. WOUND GUM. A gum-like substance, termed wound gum, is frequently found in the tracheae of plants, in the immediate neighbour- hood of wounds, and stopping up the lumina ; it is secreted by the surrounding living cells. Wound gum does not swell in water, and is insoluble in sulphuric acid and in caustic soda. On oxidation with nitric acid it yields both mucic and oxalic acids, and it responds to lignin tests ; e.g. on treatment with phloroglucinol and hydrochloric acid a bright red coloration results. The origin of gums is as yet unknown ; by some authors they are regarded as decomposition products of cellulose, * V. Fellenberg : " Biochem. Zeit.," 1918. 85, 118. I90 THE CARBOHYDRATES produced either by over-nutrition of certain cells or by bacterial action ; * according to Wiesner, all gums are pro- duced by a diastatic ferment acting on cellulose ; although it is not possible to express any definite views on the subject, it would appear not improbable that in many cases the formation of gums and gum-like substances in the plant is a morbid condition. Mohl was able to show in the case of tragacanth gum that this substance was produced by the metamorphosis of the cells of the medullary rays. MUCILAGE. The term mucilage is applied to those substances which with water produce a slimy liquid. Mucilage is widely dis- tributed, and occurs in all or nearly all classes of plants. Mucilage-secreting hairs, or comparable structures, occur in various Muscinese, Filices, and especially in the Phanerogams ; mucilage sacs or canals are found in certain Muscineae, e.g. Anthoceros, Marattiaceae, some Cycadaceas, and Phanerogams ; further, the external walls of plants may be generally mucila- ginous ; e.g. in very many Algae, the hibernaculas of some aquatic Phanerogams, like Utricularia and Myriophyllum, and finally in the coats of seeds and fruits, such as Lepidium and Sterculia scaphigera respectively, in which cases the superficial cell walls are mucilaginous. Mucilage is not infrequently associated with other substances ; thus in the case of mucilage- secreting hairs, it is sometimes associated with tannin, and in many plants, especially in the mucilage sacs of many Mono- cotyledons, calcium oxalate is found. Employed in the morphological sense the term mucilage includes a number of chemically distinct substances ; thus while the mucilages from linseed, many of the Liliacese, and also salep yield only sugars on hydrolysis, many of the mucil- ages contained in seaweeds yield in addition to sugars, ash constituents which, previous to hydrolysis, were chemically combined with the carbohydrate residue. The high sulphate content of the ash of carragheen mucilage f (obtained from * See Greig Smith : " J. Soc. Chem. Ind.," 1904, 105, 972. t Haas and Hill : " Ann. App. Biol.," 1921, 7, 352 ; Haas, " Biochem. Journ.," 1921, 15, 469. GUMS 191 Chondrus crispus) and of agar * is accounted for by the fact that these substances have their carbohydrate residues com- bined with calcium sulphate in the form of an ethereal sul- phate represented by the formula — • o— SO,— Ov ^o— so,— O^ in which R represents the polysaccharide residue. Substances of this type have been shown to be colloidal electrolytes which exert measurable osmotic pressures ; their solutions contain calcium ions, but the sulphate complex is masked and is only set free after hydrolysis : — yosop-. .on R<^ pCa + HjO ^ R\ + CaSO, + H2SO4 ^OSO^O-^ ^OH It will be seen that this hydrolysis involves the liberation of a molecule of sulphuric acid, a fact which accounts for the charring of this material which frequently occurs, owing to spontaneous hydrolysis, when the material is heated in a steam oven and even, sometimes, in the cold. Mucilages of this type have been shown to occur in a number of marine algae f both red and brow^n. Function. Mucilage, when it is a definitely secreted product or of a definite and constant occurrence in a plant, may perform several functions, but how far these are primary functions cannot yet be stated. When it occurs in tubers, as in the OrchidaccEe, mucilage is generally looked upon as a reserve food-material ; it may serve as a check against too rapid transpiration, especially when produced in connection with developing organs, such as vegetative buds, young leaves, in the epidermis of mature leaves, the sporangia of Cryptogams, etc. ; in the case of aquatic plants, such as Algae, the hibernaculae of Myrio- phyllum, etc., its presence may prevent a too rapid diffusion ; * Neuberg and Ohle : " Biochem. Zeit.," 1921, 125, 311. t Haas and Russell-Wells : " Biochem. Journ.," 1923, 17, 696 ; also Harwood ; " J. Chem. Soc," 1923, 123, 2254. 192 THE CARBOHYDRATES the calcareous incrustation of certain Algae, e.g. Neomeris dumetosa, is dependent on the presence of mucilage ; mucilage provides a water-storage mechanism in plants subjected to xerophytic conditions, e.g. Cassia obovata, Malva parviflora, Theobroma cacao, and Pterocarpiis saxatilis ; finally, it may be an important aid in connection with seed-dispersal and ger- mination, as in some species of Salvia and Lepidium. Related to the gums and mucilages are the substances known as galactans occurring in the seeds of Leguminosae {Lupinus, Medicago, etc.) ; wood gum or xylan, occurring in wood, etc. These substances have already been dealt with. PECTIC BODIES. The term pectin was first applied by Braconnot * to the mucilaginous substance which he precipitated by means of alcohol from the juices of many fruits and from aqueous extracts of fleshy roots such as beet, carrot, swede, etc. Similar substances were later found to obtain in a great variety of plants such as onion, pea pods, leaves and stalks of cabbage, rhubarb, and flax, and also in young cells such as the root hairs of cabbage, cucumber, bean, and other plants. f In all these cases the pectin occurs in a state of solution in the cell sap or in association with the cellulose of the cell walls of parenchymatous tissues. The name pectin was chosen because it was recognized that these substances were in some way connected with the jellying properties of fruit juices, Tre/cru? being the Greek for jelly. Fremy J was the first to show that unripe fruits contain an insoluble precursor of the soluble pectin to which he gave the name pectose ; as the fruit ripens the insoluble pectose is gradually converted into soluble pectin, a change which is revealed under the microscope by the swelling of the thickened walls which become translucent and exude a mucilaginous pectin. Somewhat similar changes are brought about by boiling * Braconnot, " Ann. d. Chim. et Phj^s.," 1824, [2], 28, 173. t Howe : " Bot. Gaz.," 1921, 72, 313. I Fremy : " J. de Pharm.," 1840, [2]. 26, 368 ; and " Ann. d. chim. et d. Phys.." 1848. [3]. 24, 5. PECTINS 193 unripe fruit, whereby the acid juices exercise a hydrolytic effect upon the insoluble precursor and soluble pectin results. Prolonged boiling alters the pectin, with the result that its power to form a jelly is reduced ; similarly, over-ripe fruit loses its coherence owing to the loss of the jellying qualities characteristic of the soluble neutral pectin. When the water-soluble pectin is treated with sodium hydroxide it undergoes hydrolysis almost instantaneously, giving off methyl alcohol and leaving the sodium salt of an acid from which, on the addition of a mineral acid, the insoluble pectic acid is precipitated ; this latter substance has lost all power of forming jellies which was the characteristic of the soluble pectin. It thus becomes possible to distinguish three stages in the history of the pectins, which are represented in the following classification adopted at the Pectin Symposium of the American Chemical Society in 1925 : — 1. Protopectin (equivalent to the older term pectose of Fremy). This represents the insoluble precursor of the true pectins and is the form in which these substances occur in the unripe material. 2. Pectin is the soluble substance capable of forming jellies which occurs free in the plant or is produced from protopectin in ripening or by chemical hydrolysis. Pectin is the methyl ester of pectic acid. 3. Pectic acid is demethylated pectin and is incapable of forming a jelly. Isolation of Pectins from the Tissues. Two methods of separating pectins from tissues have been adopted : Extraction by means of ammonium oxalate, and extraction by means of hot water. [a) Ammonium Oxalate Method.— This is the method fol- lowed by Schryver and his fellow-workers : the material se- lected—turnips, strawberries, rhubarb petioles, apples, onions, and cabbage — is first ground and pressed to remove soluble pectins ; the residue, after drying and further grinding, is extracted with warm 0-5 per cent ammonium oxalate solution 13 194 THE CARBOHYDRATES at about 80-90° ; on addition of 2-3 times its volume of 95 per cent alcohol, the solution gives a precipitate of the pectin in a yield of approximately o-i per cent, or, in the case of the turnip, almost double this quantity. Besides being extracted by ammonium oxalate, pectin may also be extracted by means of warm solutions of sodium or ammonium salts whose anions form insoluble salts with calcium, such as sodium carbonate or ammonium tartrate. If the dried and ground tissues are extracted with 8 per cent sodium hydroxide, free from carbonate, previous to extraction with the ammonium oxalate, the sodium hydroxide solution will be found to contain no pectin (provided the caustic soda used was free from carbonate) ; it contains instead a mixture of substances which can be precipitated by an equal bulk of 95 per cent alcohol ; this material reduces Fehling's solution only after hydrolysis and is coloured blue by iodine ; the substances comprising this mixture yield furfural equivalent to pentose contents ranging from 40-85 per cent ; they are presumably hemicelluloses and are described as Cyto- pectins * ; though extracted from the tissues by alkali, they are not all precipitated from these solutions on addition of acid. If the residue remaining after extraction of the tissues with caustic soda is washed free from alkali and extracted with warm 0-5 per cent ammonium oxalate, the resulting extract, on treatment with hydrochloric acid, yields a precipitate of pectic acid. {b) Hot Water Method. — Ehrlich extracts sugar beet residues, which contain about 25 per cent of pectin, by heating with water in an autoclave under 1-2 atmospheres pressure; this treatment yields a solution of what is described as hydro- pectin. Hydropectin when extracted with 70 per cent alcohol yields an extract which contains an araban, while the residue insoluble in alcohol is a water-soluble calcium magnesium salt of pectic acid ; a careful study of the products of the hydrolysis of this substance has shown it to be a galacturonic acid derivative (see p. 196). * Clayson, Norris, and Schryver : " Biochem. Journ.," 1921, 15, 643. PECTINS 195 Until some insight had been obtained into the chemical nature of these substances, much confusion arose owing to the tendency of different authors to describe identical products under different names ; the following brief summary of the development of the subject may help to clear the situation. Fremy, acting upon pectin with acids or alkalis, obtained a number of intermediate products of hydrolysis to which he gave the names of parapectin, metapectin, pectic acid, as well as para- and metapectic acid ; many of those substances were, however, insufficiently characterized and their existence is no longer credited. Although the analyses by Tromp de Haas and Tollens * of the pectins derived from a number of different sources appeared to agree fairly well with the formulae (CgHioOgj^ or 2(C6Hio05) . HgO, Tollens f suspected that the pectins con- tained carboxyl groups ; this view was finally shown to be correct when the acidic nature of these substances was estab- lished by Schryver and Haynes,J who prepared from pectin, by alkaline hydrolysis, a pectic acid to which they assigned the formula C17H24O16. Ehrlich § has shown that the residues from sugar beet re- maining after the extraction of the sugar, provide a convenient source for the extraction of pectin ; after washing with warm water to remove soluble impurities, the residue is extracted with boiling water and the filtrate after evaporation yields the pectin, though, in all probability, not in the form in which it occurred in the plant but partly hydrolysed as hydropectin (see above) ; extracted with 70 per cent alcohol, it yields to this solvent about 30 per cent of an araban or polymerized arabinose, while the residue consists of a water-soluble pectin. The fact that no araban is extracted by boiling beet residues for some hours with 70 per cent alcohol indicates that in the tissues the araban is combined with the water-soluble pectin, and is only hydrolysed during the boiling with water. Acid * Tromp de Haas and Tollens : " Annalen," 1895, 286, 278. t Tollens : id., 1895, 286, 292. t Schryver and Haynes : " Biochem. Journ.," 1916, 10, 539. § Ehrlich : " Chem. Zeit.," 1917, 4»» ^97 : " Zeit. angew. Chem.," 1927, 40, 1305. 13* 196 THE CARBOHYDRATES hydrolysis of this soluble pectin yields galacturonic and acetic acids as well as methyl alcohol, arabinose, and galactose according to the equation — C43H62O37 + 10H2O = 4C6Hio07 + 3CH3COOH +,2CH30H + C5H10O5 + QHuOe Galacturonic Acetic Methyl Arabinose Galactose acid acid alcohol On the other hand, alkaline hydrolysis by means of calcium or barium hydroxides yields methyl alcohol, acetic acid, a galacto-araban, and a tetragalacturonic acid ; the latter acid being regarded as a condensation product arising from the ehmination of 3 molecules of water from 4 molecules of galacturonic acid as follows : — 4CHO(CHOH)4 . COOH— 3H2O = C^JiaiO,^ Galacturonic acid Tetragalacturonic acid The galacto-araban set free by alkaline hydrolysis must be distinguished from the araban separated from the original plant material by boiling with water. Combining the information obtained from acid and alkaline hydrolysis, Ehrlich concluded that the original pectin was a triacetyl-arabino-galacto-dimethoxy-tetragalacturonic acid. The pectins of currants and strawberries and also of orange peel are generally similar but show a rather higher content of galacturonic acid, i.e. 73-78 per cent, as compared with 68 per cent for sugar beet and 61 per cent for flax. Whereas the fruits mentioned contain about 40-50 per cent of the total pectin dissolved in the juice, the beet and orange peel contain only about 5-10 per cent in soluble form. As has been stated above, some difficulty is experienced in recon- ciling the various results obtained by different investigators. Thus von Fellenberg,* who first established the presence of methylated carboxyl groups in the pectins from various fruits, came to the conclusion that the completely demethylated acid corresponding to pectin was a pectic acid of the formula C70H104O68, and that the formula of the neutral pectin was — (C^HsOJa (CeHjoOs)^ (C,H,0, . €00^3)3 . 2H2O Arabinose Methyl Galacturonic acid pentose and Galactose i.e. a condensation product formed from 2 molecules of * V. Fellenberg : " Biochem. Zeit.," 1918, 85, 141. PECTINS 197 arabinose, i molecule of methyl pentose, i molecule of galac- tose, and 8 molecules of galacturonic acid. On the other hand, Schryver * and his colleagues, who worked on turnips, onions, and pea pods, hold somewhat divergent views from those of the previous authors. Thus they disagree with von Fellenberg's statement that pectins contain any methyl pentose residue — a view which is sup- ported by Nanji, Paton, and Ling.f Moreover, the insoluble form of pectin as it occurs in the cell wall, to which they give the name pectinogen rather than protopectin, they regard as a pectic acid in which three carboxyl groups are methylated and one is in loose combination with calcium ; they obtain their pectinogen by extracting the cell wall material with 0-5 per cent solutions of either ammonium oxalate or oxalic acid ; the methoxyl content of their extracts varies according to the period of extraction ; the shorter the time required the more nearly does the composition correspond to that of the pectinogen as it occurs in the cell wall. Alkalis, such as lime water, convert pectinogen into pectic acid with Hberation of methyl alcohol, but accompanying this hydrolysis there is also the separation of a second substance of the nature of a hemicellulose ; no mention is made of the araban obtained by Ehrlich in the sugar beet pectins, and it is not clear whether this is identical with the above hemicellulose. As is stated previously, the formula assigned to the pectic acid obtained by Schryver and Haynes J by the action of caustic soda on pectic substances, is C17H24OX6, while that assigned by von Fellenberg to his acid was C70H104O68. The discrepancy may be explained by assuming that the latter formula is that of the normal acid containing eight carboxyl groups, while the formula of Schryver and Haynes represents an anhydride of this acid resulting by the elimination of 4 molecules of water, thus § : — 4Cj,H240ie or QgHggOg^ ^ C7oHi„4C)68 ~ 4H2O or CjgHggOgi [Schryver & [v. Fellenberg] Haynes] * Norris and Schryver : " Biochem. Journ.," 1925, 19, 676. t Nanji, Paton, and Ling : " J. Soc. Chem. Ind.," 1925, 44, 253T. I Schryver and Haynes : " Biochem. Journ.," 1916, 10, 539. Carre and Haynes : id., 1922, 16, 60. § Carr6 : " Ann. Bot.," 1925, 36, 818. 198 THE CARBOHYDRATES As regards the configuration of the pectic acid molecule, it has been suggested by Nanji, Paton, and Ling * that it contains a six-sided ring, each side of which is occupied by an appropriate carbohydrate residue as under : — COOH Ga Ga / \ l\Ga g/ HOOC '^^ y' \ COOH G = Galactose. A = Arabinose. Ga = Galacturonic acid. a formula f which bears a striking resemblance to the basal nucleus suggested for starch (p. 157). The empirical formula of this acid is C35H50O33, i.e. approximately double that pro- posed by Schryver and Haynes, namely C17H24O1B. Properties of Pectins. 1. Neutral pectins are soluble in warm water, without boiling, up to 2 per cent, yielding a viscous solution ; such solutions do not give a jelly unless boiled with sugar and tartaric acid. 2. A 2 per cent solution of pectin is mixed with one-tenth its volume of a freshly prepared solution of pectase and a pinch of calcium carbonate ; in from one to two hours the reaction will have become acid and the solution should have set to a gel. 3. Treated with caustic soda, pectins are saponified with formation of sodium pectate which, on addition of acetic acid and calcium chloride, gives a precipitate of calcium pectate. 4. About 0*5 gram of pectin is placed in a 150 c.c. distilla- tion flask with 20 c.c. of water ; as soon as solution is effected, * Nanji, Paton, and I-ing : " J. Soc. Chem. Ird.," 1925, 44, 253 T. t See also Norris and Schryver : " Biochem. Journ.," 1925, 19, 685. PECTINS 199 5 c.c. of 10 per cent caustic soda are added ; after gently agitating the flask, the cork is inserted and the mixture is left for five minutes ; now add 2-5 c.c. of dilute sulphuric acid, gently agitate once more, and distil over 17 c.c. of liquid ; this is once more distilled, about 11 c.c. being collected. The resulting hquid is tested for methyl alcohol * 5. Pectin solutions are precipitated by copper sulphate or lead nitrate but not by barium chloride, ferric chloride, or lead acetate. Also they are precipitated by baryta or lime water or basic lead acetate. Alkalis readily attack pectins with liberation of methyl alcohol and formation of the alkali metal salt of pectic acid ; neutral calcium and barium salts do not precipitate unless alkali is present. 6. Aqueous solutions of pectins are precipitated by alcohol, but the precipitate can be redissolved in water. 7. Pectins are insoluble in ammoniacal solution of copper hydroxide. Microchemical Reactions. The fact that these pectic substances are akin to cellulose, and occur in conjunction with it, renders its identification by microchemical means somewhat difficult. Mangin f gives the following methods : — 1. Methylene blue, Bismarck brown, and fuchsin stain pectic substances, lignified and suberized walls, but not pure cellulose. If sections thus stained are treated with alcohol, glycerine, or dilute acids, the lignified or suberized walls retain their coloration, whilst the pectic substances are de- colorized with rapidity. 2. Crocein and nigrosin stain lignified and suberized walls, but do not stain pectic compounds. 3. Crocein, naphthol black, and orseille red stain pure cellulose, but do not stain pectic substances ; similarly, pectic compounds are unstained by congo-red and azo-blue, whilst cellulose and callose are. * Particulars of this test will be found in Sucharipa : " Die Pektin- stoffe," Braunschweig, 1925. t Mangin : " Compt. rend.," 1889, 109, 579 ; 1890, no, 295, 644. 200 THE CARBOHYDRATES 4. The middle lamella, which consists of compounds of pectic acid, may be differentiated from the other pectic sub- stances which are mixed with the cellulose of the cell walls by the following method : A thin section is placed in a 20-25 per cent solution of hydrochloric acid in alcohol for twenty-four hours ; the section is then washed with water and treated with methylene-blue or phenosafranin. The middle lamella stains much more deeply than the rest of the wall. 5. If, after the above treatment with acid alcohol, the section be washed in a 10 per cent solution of ammonia, it is found that the cells separate with ease one from the other. According to Mangin, the combined pectic acid is freed from its bases by the treatment with acid alcohol, and is then dissolved by the ammonia. A recombination of the pectic acid may be brought about by treatment with baryta water, and after this process the cells will not separate one from the other. 6. Mehta * finds that pectic compounds stain deeply with the following dyes : Alcoholic malachite green, aqueous Congo red, alcoholic eosin, alcoholic safranin. aqueous gossy- pimin, aqueous iodine green, and aqueous ruthenium red. None of the stains, however, is specific for pectic substances ; thus ruthenium red stains oxycelluloses, hemicellulose, gums, galactans, etc. The procedure adopted by Mehta is to dissolve out the various constituents of the cell wall with appropriate reagents and then to compare their staining reactions. The sections are placed in a test tube with the reagent which is then heated for six to eight hours in a boiling water bath, the liquid being decanted off every hour and replaced by fresh reagent. The sections are then washed with hot distilled water, stained for two hours, washed with 90 per cent alcohol to remove excess of stain, dehydrated with absolute alcohol, and mounted in cedar-wood oil. The reagents used were 0-5 per cent ammonium oxalate, 0-5 per cent ammonium oxalate in 3 per cent ammonia solution, 4 per cent sodium hydroxide, 3 per cent hydrochloric acid, or 95 per cent alcohol. * Mehta : " Biochem. Journ.," 1925, 19, 979. PECTINS 201 7. Pectins are insoluble in ammoniacal solution of copper hydroxide (cuprammonia). Estimation of Pectins. The fact that pectins are readily saponified by caustic soda has been adapted by Carre and Haynes * as a means for their estimation. A dilute solution of the pectin is allowed to stand with N/io NaOH overnight, and is then treated with N acetic acid and after five minutes with M calcium chloride ; the mixture is allowed to stand for an hour and is then boiled for a few minutes and filtered ; the precipitated calcium pectate is washed repeatedly until free from chloride and weighed after drying at 100° C. The composition of the pre- cipitate was represented as Ci7H220i6Ca, containing 7-66 per cent of calcium ; but if the newer formula for pectic acid is accepted, it would be C35H4^033Ca2, containing 7-45 per cent of calcium. The method has been employed by Carre f for investigating the changes which occur in the pectic constituents of fruits during ripening. Action of Enzymes on Pectins. Pectase is the name given by Fremy to an enzyme which he found was able to effect the coagulation of pectin solutions. Pectases are very widely distributed in the plant world and are found in the leaves of very many green plants ; the activity of the enzymes from different sources is, however, not the same as may be seen from the following table % giving the time re- quired for gelatinizing a 2 per cent pectin solution Solanum Ivcopersicum . Vitis vinifera (fruit) Ribes Rheum, rhaponticum Marchantia polymorpha D uncus (mature) Delphinium Gitigko biloba 4S hrs. 24 .. 15 .. 12 ,, 2j „ 2 T 1 35 mins. Daucits (young) Zea Mais Iris florentina Trifolium pratense Medicago sativa PI ant ago Byassica napus Lolium perenne 15 mms. Less than I min. * Carre and Haynes: " Biochem. Tourn.," 1922, 16, fio ; also Eramett and Carre : id., 1926, 20, 6. t Carre : id., 1922, 16, 704 ; " .\nn. Bot.," T925, 39, 811. t Bertrand and Mallevre : " Compt. rend.," 1S95, 121, 726. 202 THE CARBOHYDRATES The optimum hydrogen ion concentration of the pectase from currants was found by Euler and Svanberg * to be Ph4-3- While Bertrand and Mallevre showed that the presence of calcium salts was essential for the production of a gel, Goyaudf came to the conclusion that the activity of the enzyme was in no way dependent upon the presence of calcium salts inasmuch as it was able to break down the pectin to pectic acid even in solutions which had been deprived of calcium salts by the addition of potassium oxalate ; the addition of calcium salts to such solutions, however, at once produced a gel owing to the precipitation of the insoluble calcium pectate. It is concluded from this that the function of calcium in this connection is only to reveal the products of the activity of the hydrolytic enzyme ; if this be the true explanation, it is a very remarkable fact that pectase should in many cases be able to effect hydrolysis of pectin in less than one minute, although it must be borne in mind that the hydrolysis by means of caustic soda likewise is completed in a very short time (cf. p. 198). Another enzyme, pectinase, was first described by Bourquelot and Herissey % as occurring in malt and was later found by Ehrlich in takadiastase ; this enzyme also acts hydrolytically upon pectins but breaks them down further than pectase, past the pectic acid stage to the yielding of reducing sugars. This enzyme is said to be the one secreted by Granulohacter pectinovorum, and Bacillus carotovorus. An enzyme having similar properties has been described by Kylin § as occurring in marine algae. Pectins are also subject to attack by enzymes secreted by various fungi and other bacteria, though the exact nature of the products of their activity has yet to be studied. Thus it was shown by de Bary that Peziza sclerotiorum destroys the host plant by disintegrating the cell walls owing, presumably, * Euler and Svanberg : " Biochem. Zeit.," igig, 100, 271. jGDyaud: "Compt. rend.," 1902, 135, 537. t Bourquelot and Herissey : " J. Pharm. et Chim.," 1898, [6], 8, 145 ; 1899. [6], 9, 563, and 10, 5. Also Verdon : " J. Pharm. Chera.," 1912, 5. 347- § Kylin : " Zeit. physiol. Chem.," 1915, 94, 412. PECTINS 203 to a solution of the middle lamella ; a somewhat similar effect was shown by Brown * to occur when an enzyme extract of Botrytis cinerea was allowed to act upon tissues of potato, turnip, beet, and apple. It was, moreover, shown by Wino- gradsky f that the retting of flax was due to the solution of pectic substances by enzymes secreted by bacteria. Origin and Constitutional Relationships of the Pectins. The fact that galacturonic or similar sugar aldehyde acids occur in pectins, hemicelluloses, and gums suggests that all cell wall constituents are more or less related to each other. It must be borne in mind that whilst the cell wall of un- lignified elements is composed of cellulose together with pectin, in hgnified elements lignin occupies the place of pectin ; for this reason pectin is regarded as the precursor of lignin, and the fact that both pectins and lignin contain acetyl % and methoxyl groups would tend to support this view. Ehrhch § even claims to have isolated from the lignified tissues of the flax plant a substance which he describes as a resin-Hke lignic acid ; this he considers to represent a tran- sition stage between a typical unaltered pectin and lignin. Fuchs II considers that lignin may have been produced from pectins by loss of water and oxygen. It has been suggested by Nanji, Paton, and Ling that pectins themselves arise from the condensation of galactose to a hexagalactan which is then oxidized to uronic acids and methoxylated. Yet another suggestion is that of Smolenski,^ who regards pectins as intermediate stages in the conversion of hexoses into pentoses. The Changes taking Place in Ripening. The view originally put forward by Fremy was that softening of the tissues of fruits on ripening was due to a * Brown : " Ann. Bot.," 1915, 29, 313 ; 1917, 31, 489. t Winogradsky : " Compt. rend.," 1895, 131, 742. X But see also Nelson : " J. Amer. Chem. Soc," 1926, 48, 2945. § Ehrlich : " Zeit. angew. Chem.," 1927, 40, 1305. II Fuchs : " Brennstoflf Chem.," 1926, 7, 302. ^ Smolensk! ; " Chem. Soc. Abstracts," 1924, i, 16. 204 THE CARBOHYDRATES conversion of the insoluble pectose, or protopectin as it is now termed, into soluble pectin, a change which he thought was due to the hydrolytic action of the fruit acids. Evidence has, however, been obtained to show that this change is due to enzyme action.* That the middle lamella is not involved in the earlier stages of this change has been shown by Carre, f who found that the middle lamella pectic substance remained at a constant level throughout the process of ripening, and it was only in the over-ripe condition that the amount begins to decrease and finally vanishes when, owing to the absence of cementing material, the cells are entirely separated from one another. In the absence of exact knowledge concerning the nature of the relation between soluble pectin and proto- pectin, it is not possible to be certain what happens when the one is formed from the other. The view put forward by Mangin $ that protopectin is a loose form of combination between pectin and cellulose is supported by von Fellenberg, who suggests that the production of soluble pectin from proto- pectin involves hydrolysis of this compound ; the same con- clusion has been arrived at by Sucharipa § ; possibly the softening of fruit on ripening is due to this same separation of soluble pectin from its combination with cellulose ; it is only in the last stages of over-ripeness that still further hydrolysis of the pectin would occur with consequent dis- integration of the tissues as indicated above. Appleman and Conrad, || working on peaches, find that the transformation of protopectin into pectin appears to be the only pectic change during ripening and softening. The sum of pectin and protopectin was practically constant at all stages of ripening, but both constituents disappear slowly in over-ripe peaches. The everyday significance of pectins as the basis of fruit jellies and jams justifies a reference to their use in this con- * Thatcher : "J. Agric. Res.," 1915, 5, 103; Carre, "Ann. Bot.," 1925. 39» 811. t Carre : loc. cit. % Mangin : " Comp. rend.," 1888-1893. • § Sucharipa : " J. Amer. Chem. Soc," 1924, 46, 145. II Appleman and Conrad : " Univ. Maryland Agr. Exp. St. Bull.," 283. July, 1926. CELLULOSE 205 nection. At the outset it is important to distinguish clearly between the irreversible pectin gels formed by the action of pectase, and the reversible gels concerned in the formation of fruit jellies or jams. As pointed out above, the former are most probably composed of insoluble calcium pectate which when once formed cannot be got into solution again. On the other hand, it is known that soluble pectin forms in water not a true solution but a sol which in about 2 per cent concentration is fairly viscous but does not set to a gel ; in order to produce a gel from such a solution it requires to be boiled in water containing about 60 per cent of cane sugar and approximately I per cent of tartaric acid ; on cooling the resulting mixture sets to a gel which, according to Sucharipa, is due to the fact that the pectin is insoluble in such a solution of cane sugar. Care must be taken not to boil for too long as otherwise hydrolysis may set in which will entail loss of methyl alcohol ; it appears from the work of Nanji and Norman * and others that the jellying power of a pectin is a function of its methoxyl content ; the authors mentioned have worked out a micro-method for the determination of methyl alcohol. CELLULOSE. The term cellulose should be taken in general to connote a group of substances rather than a single chemical compound ; used in this generic sense, it comprises a number of substances of somewhat different origin and characters, whose chief common properties are their physiological origin and their function in forming the basis of the material which is isolated by the protoplasm of the living cell for the purpose of form- ing the wall or periphery of that cell. Though met with chiefly in the vegetable kingdom, its occurrence in the animal kingdom is not unknown, since a substance described as tunicin, said to be identical with cellulose, has been found in the cell walls of certain tunicates and insects. In the course of time the cellulose originally formed is altered by the addi- tion to it of various secondary products known as encrusting * Nanji and Norman : " J. Soc. Chem. Ind.," 1926, 45, 337 ; Baker : " J. Ind. Eng. Chem.," 1926, 18, 89. 2o6 THE CARBOHYDRATES substances ; thus the process of Hgnification consists in the conversion of cellulose into ligno-cellulose ; accompanying this change is a gradual disappearance of the protoplasm. Thus the protoplasm within the cell produces a number of different substances which are deposited in the cell wall, the nature and properties of the resulting fibre depending on the nature of these substances. CLASSIFICATION OF CELLULOSES. The naturally occurring celluloses were originally classified by Cross and Bevan in the following manner : — I. Typical or Normal Celluloses of the Cotton Type. — These were exemplified by the cellulose obtained from cotton, flax, hemp, etc. n. Compound Celluloses of the Wood Cellulose, Jute and Cereal Grass Types. — The natural celluloses occurring in jute, cereal straws, esparto grass, etc., were regarded as consisting of some form of combination of cellulose with a non-cellulose constituent, either of the nature of lignin in the case of lignocelluloses, or a pectic or gummy substance in the case of pectocelluloses, or a fatty substance in the case of adipocellu- loses. This group was therefore subdivided into : — {a) Lignocelluloses, e.g. jute fibre. {h) Pectocelluloses, e.g. flax. [c] Adipo- or Cuto-celluloses, e.g. cork. HI. Hemi-, Pseiido- or Reserve Celluloses. — This hetero- geneous collection of substances differ structurally from the fibrous celluloses, and occur in the cell walls of the seeds of various plants such as Coffea arabica, Soja hispida, Lupinus luteus, Cocos nucifera, TropcBolum. majus, Impatiens balsamifera, Pceonia officinalis, and in peas and beans. In this group of celluloses were also included those which, according to the researches of Brown and Morris, are dissolved by the enzymes secreted by the germinating seed ; these are sometimes referred to as reserve cellulose, though the name seems ill-chosen, inasmuch as they would not appear always to function as reserve material. CELLULOSE 207 Associated with this classification was the conception that there existed in the plant several distinct varieties of cellulose. Thus Cross and Bevan found that the cellulose obtained after delignification of the straw of cereal grasses and of esparto when distilled with hydrochloric acid gave considerable quantities of furfural, from which they concluded such cellulose to be possessed of furfural-producing groups which they termed furfuroids. It has, however, been shown by Irvine and Hirst * that esparto cellulose consists of a mixture of ordinary cellulose with a pentosan, xylan, in the proportion of, approximately, 80 to 20, and that by repeated treatment with alkali the xylan could be dissolved out. The same state of affairs has been shown to hold for straw cellulose by Heuser and Haag,t and in view of the proved existence of pentosans in such cases Heuser and others consider the identity of cellu- loses from different sources to be established, and regard Cross and Bevan's assumption of the existence of furfuroids to be unnecessary. One of the richest sources of cellulose in nature is the cotton plant. The following table, taken from Bowman, | re- presents approximately the composition of cotton fibre from various sources : — Source of Cotton. Surat. American. Egyptian. Per cent. Per cent. Per cent. Cellulose ..... 91-35 91-00 90-8 Wax, oil, and fat ... •40 •35 •42 Protoplasm and derivatives (Pectose) •53 •53 •68 Mineral matter, i.e. salts of K, Na, Ca, Mg, Fe, and Al . •22 •12 •25 Water 7-50 8-00 7-85 Although the raw cotton wool is amongst the purest of mature structures with respect to its cellulose, it requires treatment before it can be regarded as approximating to a * Irvine and Hirst : " J. Chem. Soc," 1924, 135, 15. •j- Heuser and Haag : " Zeit. angew. Chem.," 1918, 31, 99, 103, 166, 172 ; also Heuser and Aiyar : id., 1924, 37, 27. t Bowman : "The Structure of the Cotton Fibre," London, 1908, p. 147. 2o8 THE CARBOHYDRATES condition of chemically pure cellulose. For this purpose * the cotton wool has to be extracted successively in a Soxhlet for six hours with 96 per cent alcohol and with ether to remove fats and waxes ; it is then boiled for several hours with i per cent caustic soda under specified conditions involving the rigorous exclusion of air in order to avoid oxidation. After washing with water and acetic acid, and finally with water, it is dried. The resulting product, known as standard cellu- lose, should have the following composition :— a-Cellulose ..... 99-8 Ash ....... 0-03-0-06 The term a-cellulose is applied to cellulose which is in- soluble in 17-5 per cent caustic soda. In other plant material such a-cellulose is accompanied by varying proportions of two other modifications known respectively as j8- and y- cellulose ; these forms are less resistant to chemicals than the a form, but may only differ from it in the degree of polymeri- zation, of dehydration or even of dispersion. f The relative proportions in which these forms occur in a given sample of cellulose may be determined by extraction with 17-5 per cent caustic soda whereby a-cellulose remains undissolved while the jS and y modifications go into solution ; on acidifying the filtrate with acetic acid, the jS-cellulose is precipitated while the y- remains dissolved. The evaluation of a cellulose for its suitability for technical purposes is largely dependent upon the results of such an analysis. PROPERTIES OF CELLULOSE. Pure cellulose is a white hygroscopic substance, which absorbs about 6-12 per cent of water, which it loses again when heated to 100° ; it is insoluble in water at ordinary pressure, but when heated with water in sealed vessels at 500° F. it is dissolved completely with decomposition. SOLUBILITY OF CELLULOSE. Cellulose is insoluble in all ordinary solvents, but when treated with zinc chloride in the presence of water it is con- * Corey and Gray : "J. Ind. Eng. Chem.," 1924, 16, 853, 1130. t See Schwalbe and Becker ; " J. prakt. Chem.," 1919, lOO, 19. CELLULOSE 209 verted into a gelatinous hydrate which, after prolonged treat- ment, goes into solution. A solution of 6 parts of zinc chloride in 10 parts of water heated to 60-100° is thoroughly stirred up with i part of cellulose, and then digested for some time at a gentle heat. When the cellulose is gelatinized, its solution is completed by heating over a boiling water bath, and adding water from time to time to replace that lost by evaporation. Two other salt solutions are known which dissolve cellu- lose : — {a) Zinc Chloride and Hydrochloric Acid. — A solution of zinc chloride in twice its weight of hydrochloric acid dissolves cellulose rapidly in the cold. [h] Ammoniacal Cupric Oxide [Schweitzer' s Reagent). — The solution is most conveniently prepared by drawing a current of air through a Wolff bottle containing o-88o ammonia and some copper turnings, until a deep blue solution is obtained. Cellulose dissolves in this solvent and on the addition of acid is reprecipitated. ACTION OF VARIOUS CHEMICALS ON CELLULOSE. I. Alkalis. — Solutions of caustic soda of 1-2 per cent strength have no action on cellulose at temperatures con- siderably above 100°. When cotton fibres are immersed in a 17-5 per cent solution of caustic soda they shorten, swell up, and the lumen becomes obliterated ; the physical process of swelling is accompanied by a chemical change involving the formation of an unstable sodium compound CgHgOgNa ; on washing with water the sodium is removed, but the recovered cellulose has, as a result of its swelling, acquired a greater affinity for dyes. This observation was first made by Mercer in 1844 and technically exploited by him for dyeing cotton. Later, in 1890, it was discovered by Lowe that if the alkali treatment is carried out while the cotton was under tension the fibres acquired a lustre, a process known as mercerization. When fused at 200-300° with a mixture of sodium and 14 2IO THE CARBOHYDRATES potassium hydroxides, cellulose undergoes complete decom- position with the formation of oxalic and acetic acids. The so-called alkaU cellulose obtained by treating cellulose with 17-5 per cent caustic soda reacts with carbon disulphide to form xanthogenates ; * these compounds are used in the manufacture of viscose (see below). 2. Acids. — Nitric acid (sp. gr. 1-25) at 180° converts cellu- lose into oxycellulose, a substance of a weak acidic character, which reduces Fehling's solution (see below). Concentrated nitric acid, or a mixture of this acid with concentrated sul- phuric acid, converts cellulose into nitrates, the composition of which varies with the conditions of the experiment ; di-, tri-, tetra-, penta-, and hexa-nitrates, which are of considerable technical importance, are known. If dilute sulphuric acid is allowed to act for some hours at 100° C. on cotton, it does not alter the structure of the fibre, but makes it friable. This was at one time thought to be due to the formation of a definite substance, hydrocellulose. That this material is not a simple substance may be shown by the fact that it has acquired reducing properties, the sub- stance responsible for which may be extracted with alkali leaving behind unchanged cellulose. The fact that the alkaline extract is yellow in colour suggests the presence of an alde- hyde, possibly glucose. For these reasons it is considered that the term hydrocellulose implies a stage in the hydrolysis of cellulose rather than a definite chemical substance ; it may be a mixture of cellulose, cellulose dextrins, and glucose. Cellulose, when treated with concentrated sulphuric acid, undergoes considerable swelling, and goes into solution with the ultimate formation of dextrose. This is made use of in the preparation of vegetable parchment for which purpose paper is rapidly drawn through a mixture of 4 parts of sulphuric acid with I of water ; the paper is then thoroughly washed with water until free from acid. If, on the other hand, cellulose is left in contact with concentrated sulphuric acid for a time sufficient to dissolve it, and the solution is immediately * Cross, Bevan, and Beadle : " Ber. deut. chem. Gesells.," 1893, 26, 1090 ; and Cross and Bevan : id., 1901, 34, 1513. CELLULOSE 21 r diluted, a gelatinous hydrate is precipitated ; this substance is known as amyloid, since it resembles starch in giving a blue colour with iodine. The same substance is formed by the action of chlorzinc iodide, the reaction being used as a test for cellulose. Cellulose on hydrolysis yields glucose only. Several claims to have effected the quantitative conversion of cellulose into glucose were made on the basis of observations of the change in optical activity, but the first to obtain an approximately quantitative yield of crystalline glucose from cellulose was Monier Williams * who left the cellulose in contact with 72 per cent sulphuric acid for a week and then after dilution boiled the mixture for fifteen hours. The combined action of glacial acetic acid and acetic anhy- dride in the presence of concentrated sulphuric acid or zinc chloride converts cellulose into acetyl cellulose, which is insol- uble in water but soluble in several organic solvents. Acetyl cellulose is also used in the manufacture of artificial silk. Cellobiose,f C12H22O11, is a disaccharide obtained in the form of its acetate by acting on cellulose with acetic anhy- dride and concentrated sulphuric acid. It stands in the same relation to cellulose as does maltose to starch. 3. Oxidizing Agents. — Dilute solutions of alkaline hypo- chlorites have very little action on typical cellulose, and can therefore be employed for bleaching this material ; with con- centrated solutions of hypochlorites, however, a general decom- position ensues. As already mentioned, nitric acid (sp. gr. 1-25) at 180° converts cellulose into a series of oxidation products known as oxycellulose, and similar substances are produced by the action of other oxidizing agents, such as chromic acid, potassium chlorate in the presence of hydro- chloric acid, etc. Oxygen containing 2 per cent of ozone at once attacks dry cotton with the formation of a cellulose peroxide % and an acid substance ; the latter, when boiled with water, dissolves, * Monier Williams : " J. Chem. Soc," 1921, II9» 803. I Skraup and Konig : " Ber. deut. chem. Gesells.," 1901. 34, 1115 ; Schliemann : " Annalen," 191 1, 378, 366. JDoree: " J. Chem. Soc," 1913, 103, 1347. 14* 212 THE CARBOHYDRATES leaving a neutral product which resembles a typical aldehydic oxycellulose. This is regarded as being due to the oxidation of an alcoholic group into cellulose molecule (see formulae, p. 214). 4. Action of Ferments. — It has been shown by Brown and Morris, in the case of barley, rye, oat, and other cereals, that the cell wall of the endosperm cells which contain nutrient material are broken down by a cellulose-dissolving ferment, a cyto-hydrolyst, before the embryo can procure the food- stuff contained in these cells. This enzyme, which is de- veloped during the germination of the seed, can be extracted from the malt by cold water, and precipitated from this solu- tion by alcohol. A cytase capable of hydrolysing hemicellulose has been extracted from Aspergillus OryzcB, from the cotyledons of Lupinus albus, and Phoenix dactylifera* Cytase splits hemi- cellulose into glucose, mannose, galactose, and pentose, Cellulase is an enzyme which attacks ordinary cellulose converting it into cellobiose. It occurs in Aspergillus celluloses^ and in certain bacteria.J It is well known that many fungi, Actinomyces, Aspergillus, Coprinus, Penicillium, and Tricho- derma, § for example, have the power of breaking down cellulose. In this activity they may be of even greater signi- ficance in the soil than the cellulose-splitting bacteria || which belong to the aerobic and the anaerobic forms : of the former Spirochceta cytophaga ^ and Microspora agarlique- faciens ** may be mentioned ; and of the latter those re- sponsible for the subaquatic decomposition of cellulose with the evolution of marsh gas or, in other forms, of hydrogen. OXYCELLULOSE. When cellulose is exposed to the action of oxidizing agents there results a product which contains a greater proportion of oxygen than the original and is therefore known as oxycellu- * Newcombe : " Ann. Bot.," 1899, 13, 49. t EUenberger : " Zeit. physiol. Chem.," 1915, 96, 236. % Pringsheim : id., 191 2, 78, 266. § Waksman : " Soil Sci.," 1916, 2, 103. II See Russell : " The Micro-organisms of the Soil," London, 1923. 11 Hutchinson and Clayton : " J. Agric. Sci.," 1919, I9> i43- ** Gray and Chalmers : " Ann. Appl. Biol./' 1924, 11, 324. CELLULOSE 213 lose ; the composition of this substance varies according to the conditions under which oxidation is effected.* The fact that many oxidizing agents act in an acid medium makes it impossible to effect oxidation without a certain amount of hydrolysis, and for this reason the term oxycellulose must be taken not to signify a definite chemical individual but an indefinite mixture of oxidized cellulose, hydrocellulose, and unaltered cellulose. Birtwell, CHbbens, and Ridge f claim that, from a technical point of view, two distinct types of oxycellulose must be recognized ; those having great affinity for methylene blue and a low reducing power, and those having a high reducing power, or so-called copper number, and a marked solubility in alkali. Properties of Oxycellulose. The outstanding characteristics of oxycellulose are the possession of (i) aldehydic properties which are shown by the ability to react with Schiff's reagent, the production of a yellow colour on warming with alkali, and the power of reducing Fehling's solution ; (2) acidic properties ; (3) greater reactivity as shown by its being more easily acety- lated, nitrated, etc., than cellulose; (4) greater affinity for methylene blue ; (5) the ability to give off furfural when distilled with hydrochloric acid ; this may be explained according to Schorger J by assuming the formation of glu- curonic acid (which is known to give furfural with hydro- chloric acid) by the following scheme : — • A B CD CH.,OH CHO COOH COOH I ' I II CH CH CH CHOH I O I O I H,0 I CH -> CH -> C -> CHOH I I 11 (CHOH)2 (CHOH)2 {CHOH)2 (CHOH)^ L- L - — — CH — — CH CH— — CHO F F F Glucuronic acid * Hibbert and Parsons : " J. Soc. Chem. Ind.," 1925. 44, 473 T. t Birtwell, Clibbens, and Ridge : " J. Text. Inst.," 1925, 16, 137. J Schorger : " The Chemistry of Cellulose and Wood," London, 1926, p. 289. 214 THE CARBOHYDRATES AF represents the glucose anhydride unit of cellulose, the dashes representing oxygen linkages ; by oxidation the alde- hydic group B is developed from A and on further oxidation gives the carboxyl group C ; hydrolysis at F then sets free the glucuronic acid. Microchemical Detection of Oxycellulose. — The investiga- tions of Wood * and of Mehta f have shown that oxycellulose occurs naturally in the cell wall of a great variety of plant materials, and may be detected by the following means : — The material is washed with acid and then with water to remove all acid and then is stained with congo red ; by treating again with acid, the red colour is changed to blue ; on washing with water until the background becomes red, any oxycellulose present will appear dark blue or black. Oxycellulose, in common with pectic substances, hemicellu- loses, and gums is stained by ruthenium red. CONSTITUTION OF CELLULOSE. While it is agreed that cellulose is built up from a number of glucose anhydride groups C^^qO^ opinions differ as to the constitution of this unit group. The formulae proposed by Cross and Bevan, Vignon, and Green are given below : — CH COH CO CHOH— CH— CHOH CHOH CHOH CHOH CHOH CHOH CHOH 9 ^ CHOH CHOH CHOH CHOH °' CHOH CHOH ^HOH-CH— CH, COH CH CH, Cross and Bevan's formulae.i Green's formula.^- O CH— CHOH I I O CHOH CHj— CH— CHOH Vignon's formula.Jl Cross and Bevan's formula implies the presence of a ketonic group for which there is no evidence ; furthermore it contains * Wood : " Ann. Bot.," 1924, 38, 275 ; 1926, 40, 547. t Mehta : " Biochem. Journ.," 1925, 19, 979. t Cross and Bevan : "J. Chem. Soc," 1901, 79, 366. § Green and Parkin : id., 1906, 81, 811. II Vignon : " Bull. Soc. Chim.," 1899, 21, 599. CELLULOSE 2 i 5 four hydroxyl groups, whereas it is known that the highest nitrate obtained from a cellulose molecule containing six carbon atoms is a trinitrate. By the nitration of cellulose, it is possible to obtain a whole series of esters representing different degrees of nitration. These various compounds may be described as mono-, di-, tri, etc., up to deca- or possibly dodeka-nitrates of a cellulose molecule containing twenty-four carbon atoms. What is commonly called cellulose hexa- nitrate, the substance employed in the manufacture of gun- cotton, is calculated on a C12 molecule which, therefore, corre- sponds to a trinitrate of a Cg molecule. Formulae such as Green's or Vignon's receive some support from the behaviour of cellulose on distillation, and from the ease with which cellulose gives rise to brommethylfurfural on heating with hydrobromic acid. On the other hand, the formula suggested by Hibbert * for the cellulose nucleus brings out clearly the relationship of cellulose to glucose as may be seen from a comparison of the two formulae : — CH„OH 6CH2OH I I. CH CHOH I CHOH O CHOH CHOH — 5CH- -c'h CHOH O 3 I 2CHOH I -CH Glucose CgH^^Ofi Hibbert's formula CgHjoOs Denham and Woodhouse, by exhaustive methylation of cellulose and subsequent hydrolysis, were able to show that 2:3:6 trimethylglucose resulted, from which it would appear that carbon atoms i and 5 are occupied in the original cellulose molecule in uniting together the various unit groups. The fact that the acetolysis of cellulose gives rise to cellobiose and glucose caused Irvine f and co-workers to sug- gest a trisaccharide constitution for cellulose as represented by the formula — * Hibbert : " J. Ind. Eng. Chem.," 1921, 13, 256, 334. t Irvine and Robertson : " J. Chem. Soc," 1926, 129, 1488. 2i6 THE CARBOHYDRATES I "" ~l -CH . (CHOH O -CH O CH . (CHOH) 2 . CH . CH . CH . CH^OH (CHOH) 2 O CH O CH . (CHOH) 2 . CH . CH . CH^OH I -O- I CH CHjOH On the other hand, Karrer * favours a disaccharide basis, such as — i — ^ 1 CH,OH . CH . CH . (CHOH), . CH I A O o CH— (CH0H)2 . CH . CH . CHj.OH I o-J but much work remains to be done before a final decision is possible. MICROCHEMICAL REACTIONS. 1. With a dilute solution of iodine a yellow coloration results. 2. After staining well with iodine, the addition of strong sulphuric acid causes the cellulose walls to swell considerably and to turn blue. 3. Chlorzinc iodide causes swelling, accompanied by the assumption of a blue colour. 4. Calcium chloride iodine solution turns pure cellulose dull pink to violet without swelling. Zimmermann gives the following directions for making this reagent. A concentrated solution of calcium chloride is made, and for each 10 c.c. of this solution there is added •5 gram of potassium iodide and -i gram of iodine. The mixture is then gently heated and filtered through glass-wool. 5. Pure cellulose is easily soluble in cuprammonia. LIGNIFIED MEMBRANES. Wood, for the most part, is the material used in the study of lignified tissues, and is best employed as a source of hgnin and associated substances. It is well, therefore, to recall to * Karrer and Smirnoff : " Helv. Chim. Acta," 1922, 5, 187. LIGNIN 217 memory the more salient facts of its structure. Xylem, or wood, is not a homogeneous material but a tissue made up of various elements which differ in their structure and function, and which occur in varying amounts in the wood of different plants. These structural units are the tracheae, the water- conducting elements, which may comprise both vessels and tracheides ; the fibres which have a mechanical function ; and the parenchyma, the only living cells of the wood, and which is mainly concerned with the storage of food, chiefly in the form of starch and fat, and is in communication with the outer tissues by means of the rays. These elements have their origin in merismatic tissue and all, in the first instance, are living cells with thin walls composed of cellulose and pectin. In the course of their development into permanent tissue elements, the walls of some of these cells may remain unaltered, but in the majority of those plants which undergo an extensive secondary thickening, or attain a large size, the cell walls undergo a great change, a reinforcement of the original mem- brane by the incorporation of a number of substances known collectively as encrusting substances, the chief of which is lignin. This lignification never occurs uninterruptedly over the whole area of the wall ; pits, either simple or bordered, are left for intercommunication between contiguous elements, whilst in the first differentiated tracheae the lignification may only occur on a relatively small area of the wall. The rate of lignification is very variable, depending on the conditions of growth and the specific physiology ; Burgerstein * obtained evidence of its inception in cells but two days old. Beckmann, Liesche, and Lehm.ann,f in an extensive study of rye, traced the variation in the lignin content with increasing age ; they found that lignin from young tissues contained a much lower percentage of methyloxyl groups than that of older tissues, and the fact that there is a considerable variation in the amount of these groups in the lignin of heart wood, or duramen, and sap wood, or alburnum, of the same tree has been shown by * Burgerstein : " Sitz. Kais. Akad. Wiss. Wien.," 1874, 70, i, 238. t Beckmann, Liesche, and Lehmann : " Zeit. angew. Chem.," 1921, 34, 285 ; " Biochem. Zeit.," 1923, 139, 491. 2l8 THE CARBOHYDRATES Ritter and Fleck.* Lignification, therefore, is a progressive change. Those cells which are destined to become tracheae and sclerenchyma lose their living contents, all of which are used up in the making of the encrusting substances. Those which develop into parenchyma, on the other hand, retain their hving contents notwithstanding the fact that their walls may be considerably lignified. Not infrequently the older wood ceases its normal functions, and passes over into heart wood where the vessels may become repositories of sundry waste metabohc products such as tannins, colouring matters, and inorganic salts such as calcium carbonate and calcium oxalate. Lignification gives the cell a greater power of re- sistance to pressure, and a diminished power of resistance to torsion.f The wood of gymnosperms differs from that of angio- sperms in the fact that vessels are absent. Further, the xylem of many gymnosperms is characterized by the presence of resin ducts which are charged with resins and terpines which form the source of terpentine and colophony of commerce. It will be apparent from this brief consideration that the analysis of wood gives very different results, according to the nature of the material, its origin, and age. The following table gives an approximate composition of the wood of spruce : — 53-55 per cent. 3-o6 12-25 30-00 2 00 i-oo The composition of hard angiospermic wood differs from the above in generally containing slightly less lignin ; the amount and nature of the hemicellulose content is also different. Hard woods are characterized by containing considerably more wood gum or xylan, the amount being anything from 15-24 per cent, whereas from 8-9 per cent is an average figure for gymnosperms. On the other hand, hexosans are much * Ritter and Fleck : " J. Ind. Eng. Chera.," 1923, 15, 1055. t Sonntag : " Ber. deut. bot. Gesells.," 1901, 19, 138. Leon : " Zeit. Verein. deut. Ing.," 1918, 62, 341. Cellulose Hemicelluloses : Hexosans Pentosans Lignin .... Fats and resin Protein LIGNIN 2IO better represented in the soft woods where they occur to the extent of about 13 per cent, whereas 3-5 per cent is the average for hard woods. Amongst the hexosans of the gymnosperm are mannan, which varies very considerably in amount averaging from 4'5-8 per cent, and galactan, which varies from 8 up to as much as 17 per cent ; the occurrence of a high percentage of galactan is characteristic of coniferous wood, and actually this material has been commercially ex- ploited as a source of mucic acid by oxidation with nitric acid.* A method for the analysis of the various constituents of wood mentioned above has been worked out by Dore.f CHEMISTRY OF LIGNIN. Lignification is easily detected by certain colour reactions which readily distinguish lignified from unlignified tissue ; of these the two following may be regarded as the most generally useful : — (a) The yellow colour produced when lignified tissue is treated with a solution of i per cent aniline sulphate or hydro- chloride acidified with the corresponding acid. (b) The bright crimson produced on treatment of lignified tissue with a dilute alcohoHc solution of phloroglucinol fol- lowed by a little concentrated hydrochloric acid. The aniline employed in the first test may be replaced by a great many other primary or secondary amines or even by heterocyclic nitrogen bases such as pyrrol or indol, while the phloroglucinol of the second test may be replaced by a number of phenols both monohydric and di- or tri-hydric, but the colours obtained are not all the same and vary in intensity. Some wood, notably that of the cherry, when moistened with hydrochloric acid alone gives a red or violet colour ; the fact that an aqueous extract of this wood gives with lignified tissue a red colour on addition of acid, suggests that cherry wood contains some phenolic substance capable of replacing phloroglucinol in the test mentioned above ; it * Schorger and Smith : " J. Ind. Eng. Chem.," 1919, II, 556 ; 1920, 12, 264, 472, 476, 984. + Dore : id., 1919, 1 1, 556; 1920, 12, 264, 472, 476, 984. 220 THE CARBOHYDRATES was suggested by Wiesner that the substance was actually phloroglucinol, but the presence of this substance in wood has not been established, and it is more likely to be substance of a tannin-like nature which, after all, is closely related to phloroglucinol, which is responsible for the reaction. The opinion generally held is that the colour reactions for Hgnified tissue are not due to lignin itself — which forms about 50 per cent by weight of wood — but to small quantities of substances of an aldehydic nature which may have been adsorbed upon the surface of the lignin from the cambial sap, but their exact nature is still largely a matter of surmise. That the colour reactions are not due to hgnin itself but to small quantities of substances accompanying lignin * is sup- ported by two facts, firstly, as shown by Wichelhaus and Lange,t pine or firwood when distilled with superheated steam at 180-200° yields a distillate which gives all the characteristic colour reactions of the original wood, and secondly, that lignin once isolated from its association with cellulose in wood generally no longer gives the colour reaction. In attempting to find an explanation of the nature of this substance, suspicion at first fell upon vanillin and coniferyl aldehyde, since both these substances give the reaction with phloroglucinol and hydrochloric acid, but no proof has been furnished for the universal occurrence of these substances in hgnified tissues. It is true that coniferin, a glucoside giving rise to coniferyl alcohol, occurs in the cambial sap of most conifers and that it is easily oxidized to vanillin. OH OH ^OCHs /^\oCH3 ^^CH = CH— CH2OH -^^Q Coniferyl alcohol Vanillin Also vanilhn itself is widely distributed, having been reported in many resins, in dahlia tubers, potato peel, asparagus shoots, * vSee Cross and Doree : "Researches on Cellulose, IV.," London, 1922, p. 153. t Wichelhaus and Lange : " Ber. deut. chem. Gesells.," 1916, 49, 2001 ; 1917, 50, 1683. Wichelhaus : " Chem. Zeit.," 1923, 47, 865. LIGNIN 221 and also in the bark of the lime and in decayed oak wood ; nevertheless, there is considerable doubt as to whether these substances are universally present in lignified tissues other than wood, although in the opinion of Klason lignin itself is a condensation product of coniferyl alcohol (see below). It was first suggested by Nickel * that the colour reactions of lignified tissue were due to the presence of an aldehyde group in the lignin complex, more especially as the colour reactions were not given by wood which had been treated with sodium bisulphite and other reagents which would mask its aldehydic properties. Czapek,t by a somewhat drastic treatment of wood with stannous chloride, isolated a substance which was both an aldehyde and a phenol ; to this substance, which gave the colour reaction with phloroglucinol and hydro- chloric acid, he gave the name of hadromal, without assigning any constitution to it ; his views did not attain general acceptance and were discredited especially by Grafe J ; since then, however, Hoffmeister,§ modifying Czapek's original con- ditions, has isolated from oak sawdust a substance of the formula CjoHjoOs, which proved to be coniferyl aldehyde of the formula — CH = CH . CHO -OCH3 OH This substance, whose constitution was definitely estab- lished by synthesis from vanillin and acetic aldehyde, gives the colour reaction with phloroglucinol and hydrochloric acid and would thus appear to be identical with Czapek's hadromal. According to the view of Hoffmeister this substance occurs to the extent of about 3 per cent as a cellulose ester in wood. * Nickel : " Chem. Zeit.," 1887, 11, 1520. f Czapek : " Zeit. physiol. Chem.," iSgg, 27, 141. I Grafe : " Monatshefte," 1904, 25, 987. § Hoffmeister : " Ber. deut. cfiem. Gesells.," 1927, 60, 2062. 222 THE CARBOHYDRATES Whilst it may be generally accepted that the commonly employed colour reactions for lignified tissue are given not by lignin itself but by a substance, most probably coniferyl aldehyde, which accompanies lignin, there are nevertheless two colour reactions which may be regarded as being pro- duced by the lignin complex itself ; these are known as the lignone chloride reaction of Cross and Bevan * and of Maiiie's reagent. The former depends on the formation of a yellow colour when lignified material is exposed to moist chlorine gas or bromine, and which on addition of sodium sul- phite changes to red ; Maiiie's reaction also consists in the pro- duction of a red colour when wood is treated successively with potassium permanganate, hydrochloric acid, and ammonia. Probably this is a modified form of the Cross and Bevan re- action, since permanganate followed by hydrochloric acid evolves chlorine. The colour obtained is, however, not uni- form and tends, in the case of wood of deciduous trees, to be brown instead of red. The Isolation and Constitution of Lignin. As stated above, the process of lignification consists in the incorporation into the cell wall of a substance known as lignin ; but opinions are divided as to whether such hgnin is chemically combined with the cellulose or only physically adsorbed. The facts that lignified cellulose, or lignocellulose as it is called, is not soluble in cuprammonia solution and is also incapable of entering into such chemical reactions as can cellulose with carbon disulphide and caustic soda, for example, suggest that there is some kind of chemical union between lignin and cellu- lose. The very great technical importance of cellulose free from lignin necessitated the provision of methods for separating these two substances, the two best known and most widely employed being those of heating the wood with caustic soda or with calcium disulphite ; both these methods are somewhat drastic, and it is reasonable to suppose that the lignin so isolated will differ somewhat from the form in which it existed in the original material ; in the case of the bisulphite method * Cross and Bevan : " J. Chem. Soc," 1889, 55, 199. LIGNIN 223 this is conspicuously so, since the material isolated from the so-called sulphite liquors contains sulphur and is, in fact, a sulphonic acid derivative of lignin ; in the case of the alkaline process, the lignin is less obviously altered. Most of the attempts made to elucidate the constitution of lignin have been carried out on material isolated by these two methods or else by concentrated hydrochloric acid. At first sight it appears strange that attempts to determine the constitution of such a complex substance as lignin should be made upon material which had undergone such drastic treatment, but this is explained by the fact that as yet no gentler methods have succeeded in separating lignin from cellulose. In spite of the considerable literature on the subject com- paratively little is definitely known regarding the constitution of this substance. The following facts are generally accepted : the presence of hydroxyl methoxyl and acetyl group ; the presence of an aldehyde or ketone group as is shown by the ability to react with hydroxylamine, while evidence of un- saturation is revealed by its ability to absorb iodine or bromine and the readiness with which it is oxidized by ozone, nitric acid ; furthermore, it appears fairly certain that some of the methyl groups are attached to phenolic hydroxyl groups and some are not. Lignin Isolated by the Bisulphite Process. Klason,* who worked on lignin isolated from wood by the sulphite process, and which consequently contained more or less combined sulphur, came to the conclusion that lignin exists in two forms in spruce wood, namely a-lignin which contains the acrolein group — CH = CH . CHO, and jS-lignin which contains the corresponding acrylic acid group — CH — CH . COOH in the proportions 63 : 37 per cent, Klason has shown that a-lignin contains two methoxyl groups, one phenolic and one alcoholic hydroxyl, and comes to the conclusion that it has been formed from 2 molecules of coniferyl aldehyde to produce a compound of the constitution * Klason : " Ber. deut. chem. Gesells.," 1923, 56, 300. 224 THE CARBOHYDRATES CH CH ^\ /\ CHO . CH = CH . C C CH .OH CH C . OH I II I II I CH C CH CHjs— C C.OCH3 CO CH I OCH3 The above formula is closely related to that assigned to Gambier-catechin by Freudenberg,* and lends support to the view that lignin may be related to the tannins. Lignin Isolated by the Action of Alkali. A study of the lignin of flax isolated by heating with 8-12 per cent caustic soda for six to ten hours at 140-160°, led Powell and Whittaker f to compare the resulting product with that isolated from various woods including pine, spruce, ash, birch, and poplar ; they conclude that jute lignin is essentially different from flax lignin, to which they assign the formula C45H48O10 which differs considerably from Klason's formula, CaoHaoOe, for pine lignin. Flax lignin has four methoxyl groups, and five hydroxyl groups capable of acety- lation, three of which are phenolic. To the parent hydroxyl compound free from CH3 or COCH3 groups, they assign the name lignol, C41H40O16, and the formula — C3eH3„04(CO)2CHO(OH), Powell and Whittaker disagree with Hagglund's | state- ment that lignin contains 5 per cent of a furfural yielding carbohydrate as an integral part of the molecule ; purified lignin contained only 0-3 per cent pentosan and still further purification gave no furfural at all. Beckmann, Liesche, and Lehmann § in an investigation upon the lignin content of winter rye straw, used a 2 per cent aqueous alcoholic solution of caustic soda acting in the cold * Freudenberg : " Ber. deut. chem. Gesells.," 1920, 53, 1416. t Powell and Whittaker : " J. Chem. Soc," 1924, 125, 337 ; 1925. 127, 132. X Hagglund : " Cellulosechemie." 1923, 4, 73. § Beckmann, Liesche, and Lehmann: " Zeit. angew. Chem.," 1921, 34» 285. LIGNIN 225 for forty-eight hours ; they obtained a substance of molecular weight 800 to which they assigned the formula C4oH440ig which they claimed contained four methoxyl and four hydroxyl groups. Mehta,* in attempting to devise a method for the quantita- tive estimation of lignin, recommends heating in the autoclave with 4 per cent caustic soda under a pressure of 10 atmospheres for one hour. On precipitating with acid a lignin was obtained which, when purified by extraction with alcohol, was an amorphous, faintly acid substance with a pleasant aromatic odour and melting at 170° ; its iodine value was found to be 1397- Doree and Barton-Wright, f employing the above con- ditions and working with spruce wood previously extracted with benzene, alcohol, and water, isolated a substance with melting-point 186° to which they assign the fornmla CaoHaoOg which, though agreeing with that assigned by Klason to a-lignin, has approximately half the molecular weight of the formulse suggested by earlier workers. This substance, which they propose to call meta-lignin, has one hydroxy], two methoxyl, and two carboxyl groups, one alde- hydic, and the other ketonic. They suggest that meta-hgnin is the unit upon which the natural lignins are based and that, whilst the usual type isolated is of the order C40, it may exist in the plant in an even more polymerized form. They suggest for meta-lignin the extended formula — CioH,jO(OCH3)2 . OH . CO . CHO Doree and Barton-Wright disagree with Klason's formula on the ground that no aromatic compounds are obtained from lignin on oxidation, and they suggest, as an alternative, that lignin contains hydroaromatic nuclei which would account both for the unsaturated properties of the substance and for the profound disruption undergone by the molecule with formation of oxalic and carbonic acids. * Mehta : " Biochem. Journ.," 1925, 19, 958. t Doree and Barton-Wright : id., 1927, 21, 290. 15 226 THE CARBOHYDRATES Lignin Isolated by Acid Treatment, Methods for the isolation of hgnin from Hgno-cellulose have also been devised which depend on the solubility of cellulose in strong acids, the lignin remaining undissolved ; for this purpose Ost and Wilkening * employ 72 per cent sulphuric acid, while Willstatter and Zechmeister f recommend concen- trated hydrochloric acid, either acid being allowed to act in the cold. Analyses of the lignin isolated by these methods, however, show that lignin has undergone some degree of hydrolysis, since it contains fewer methoxyl groups as compared with lignins prepared by other methods. Cross and Bevan isolated from hgnin, by the action of chlorine, a compound which contained chlorine and had the properties of a ketone ; from this and other evidence they propose the following formula for lignin : — ABC D CO 00 y\ y'\ y\ yOU Ca cellulose. HC CH - (CHaCO)^ - HC CH . CH-CH . CH<^ \ II I II ^OH lj3 cellulose. HC CO CH3OHC CH.OCH3 CH2 CO in which A is the group which is attacked by the chlorine, B gives rise to the acetic acid obtained by hydrolysis or distillation, and D is the aldehyde group to whose two-hydroxyl group cellulose a and 8 are supposed to be attached in ester- hke combination ; it is, however, not easy to see how his compound which contains Cjo should give rise to a lignone chloride containing Cjq. Estimation of Lignin. The methods for estimating lignin are based upon the use of mineral acids under various conditions with the object of • Ost and Wilkening : " Chem. Zeit.," 1910. 34, 461. I Willstatter and Zechmeister: " Ber. deut. chem. Gesells.," 1913. 46, 2401. LIGNIN 22/ dissolving out the hydrolysable carbohydrate and weighing the residual lignin ; the latter substance, however, is also attacked to some extent by the acid with consequent loss of methoxyl and acetyl groups ; moreover, it is liable to retain a certain amount of carbohydrate. The action of 42 per cent hydrochloric acid upon the lignified material for eighteen hours at the ordinary temperature was first suggested by Will- statter and Zechmeister * ; subsequently this method was modified by Hagglund ; f Konig and Rump | employ i per cent hydrochloric acid under 6 atmospheres pressure for six to seven hours. Ost and Wilkening,§ on the other hand, recommend 72 per cent sulphuric acid in the cold until a portion of the solution gives no precipitate with water ; the whole mixture is then poured into ten times its volume of water and the residual lignin is filtered off through cotton wool. Methods of Estimating Cellulose in Lignified Tissues. Cross and Bevan's || method consists in exposing the moist material to the action of chlorine for a short time,^ whereby chlorination of the lignin complex results in the formation of a lignone chloride to which they give the formula Ci9Hig09Cl4. The lignone chloride is then dissolved out by means of a 2 per cent solution of sodium sulphite whereby a pink colour is produced which may be regarded as a true colour reaction of the lignin complex ; the material is then chlorinated again, and extracted with sodium sul- phite and the process is repeated until a pink colour is no longer produced by addition of the sulphite ; the number of chlorinations required varies from two to five according * Willstatter and Zechmeister : " Ber. deut. chem. Gesells.," 191 3, 46, 2403. t Hagglund : " Arkiv Kemi, Mineral Geol.," 1918, 7, 8. t Konig and Rump : " Zeit. Nahr. Genussm.," 1914, 28, 177. § Ost and Wilkening : " Chem. Zeit.," 19 lo, 34, 461. II Cross and Bevan : " Cellulose," 1895, p. 102 ; and " J. Chem. See," 1882, 41, 94. Tl Over-exposure leads to the chlorine attacking the cellulose with formation of oxycellulose. See Heuser and Siebert : " Zeit. angew. Chem.," 1913, 26, 801. 15* 228 THE CARBOHYDRATES to the nature of the material ; hard woods require less than soft woods since they contain as a rule rather less lignin. E. Schmidt and Graumann * suggested the use of an aqueous solution of chlorine dioxide f in place of gaseous chlorine ; the original procedure was to employ a solution of approximately 0-3 per cent strength, but Heuser and Merlau $ recommend a 1-5 per cent solution. For the esti- mation 0-5 gram of wood, which has been extracted with alcohol and benzene to remove resins, etc., is placed in a glass-stoppered flask with 100 c.c. of 1-5 per cent solution of chlorine dioxide ; after forty-eight hours the residue is washed free from chlorine dioxide and then with 2 per cent sodium sulphite until the filtrate is no longer coloured ; a second similar treatment is generally sufficient to remove all the lignin ; the residue is washed and dried and may be weighed as cellulose. According to Schmidt and Graumann, chlorine dioxide solution has no action on the carbohydrate con- stituents of the cell wall, whereas small quantities of incrustive substance are readily attacked ; it is thus possible to estimate quantitatively the percentage of incrustive and tissue sub- stance in portions of plants ; thus in Piniis sylvestris they found 63-28 per cent of tissue substance and 3672 per cent of lignin, whereas Willstatter and Zechmeister found only 27-25 per cent of the latter. The Nature of the Union Between Lignin and Cellulose. Opinions are divided as to the nature of the association between lignin and cellulose ; the view formerly held by Cross and Bevan § was in favour of some form of chemical union, but later they admit the possibility of there being only a physical association, while Klason, who formerly beheved in physical union, now favours combination. j| According to Konig and Rump ^ the fact that wood when treated with * Schmidt and Graumann : " Ber. deut. chem. Gesells.," 1921, 54, B., i860. t Prepared from potassium chlorate and oxalic acid. X Heuser and Merlau : " Cellulosechemie," 1923, 4, loi. § Cross and Bevan : " Ber. deut. chem. Gesells.," 1893, 26, 2520. II Klason : id., 1923, 56, 300. ^ Konig and Rump : loc. cit. LIGNIN 229 72 per cent sulphuric acid to remove lignin, still retains its original organised structure, shows that lignin is merely mixed with or incorporated with cellulose ; the argument, however, is not convincing since cotton cellulose can be nitrated without destroying the structure of the fibres. The view of Wislicenus * on the mode of origin of lignified cell walls is that the original cellulose wall is a colloidal hydrogel which adsorbs from the sap other colloidal materials that produce lignin ; the union between the lignin and the cellulose is accordingly only one of physical adsorption, possibly reinforced by supplementary valencies of oxygen atoms. Robinson f also favours the view of physical mixture from observations on the microscopical features of mechanical strains in timber which he explains as being due to displacement of films of lignin overlaying the ground cellulose of the tracheids. On the other hand, Schmidt and others J have published some theoretical specu- lations on the relation of cellulose to the incrustation and conclude that they are united in ester-like combination, while Mehta § considers that lignin is combined with cellulose as an aromatic glucoside. MICROCHEMICAL REACTIONS. Lignified tissues give the following reactions :— 1. A brownish-yellow colour is given with iodine. 2. A brown colour is obtained with the use of chlorzinc iodide. 3. Calcium chloride iodine solution turns lignin yellow to yellow-brown. 4. Insoluble in cuprammonia. 5. Aniline sulphate or aniline chloride in aqueous solution and acidified with the corresponding acid turns lignified walls a bright yellow. 6. If the sections be soaked for about a minute in an alcoholic solution of phloroglucin and then mounted in a drop * Wislicenus : " Kolloid Zeit.," 1910, 6, 17 and 87 ; " Cellulosechemie," 1925. 6, 45. t Robinson : " Trans. Roy. Soc," B., 1920, 210, 49. J Schmidt, Haag, and Sperling: " Ber. deut. chem. Gesells.," 1925, 58, 139. § Mehta : " Biochem. Journ.," 1925, 19, 958. 230 THE CARBOHYDRATES of strong hydrochloric acid, the lignified walls are turned a bright red. 7. A concentrated solution of thallin sulphate in 50 per cent alcohol gives a yellow to orange-yellow coloration. The sections should be treated first with alcohol, and the thalhn sulphate solution should be freshly prepared. 8. If lignified tissues be treated with chlorine water and then with sodium sulphite, a deep magenta colour is produced. 9. Lignocelluloses induce the formation of Prussian blue in the greenish-red solution produced by mixing ferric chloride with potassium ferricyanide. CUTINIZED MEMBRANES. The surface of the subaerial parts of the majority of vascu- lar plants is covered by a secretion of the epidermis. This secretion is known as cutin and forms a continuous trans- parent layer, the cuticle, which may be so well developed as to give a shining surface to the plant member, the upper surface of a holly leaf, for example. The cuticle may be quite distinct from the underlying cellulose membrane of the epidermal cells, to which it is closely applied, as in the leaf of the hellebore ; in other cases the distinction between the cutinized and non-cutinized parts is not sharply defined, as in Selaginella, the one merging gradually into the other. When thick, the cuticle not infrequently shows stratification, and wax-Hke substances may be present. The thickness of the cuticle varies much in different plants and with the conditions of growth. Its greatest development is found on leaves and shoots which are exposed to arid conditions such as high insolation, the prevalence of dry winds, growth in soils poor in available water, together with other factors. Its chief physical property is its high degree of impermeability to water vapour and gases, its presence, therefore, impedes the evapora- tion of water from the surface of the plant. Lee and Priestley * conclude that cuticle is formed by the migration of fatty substances liberated at the surface of the * Lee and Priestley : " Ann. Bot.." 1924, 38, 525. Priestley : " New Phyt. " 1921, 20, 17. Lee : " Ann. Bot.." 1925, 39, 755. CUTIN 231 protoplast, the thickness of the cuticle being proportional to the amount of fat secreted by the plant. Thus heath plants, which synthesize much fat, are characterized by the presence of thick cuticles. The authors explain the distribution of cuticle in various plants by speculations regarding the in- fluence of external factors such as the relative proportion of potassium and calcium, light and humidity. The presence of hydroxy-acids in cuticle would appear to be established, from which the authors conclude that aeration is an important factor. The fact that the iodine value of the fat extracted from the shoot tips of Vicia Faha was 73 as compared with 114 for the root apices, and that the iodine values of 90 and 54 were obtained for the cuticle fat of indoor forced and outdoor grown rhubarb respectively, suggest that fats exposed to the oxidizing and drying conditions of the open air become saturated more quickly than those exposed to the atmosphere of forcing sheds. Further, the authors found that the outdoor rhubarb contained twice as much hydroxy-fatty acid as forced rhubarb. With respect to the chemistry of cutin, it is concluded that cutin is a complex mixture of fatty acids, both free and com- bined with alcohols, that have undergone condensation and oxidation ; soaps of fatty acids together with unsaponifiable material which probably contains some higher alcohols, and resinous substances. Cutin, unlike suberin, contains no phel- lonic acid, phloionic acid (see p. 233), or glycerol. From these observations it will be seen that the term cutin does not represent a chemical individual but an aggre- gate of substances varying in specific composition but occurring at the same place in the plant and having the same general characters. Other investigations of cuticle are those of Clifford and Probert * on the wax of American cotton, and of Legg and Wheeler f upon the cuticle of Agave americana. The former authors find the cuticle wax to contain some glycerol esters, a number of monohydric alcohols, hydrocarbons, and resin * Clifford and Probert : " J. Text. Inst.," 1924, 15, 8, 401. t Legg and Wheeler : " J. Chem. Soc," 1925, 127, 1412. 232 THE CARBOHYDRATES esters and alcohols. Legg and Wheeler, after saponification of Agave cuticle with alcoholic potassium hydroxide, isolated cutic acid CgeHgoOg and cutinic acid C13H22O3, which they consider to be the constituents of the acid described by Fremy and Urbain * as oleocutic acid. SUBERIZED MEMBRANES. In the majority of trees and shrubs and in many her- baceous plants, the superficial tissue, or tissues, is replaced by a secondary tegumentary system. This normally arises after the primary tissues are fully differentiated and, generally, soon after the beginning of secondary thickening in the vas- cular system. This secondary tegument is known as periderm, and its formation is instituted by the advent of a new meri- stem, the cork cambium or phellogen. In the stem, the phellogen may arise in the epidermis, as in Nerium ; in the hypodermis, as in Samhucus ; or in the deeper layers of the cortex, as in Ribes. In the root, the phellogen generally has its origin in the pericycle, although in some instances it may arise in the superficial parts of the cortex as in V alerianella. The segmentation of the phellogen results in the formation of regular serial rows of closely packed brick-shaped cells towards the exterior and, more especially when the phellogen is deeply seated, a less regular and more or less extensive series of cells towards the interior. The former undergo a gradual change, suberization, lose their living contents, and finally become cork, whilst the latter retain their living contents and form a secondary cortex, known as phelloderm. The formation of cork isolates the tissues on its outer side which thus are cut off from all supplies and die.f Phelloderm thus comprises the dead cork and the dead primary tissues on its outer side, the living phelloderm if formed, and the phellogen situated between the cork and the phelloderm. The cork of commerce is mostly derived from the cork oak, Quercus suher. * Fr6my and Urbain : " Ann. Sci. Nat. Bot.," 1882, vi., 360. t The term " bark " often is loosely used. Bark comprises all the dead tissues external to the phellogen. SUBERIN 233 In addition to this normal formation of cork, a phellogen may arise and form cork as a result of wounding, and suberi- zation, without the formation of a phellogen, may take place when non-superficial cells are exposed by the removal or destruction of superficial tissue. Further, cork formation is associated with the fall of the leaf. A mature cork cell consists of an internal suberin lamella possessed of fat-staining properties, a cellulose layer and a middle lamella both of which are more or less impregnated with fat-like bodies to which the name of suberin is given and to which is due the characteristic properties of cork, more especially relative impermeability to water and to air. It was formerly thought that cork was a compound of cellulose and suberin. The work of Gilson,* however, shows that cellulose does not enter into the composition of cork for the following reasons : — 1. Cellulose is not attacked by prolonged boiling in a 3 per cent solution of potassium hydrate in alcohol ; suberized walls, on the other hand, are dissolved. 2. Phellonic acid (C22H43O3) has been isolated from cork, and this substance, together with its potassium salt, gives a red coloration with chlorzinc iodide. This suggests that the coloration of suberized membranes with chlorzinc iodide after treatment with potash is due to the presence of potassium phellonate and not to cellulose, for, in addition, the coloration does not take place if the corky tissue be subjected to the action of boiling alcohol after treatment with potash. 3. After treatment with cuprammonia, the chlorzinc iodide gives a yellowish-brown colour ; this, according to Gilson, is due to the conversion of potassium phellonate into the copper salt, and not to the removal of cellulose, as had been sup- posed. Gilson separated from oak-cork suberic acid (CjTHsoOg) and phloionic acid (CuHaiO^) in addition to phellonic acid. He does not think that these occur as true glycerol esters, since * Gilson: "La Cellule," 1890, 6, 63. See also van Wisselingh : " Chem. Zentr.," 1892, 2, 516 ; and Schmidt : " Monatshefte," 1910, 31, 347- 234 THE CARBOHYDRATES the suberin walls are insoluble in all fat-solvents, and do not melt at a temperature below 290° C. An investigation of the chemical nature of potato cork was undertaken by Rhodes,* who obtained a chloroform extract and an insoluble residue ; the latter boiled with excess of alcohohc soda gave a solution from which he separated normal and hydroxy-acids, the latter being characterized by insolu- bility in light petroleum. The hydroxy-acids are not extracted from the suberin lamella until after saponification, showing that they occur there in some form of combination. He concludes that the suberin lamella arises by changes taking place in the fatty material rendering them no longer soluble in fat solvents ; part of the fatty substances never undergo this change, and it is this part which is chiefly responsible for the staining properties of the lamella with the ordinary fat stains. The lamella consists in the main of relatively insoluble normal and hydroxy-fatty acid complexes which can be re- leased by prolonged saponification as soluble soaps. Glycerol was found only in the chloroform extract and then only in traces, except in the case of regenerated cork layers. Microchemical Reactions of Suherized and Cuticularized Memhrayies. 1. With chlorzinc iodide, and also with iodine and sul- phuric acid, a brown or yellow colour is given. 2. Suberized and cuticularized walls are insoluble in cuprammonia and concentrated sulphuric acid. 3. Suberized walls are coloured yellow with strong potash solution ; on heating the colour deepens, and on boiling yellow oily drops exude from the membranes. 4. Suberized walls are the most resistant of membranes to Schultze's macerating mixture. 5. These membranes are stained red by treatment with alcohohc solutions of Alkannin, Sudan HI and Scharlach R. 6. If a section of the material be treated first with eau de Javelle, in order to destroy any tannins which may be present, * Rhodes : " Biochem. Journ.," 1925, 19, 454. INDUSTRIAL , USES 2 3 5 suberized walls are stained very deeply with a solution of cyanin in 50 per cent alcohol to which an equal volume of glycerol has been added. Lignified walls, on the other hand, are not stained under these conditions. INDUSTRIAL USES OF CELLULOSE AND CELLULOSE PRODUCTS. One of the industries which consumes the largest amount of cellulose is that of paper manufacture. Formerly the chief sources of cellulose for this purpose were cotton or hemp fibres but with the increased consumption of paper other sources had to be found. Although straw contains cellulose which has been only slightly lignified, it is found to be unsuitable for the preparation of pure cellulose, owing to the fact that it contains a considerable quantity of siHca. The employment of wood as a source of cellulose became possible with the discovery of chemical methods of destroying the non-cellulose constituent lignin, i.e. the " encrusting substances," without affecting the cellulose proper. In the manufacture of paper from linen rags or cotton waste the material is cut up, cleaned, and disintegrated by boiling successively with dilute sodium carbonate and caustic soda under pressure ; the fibre is then bleached with chlorine, the excess being subsequently removed ; it is then treated with resin, soap, and alum, and spread in thin layers and dried, whereby the fibres become felted together in a peculiar manner, with the formation of paper. When wood is used the " en- crusting substances " may be removed by boiling with calcium bisulphite, whereby the lignin remains in solution and a fairly pure form of cellulose, known as sulphite cellulose, is pro- duced. In the preparation of inferior quality papers there is no chemical treatment of the disintegrated wood pulp ; the material is, therefore, known as mechanical pulp, and paper made from it gives reactions for lignocellulose. Cellulose used for the preparation of filter papers is, after the ordinary methods of purification, treated with hydrofluoric acid to remove silica. 236 THE CARBOHYDRATES COMMERCIALLY VALUABLE DERIVATIVES OF CELLULOSE. When heated in a concentrated solution of zinc chloride, cellulose is converted into a viscid syrup. This syrup, when forced through glass nozzles into alcohol, forms threads which, after being washed and carbonized, become hard and are used for electric lamp filaments ; they have also been employed for the basis of incandescent lamp mantles. Gun Cotton or Pyroxylin. — That a variety of different pro- ducts may be obtained by the action of various strengths of nitric acid, either alone or in the presence of sulphuric acid, on cellulose, has already been mentioned. The substance known as gun cotton is a hexanitrate ; it is obtained by immersing dry cotton waste, freed from grease by treatment with alkali, in a mixture of i part nitric acid (sp. gr. 1-52) with 3 parts sulphuric acid (sp. gr. 1-84) ; the resulting substance is then rapidly and thoroughly washed with water, moulded into discs, and dried on heated plates. On explosion it produces corrosive gases and therefore is not suitable for use, as such in firearms ; when, however, the gun cotton is dissolved in ethyl acetate or acetone and the solution is evaporated, a new substance is obtained which has the same composition as gun cotton, but different properties ; it explodes with less violence and produces no corrosive vapours, and is therefore employed in the manufacture of smokeless powder. Blasting Gelatine is a mixture of gun cotton and nitro- glycerine. Gun cotton mixed with a variety of other sub stances enters into the composition of numerous explosives, such as ballastite, melanite, cordite, etc., etc. Collodion is the name applied to a solution of cellulose tri- and tetra-nitrates in a mixture of equal parts of 95 per cent alcohol and ether. A substance known as artificial india-rubber * is produced by kneading together a mixture of tri- and tetra-nitrocelluloses partially dissolved in ether alcohol with castor oil. The * This substance must be carefully distinguished from so-called syn- thetic rubber, which is an artificially polymerized hydrocarbon of the for- mula (CgHe),, ; this substance, if not actually identical with natural rubber, is at any rate closely related to it, whereas the artificial india-rubber mentioned above is a nitrated cellulose. INDUSTRIAL USES 237 resulting substance may be made to have any degree of elas- ticity, according to the materials which are mixed with it. It forms a more or less satisfactory substitute for rubber and possesses a high electric resistance. Though not explosive, it is inflammable, but to do away with this inconvenience the outer surface may be denitrated by treatment with alkali, whereby it is rendered non-flammable. Artificial gutta-percha is obtained by allowing an acetone solution of tetra-acetyl cellulose to evaporate. Celluloid is produced by mixing the tri- and tetra-nitrates, as employed for collodion, with camphor. Artificial Silks. — These are produced in a variety of ways by precipitating some form of cellulose from solution. The first artificial silk was prepared by Chardonnet, who obtained it by forcing collodion through fine nozzles ; the thin stream of nitrocellulose solution on coming in contact with the air sohdifies to a thread by the rapid evaporation of the solvent. To render it non-flammable the thread is denitrated by treat- ment with ammonium sulphide. A second process for preparing artificial silk consists in dissolving bleached mercerized cotton (see p. 209) in cupram- monia solution. A fine stream of this solution is then run into a dilute sulphuric acid, whereby a continuous thread of cellulose is at once precipitated. A third process is that in which viscose solution is forced through fine nozzles, the emerging streams being coagulated either by hot air or by a bath of ammonium chloride. The fine threads which result can be spun like silk. Cellulose acetate also is used for this purpose. Viscose is obtained by acting on finely divided cellulose with soda and treating the resulting substances with carbon disulphide, whereby a cellulose thio-carbonate is produced ; this substance on exposure to air decomposes spontaneously into cellulose alkaH and carbon disulphide. Viscose solutions are employed for sizing paper and in the manufacture of wall- papers. Mixed with metallic dust and colouring matters, viscose can be converted into an artificial leather, and may also be 238 THE CARBOHYDRATES employed for rendering canvas waterproof and for making cinematograph films, etc. Viscoid, which is congealed viscose, is a hard mass obtained by mixing viscose with various substances and allowing the mixture to decompose spontaneously and harden ; it is used for mouldings, cornices, statuettes, etc. Solid Spirit. — The substance sold under this name is ob- tained by pouring a solution of cellulose acetate in glacial acetic acid into alcohol ; a white solid is produced which does not melt, and burns when ignited without leaving any ash. Cellulose acetate, in which there are approximately five acetyl groups to the Cjg cellulose unit, is soluble in acetone, and is used largely as a dressing for the fabric of aeroplane wings. Cellite is acetyl cellulose which is soluble in a mixture of ethyl acetate and ethyl alcohol. Mixed with camphor it is used in the manufacture of non-flammable cinematograph films. Willesden Paper is paper waterproofed by treatment with cuprammonia, whereby the fibres are gelatinized, and, when dry, are impervious to water. Finally, mention may be made of a few substances, which are made from cellulose as a starting-point, but which are produced only by the profound decomposition of the molecule. Thus by heating cellulose with a strong solution of caustic potash and soda, oxaHc acid is produced, and by the de- structive distillation of wood, acetic acid, acetone and methyl alcohol are obtained. FURTHER REFERENCES. Cross and Bevan : " Researches on Cellulose," London, 1895, 1901, 1906, 1912, 1922. Cross and Bevan : " A Text-Book of Paper Making," London, 1916. Cross and Bevan : " Cellulose," London, 191 8. Cross, Bevan, and Sindall : " Woodpulp and its Uses," London, 191 1. Fuchs : " Die Chemie des Lignins," Berlin, 1926. Hawley and Wise : " The Chemistry of Wood," New York, 1926. Heuser : " Lehrbuch der Cellulosechemie," Berlin, 1927. Schorger : " Chemistry of Cellulose and Wood," London, 1926. Schwalbe : " Die Chemie der Cellulose," Berlin, 1912. Worden : " Nitrocellulose Industry," London, 191 1. SECTION IV. GLUCOSIDES. A GLUCOSiDE may be defined as a substance which on hydrolysis yields a reducing sugar, wherefore, strictly speaking, di-, tri- and poly-saccharides would be included. Custom, however, restricts the term to those compounds which in addition to reducing sugars also yield one or more other substances which, not infrequently, are of an aromatic nature. The non-sugar constituent, which is sometimes termed an aglucan, may belong to various chemical classes as is seen in the following selected examples : — Glucoside. Aglucan. Salicin. The alcohol saligenin. Coniferin. Coniferyl alcohol. Amygdalin. Benzaldehyde and hydrocyanic acid. Monotropitin. Methyl salicylate. Phaseolunatin. Acetone and hydrocyanic acid. Arbutin. The phenol hydroquinone. Indigo. Indoxyl. Sinigrin. AUyl isothiocyanate. Anthocyanin. Anthocyanidin. Quercitrin. Flavonol. The tannins, fiavones, and anthocyans, owing to their special botanical significance, will be dealt with in subsequent sections. The carbohydrate constituent of the glucoside molecule is commonly glucose, but many other sugars may occur in place of the glucose ; galactose and mannose amongst the hexoses ; rhamnose and other pentoses ; gentiobiose, C12H22OH, the disaccharide of amygdahn ; and primeverose, CnHagOio, the disaccharide of monotropitin. The glucosides are generally soluble in water or dilute alcohol which solvents may be used for their extraction from plant tissues. Owing to the fact that glucosides are not pre- cipitated by lead acetate, their solutions may be purified by 239 240 GLUCOSIDES treatment with this salt, the excess of lead being subsequently removed by hydrogen sulphide. Aqueous solutions of glucosides frequently have a bitter taste and are Isevo-rotatory ; they do not reduce Fehling's solution until liberation by hydrolysis of the monosaccharide which, as has been stated, may be a hexose, pentose, or methyl pentose or a mixture of two of more of these. In the plant hydrolysis is effected by an appropriate enzyme which may be specific, or may be capable of splitting several glucosides. In the process of extraction, precautions must accordingly be taken to prevent interaction between the enzyme and its substrate ; this is best effected by treating the material with boiling alcohol, in order to destroy the enzyme, prior to the extraction of the glucoside with alcohol or water. Bourquelot's method of investigating plants for glucosides has been extensively employed ; it depends on the fact that all glucosides which are hydrolysed by emulsin are laevo- rotatory, but after hydrolysis become dextro-rotatory and acquire reducing properties. The glucoside and the enzyme may in some cases be contained in the same cell and only come into contact with each other on injury, or during certain phases in the plant's metabolism. On the other hand, the enzyme and substrate may be secreted in distinct tissues ; an example of this is furnished by the seeds of Lunaria biennis in which the cotyledons secrete the enzyme whilst the integument co^itains the gluco- side. If the seeds are skinned and the cotyledons and testas are separately ground, no smell of mustard oil is produced ; but if the two are ground together, the myrosin acting upon the sinigrin contained in the seed-leaves, liberates allyliso- thiocyanate. THE CONSTITUTION OF THE GLUCOSIDES. The constitution of the natural glucosides can be best understood by a brief consideration of the simplest known artificial glucosides which have been synthesized from glucose. The lactone formula for glucose with its asymmetric CONSTITUTION >4i terminal carbon atom accounts for the ability of glucose to react with methyl alcohol to form two isomeric a- and j8- methyl glucosides * according to the equation— r -o- CH2OH CH (CHOH)3 CHOH + CH3OH I -0 = CH2OH CH(CHOH)3 CHOCH3 + HjO The a-glucoside, which is dextro-rotatory, is hydrolysed by maltase, but not by emulsin, while the j3-glucoside, on the contrary, is unaffected by maltase, but is hydrolysed by emulsin, t there result on hydrolysis the two isomeric a- and jS-glucoses, whose constitutions are represented by the following formulae :— H . C . OH I H . C . OH I HO . C . H O HC .OH I HC HO . C . H Hi OH HOC— H O H.i OH HC CH2OH a-Glucose a^ = + 110° CH2OH j3-Glucose CTjj = + 19° The fact that either of these sugars tends to change at once into the ordinary form of glucose, the so-called equilibrium mixture having a^ = 52-9°, may be employed as a means for determining the nature of a given glucoside since the rotation of a freshly hydrolysed a-glucoside solution will tend to decrease, while that of a j3-glucoside will increase. As the result of the study of the action of maltase and emulsin upon other glucosides, Fischer divided these sub- stances into two classes known as a-glucosides and ^-glucosides, according as they are hydrolysed by maltase or emulsin re- spectively. Other examples of a-glucosidases besides maltase * A number of analogous compounds have since been prepared by Fischer and his co-workers from mannose, galactose, and fructose, the result- ing compounds being termed mannosides, galactosides, and fructosides respectively. f See also section on Enzymes. 16 242 GLUCOSIDES are mannosidase and trehalase, while j8-glucosidases are repre- sented by amygdalase and the phenolglucosidase of emulsin, cellobiase, and gentiobiase. For a complete elucidation of the constitution of a given glucoside it is necessary to determine not only the nature of the non-sugar residue but also to ascertain which of the hydroxyl groups of the sugar and of the non-sugar residue are involved in the union between the two complexes — more especially if the non-sugar residue contains more than one hydroxyl. For this purpose the glucoside is treated with methyl iodide and silver oxide whereby all the free hydroxyls in the molecule are methylated ; the resulting methylated glucoside is then hydrolysed and the methylated sugar and non- sugar residues are examined ; any free hydroxyl groups now occurring in these products must have been involved in the union of the two complexes, since if present in the original compound they could not have escaped methylation. Thus, for example, Irvine and Rose * found that salicin yielded a pentamethyl derivative which on hydrolysis gave rise to 2 : 3 : 5 : 6 tetramethyl glucose f (I.) and a methylated sahgenin (II.) containing a free phenoHc hydroxyl but having a methyl group attached to its alcoholic hydroxyl, from which it follows that the parent glucoside must have had the formula III. : — -CHOH OH CHOMe O I f XHpMe O CHOMe I CHOMe '2^ -CH O y I ,/ \— CH,OH CHOH I CHOH I CHOH I -CH CHjOMe CHoOH I. II. III. 2:3:5:6 trimethyl * Salicin glucose ♦Irvine and Rose: "J. Chem. Soc," 1906, 89, 814; Irvine: id., 1923, 123, 903. t According to Haworth's formula for glucose, the methylated sugar is the 2:3:4:6 tetramethyl derivative ; this formula has been adopted. PHYSIOLOGY 243 Similar methods * have been applied to the elucidation of the constitution of other glucosides, and as a result many of these have subsequently been synthesized. The synthesis of the glucosides of a number of alcohols besides methyl and ethyl alcohols, was investigated by Bourquelot f who by means of the enzyme emulsin produced glucosides, galactosides, and mannosides of propyl- and iso- propyl-alcohols, glycol, glycerol, and cinnamyl alcohol ; for this purpose the sugar was dissolved in the corresponding alcohol in the presence of a little water or acetone ; all these were jS-glucosides ; with the use of maltase from yeast he was also able to prepare a number of a-glucosides. It has been shown by Armstrong that enzymes can exert their synthetic action without actually being in solution, acting merely as colloids in virtue of their surface. Bour- quelot, moreover, drew attention to the fact that in the presence of enzymes, insoluble alcohols could be converted into soluble glucosides by combination with glucose ; from this he concluded that the plant has in the formation of glucosides a very efficient mechanism for rendering insoluble substances soluble. In some cases the natural glucosides have been chemically synthesized ; thus salicin has been obtained by the reduction of the corresponding aldehyde glucoside, helicin — ■ CeH„06 . O . CgH^CHO + 2H = CoHnOs . O . CgH^CH^OH the helicin itself having been synthesized from glucose and salicylic aldehyde. PHYSIOLOGICAL SIGNIFICANCE OF GLUCOSIDES. In attempting to assign the part played by these sub- stances in the economy of the plant, it must be remembered that glucosides of natural occurrence are very numerous, and, in some cases, of a diverse nature ; it is, therefore, possible that the significance of the presence of one glu- coside may be quite different to that of another, but even in the case of glucosides of the same nature there is much * Macbeth and Pryde : " J. Chem. Soc," 1922, 121, 1660. t Bourquelot : " Bull. Soc. Chim.," 1913. [iv], 13. i-xxviii. 16* 244 GLUCOSIDES diversity of opinion. They have been described, on insufficient grounds, as direct products of photosynthesis. Many consider them to be of value as food-stuffs on account of the sugar they contain ; the occurrence of certain glucosides in seeds lends some support to this view, for in the case of the bitter almond hydrocyanic acid, in the free state, may be identified when germination starts, also the observations of Treub,* who found that in the case of some plants containing cyanogenetic glu- cosides the amount of the latter decreased if the plant was placed in the dark, in order that photosynthesis could not take place. On the other hand there was an increase in quantity when the plants were exposed to light, and this increase reached a maximum at about midday. Weevers f considers that salicin, populin, arbutin and simi- lar glucosides are of the nature of reserve food-materials, for not only do these substances form a suitable means for the storage of sugar on account of their low diffusibility, but the facts of their seasonal or diurnal variation lend support to this opinion. Thus in Vaccinium Vitis-Idcea the arbutin is stored in the leaves, and when the new leaves are formed in the spring it is used up ; it is split by a suitable enzyme, the sugar being used up, and the hydroquinone remains behind and combines with more sugar, so that by the autumn the leaves once more contain arbutin. In the case of the willow, salicin is formed day by day, but during the night it is spHt by salicase into sugar and the alcohol saligenin. The glucose is translocated, and the sali- genin remains behind and is converted into salicin by combin- ing with sugar the next day. This process stops in the autumn, by which time there is relatively much sahcin in the cortex of the stem. This translocation of glucosides from the leaves of many plants — but not of all, Samhucus and Indigofera being excep- tions — is significant, and so also are the facts relating to the * Treub : " Ann. Jard. Bot. Buitenzorg," 1896, 13, i ; 1907, 21, 79, 107 ; 1910, 23, 85. + Weevers : " Kon. Akad. Wet. Amsterdam," 1902 ; " Rec. Trav. Bot. Neerl," 1910, 7, i. SINIGRIN 245 amount of glucosides in the bark and other parts of plants at different seasons of the year. Thus in Salix and Populus the glucoside (sahcin) is most abundant in the autumn and winter, and is used up in the following spring during the period of flowering and seed formation ; also in the case of Taxus the glucoside (taxicatin), which appears principally in the young shoots, is greatest in amount in the autumn and winter. In Panghim ediile and other plants the amount of cyanogenetic glucosides is greatest in young leaves, with increasing age the amount diminishes. Guignard * does not beheve that glucosides, or at any rate the cyanogenetic ones, are reserve food-stuffs, since, if in- troduced into the food-materials of a plant, glucosides have an injurious effect, owing to the aromatic residues. Combes, t however, finds that a glucoside is toxic only to plants in which it does not naturally occur ; he thinks that glucosides do not furnish carbohydrate food, since plants grown in an atmosphere free from carbon dioxide are unable to make use of these substances. Peche X holds that hydrocyanic acid is a direct product of photosynthesis ; some of it combines with sugar to form a glucoside, and some is transported in a labile form, probably in a loose combination with tannin, and stored for future use as food in various tissues. * The occurrence of certain glucosides, especially in places of active metabolism such as leaves and young shoots, may indicate that certain bye-products are fixed, either temporarily or more permanently, in this form. The exigencies of space will permit of reference only to the following examples, which are among the more important and more interesting of the glucosides. SINIGRIN. Sinigrin, or myronate of potash, occurs in the seeds of certain Cruciferse, notably Sinapis nigra. It is split by the * Guignard : " Compt. rend.," 1905, 141, 236 ; 1906, 143, 451. t Combes : " Rev. gen. Bot.," 1918, 30, 216. X Peche : " Sitz. Kais. Akad. Vienna," 1912, 121, 33. 246 GLUCOSIDES enzyme myrosin into glucose, potassium hydrogen sulphate, and allyl isothiocyanate — CioHieOsNKS^ + H^O = C,U,,0^ + KHSO, + CH^ : CHCH^NCS Sinigrin crystallizes from alcohol in needles and from water in prisms, m.p, 126-127° C. CONIFERIN. This glucoside occurs in various coniferous trees, especially in young parenchyma, and also in asparagus. With concen- trated sulphuric acid coniferin gives a violet coloration, while hydrochloric acid and phenol give a blue coloration ; it also gives a bright coloration with phloroglucinol and hydro- chloric acid (see p. 219). Coniferin crystallizes in needle-shaped crystals, m.p. 185°, and is soluble in warm water and warm alcohol. On hydrolysis by mineral acids or by emulsin it gives glucose and coniferyl alcohol — CieH^Ps + HjO = CeHi,Oe + CioH^.O:, Coniferin Coniferyl alcohol The latter is a crystalline substance melting at y2,°. Both coniferin and coniferyl alcohol when oxidized with potassium bichromate and sulphuric acid yield vanilhn, the aromatic constituent of the fruits of Vanilla planifolia. The reaction was formerly employed for the preparation of artificial vanillin, but has now been replaced by the oxidation of isoeugenol, which is obtained by the action of dilute alkalis upon eugenol, a substance contained in oil of cloves. The relationship between these three substances is as follows : — CH = CHCH2OH CHO CH = CHCH -^ /\ -<— A OCH3 [ IJ0CH3 OH I J0CH3 OH Conii eryl alcohol Vanillin I< soeugenol SALICIN. Salicin, CiaHjgO,, occurs in the bark of Salix viminalis. It has a bitter taste and crystallizes in colourless prisms and CONIFERIN 247 scales. It is sparingly soluble in cold water but is more soluble in hot alcohol, especially amyl alcohol, and may be extracted from aqueous solutions by means of this solvent. Microscopi- cally, salicin is indicated by the fact that it gives a bright red colour with strong sulphuric acid, also with Frohde's reagent * it yields a violet coloration. Salicin may be prepared by boiling the willow bark with water which will extract a certain amount of tannin, colouring, and other matters, together with the salicin ; the solution is then treated with lead acetate and after filtering the filtrate is freed from lead by hydrogen sulphide. After removing the lead sulphide the solution on evaporation yields crystals of saHcin which may be further purified by recrystallization from alcohol. Salicin is hydrolysed by the enzyme salicase contained in willow bark and also by emulsin from bitter almonds to glucose and the alcohol saligenin according to the following equation : — C13H18O7 + H^O = CgHijOo + QH^OH CH^OH Salicin Saligenin On steeping a section in a solution of emulsin, saligenin is produced which gives a blue colour with ferric chloride. By the action of sulphuric acid and potassium bichromate salicin is oxidized to salicyhc aldehyde, C6H4OHCHO ; this substance is a fragrant colourless hquid, b.p. 196°, which occurs in the essential oil of Spircea Ulmaria ; it is soluble in water, the solution giving an intense violet coloration with ferric chloride. Jowett and Potter f claim to have found a seasonal variation in the salicin content of Salix purpurea, and they regard it as a reserve product which is stored in the winter for use in the following spring ; they also claim to have established that the reserve is drawn upon to a different extent by the male and female plants owing to their special functions, but the data they quote are hardly sufficient to warrant these conclusions ; further work on this subject is * Sodium molybdate dissolved in concentrated sulphuric acid, t Jowett and Potter : " Pharm. Journ.," 1902, 15, 157. 248 GLUCOSIDES desirable. According to Clark and Gillie,* the salicin content of samples of bark of Salix sitchensis from British Columbia varied from 2-8 per cent in the autumn to 7-38 per cent in the spring. Weevers f suggests that the salicin formed in the leaves during the daytime is hydrolysed at night, the glucose being translocated away, while the saHgenin, which remains behind in the leaf, is recombined with sugar the next day ; the object of the glucoside formation would appear to be the production of a difficultly diffusible compound of sugar. MONOTROPITIN. This glucoside was discovered by Bridel % in Monotropa hypopitys, and also in the fresh roots of Spircsa Ulmaria, S. filipendula and 5. gigantea § ; the same author also showed that gaultherin, occurring in the back of Betula alba, was identical with monotropitin. The glucoside, from whichever source obtained, is hydrolysed by the same enzyme variously described as gaultherase, betulase, or primeverase, which also occurs in Monotropa, giving methyl salicylate and the glucoxy- lose primeverose. AUCUBIN. The darkening of the tissues on drying of Aucuba, Melam- pyrunt, and Rhinanthus, and many other plants, is due to one and the same glucoside aucubin, which is acted upon by emulsin, which also occurs in the plant yielding an aglucan, aucibigenin, of unknown constitution. The darkening of the tissues is due to the oxidation of the aucibigenin. The colour change may be readily brought about by wounding or ex- posing the tissues to chloroform vapour. The similar darkening occurring in Orobanche is due to direct oxidation of a non-glucosidal material contained in the plant, the darkening taking place without previous interven- tion of a hydrolytic enzyme (see below). * Clark and Gillie: " Amer. J. Pharm.," 1921, 93, 618. See also Brown : " Pharm. Journ.," 1903, 16, 588. t Weevers : " Rec. trav. bot. Neerl.," 1910, 7. X Bridel : " Bull. Soc. chim. biol.," 1923, 5, 918. § Bridel : id., 1924, 6, 679. AUCUBIN 249 Bergmann and Michaelis * have re-investigated the con- stitution of the glucoside aucubin which is known to be identical with Rhinanthin, "j* the glucoside of Rhinanthiis Crista galli, and ascribe to it the formula C15H22O9 . HgO or some multiple ; from this it would appear to be identical with the glucoside menyanthin contained in Menyanthes trifoliata and with loganin contained in Strychnos nux vomica. The glucoside aucubin, required for the investigation was prepared from the seeds of Plantago lanceolata, occurs in several species of Plantago. The melting-point of aucubin is 181° C, and its rotation aj,=— 1 64*9°. OROBANCHIN. This is a glucoside typical of the orobanchs, having been found in five species of this genus $ ; it is not hydrolysed by emulsin nor by an enzyme prepared from Rhamnus utilis seeds, but on hydrolysis with acid gives rise to glucose and rhamnose in addition to caffeic acid or 3 : 4 dihydroxycinnamic acid — /OH ^ ' CH : CH . COOH whose close relationship to coumaric acid contained in meli- lotosin is interesting (see below). The orobanchs contain no aucubin and the darkening on drying is due to direct oxidation of the glucoside orobanchin without previous hydrolysis ; the oxidation can be brought about by an extract of Russula delica, as well as by the oxidase contained in the plant itself. ASPERULIN. This glucoside which occurs in Asperula odorata, in Galium spp. and in many other Rubiaceae resembles aucubin in giv- ing on hydrolysis in addition to glucose an insoluble greenish- black substance. Asperula odorata also contains a second * Bergmann and Michaelis : " Bar. deut. chem. Gesells.," 1927, 60, 935. t Bridel and Braecke : " Bull. Soc. chim. biol.," 1925, 6, 665. Bour- dier : " J. pharm. Chim.," 1907, [6], 26, 454. I Bridel and Charaux : " Compt. rend.," 1924. 178, 1839 ; 1925, 180, 387. 2 50 GLUCOSIDES glucoside which on hydrolysis yields coumarin,* the lactone of coumaric acid — yCH : CH ^O CO a substance which occurs also in Anthoxanthum odoratum, the grass which gives hay its characteristic smell, tonka bean [Dipteryx odorata), and other plants. GEIN. Gein occurs in the roots of Geum urbanum ; on hydrolysis by means o