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Studies in the polysaccharide gums with special reference to sapote gum Kilgour, Gordon Leslie 1953

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STUDIES IN THE POLYSACCHARIDE GUMS WITH SPECIAL REFERENCE TO SAPOTE. GUM • by . GORDON LESLIE KILGOUR A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Chemistry We accept t h i s thesis as conforming to.the standard required from candidate^ f o r the degree of MASTER OF SCIENCE Members o f the Department of Chemistry THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1953 ABSTRACT Two samples of gum supposedly obtained from Sapotaceae achras and named "sapote gum" were studied using the methods of p a r t i t i o n chromatography. The two samples were proven to be e n t i r e l y d i f f e r e n t i n composition and to constitute i n fact two separate and d i s t i n c t gums. The previously unreported gum was characterized and shown to contain D-xylose, L-arabinose, D-galactose, and one or more glucuronic acids, including some methoxy-glucuronic acid. A new spray reagent was developed f o r paper chroma-tography of the sugars, and a novel technique used f o r making permanent photographic p r i n t s of the papergrams. C r y s t a l l i n e sugars were obtained from hydrolysates i n pure form by separa-t i o n on p a r t i t i o n columns of powdered c e l l u l o s e . ACKNOWLEDGEMENT The author would l i k e at t h i s time to acknow-ledge h i s indebtedness to Professor G. G. S. Dutton f o r the assistance and guidance so f r e e l y given during the course of t h i s work. Acknowledgement i s also g r a t e f u l l y made of the scholarship provided by the Powell River Co. Ltd. and summer research funds supplied by the National Research Council. In addition, thanks are due to Dr. E. Anderson of University of Arizona; Asher, Kates and Co.; Rohm and Haas; Reeve Angel and Co.; and the Forest Products Laboratories at Vancouver f o r various samples provided for t h i s project. TABLE OF CONTENTS Page INTRODUCTION 1 HISTORICAL 5 EXPERIMENTAL 15 P u r i f i c a t i o n of the Gums 16 Anderson Sapote Gum 16 Peruvian Sapote Gum 17 Peruvian Sapote Free Gum Acid 17 A n a l y t i c a l Data 18 Peruvian Sapote Gum 18" Anderson Sapote Gum 20 Chromatographic Methods 21 Paper Chromatography 21 P a r t i t i o n Column Chromatography 25 Separation of Standard sugar Mixture 29 Graded Hydrolysis of the Gums 30 Peruvian Gum with 0 . 4 N. Acid 30 Anderson Gum with 0.8" N. Acid 32 Peruvian Gum With 0.8 N. Acid 33 Further Hydrolysis; Separation and I d e n t i f i c a t i o n of Products 34 Anderson Sapote Gum 34 Mesquite Gum 35 Peruvian Sapote Gum Hydrolysis A 36 Column Run D 37 Peruvian Sapote Gum Hydrolysis B Column Runs F to J Column Run K DISCUSSION BIBLIOGRAPHY TABLES APPENDIX A - Bibliography of references to chromatography of the sugars. APPENDIX B - Diagrams showing t y p i c a l separations obtained on paper chromatograms. FIGURES Page 1 . Outline of Possible mechanism for formation of pentoses from hexoses. 2 2 . Structure of mesquite gum according to E. V. Whitel 5 3 . One proposed structure for mesquite gum according to F. Smith. 6 l+. Cellulose column with constant head apparatus for separation of the sugars. 27 5 . Rotation of 5 % Peruvian sapote gum solution on hydrolysis with 0 . 5 N. sulphuric acid. 3 1 \ TABLES I. Components of Some Water-soluble Gums. II. Analysis of Anderson Sapote Gum. III. Analysis of Barium Salts from Hydrolysis of Anderson Sapote Gum. IV. Color Reactions with a Number of Spray Reagents. V. RG Values for a Number of Sugars and Their Methyl Derivatives. STUDIES IN THE POLYSACCHARIDE GUMS WITH SPECIAL REFERENCE TO SAPOTE GUM • by GORDON LESLIE KILGOUR INTRODUCTION .The water-soluble polysaccharide gums occupy a sing u l a r l y i n t e r e s t i n g position among carbohydrate compounds. They are made up of r e l a t i v e l y small repeating units con-tai n i n g from 5 to 10 molecules of uronic acids, hexoses and pentoses. In recent years., an increasing amount of attention has been paid to the pentoses as t h e i r wide occurence i n nature i s discovered (10) and-to the uronic acids both be-cause of t h e i r role i n physiological d e t o x i f i c a t i o n and because of t h e i r suspected r o l e i n pentose production. The o r i g i n of the pentoses i n nature has long been a subject of intense interest and speculation. One of the most prominent hypotheses involves the oxidation of a hexose to i t s cor-responding uronic acid followed by decarboxylation to the pentose as shown i n f i g . 1. 2 CHO ! HCOH I HOC I HCOH I HCOH CH20H D-glucose CHO i HCOH I -> HOCH I HCOH ! HCOH I COOH D-glucuronic acid CHO ! HCOH I HOCH ! HCOH CH20H D-xylose CHO ! HCOH I HOCH I HOCH ! HCOH H20H D-galactose CHO I HCOH I HOCH I HOCH ! HCOH I COOH D-galacturonic acid CHO I HCOH ! HOCH I HOCH I CH2OH L-arabinose F i g . 1 While proof of t h i s hypothesis s t i l l awaits further work, s u f f i c i e n t has already been done on the structure of the gums to show that such a synthesis could not operate at the polysaccharide l e v e l . Any such series of reactions would require previous degradation to monosaccharides and l a t e r combination of the products. The uronic acids, and i n p a r t i c u l a r glucuronic acid, which i s the one most commonly found in,the gums, have recently been found to have important physiological a c t i v i t i e s . Considerable success has been reported i n treating some types of a r t h r i t i c condition with glucuronic acid or i t s lactone. It i s curious .that plants would appear to use the uronics f o r much the same purpose, as the gums are formed usually i n response to injury to a plant. The recent discovery ( 2 3 ) ( 2 4 ) ( 2 5 ) ( 6 ) ( 1 ) of some gums containing methoxy-uronic acids raises the question of how and why these gums d i f f e r from the others i n hav-ing one hydroxyl methylated. It i s hoped that discovery and investigation of more such cases w i l l permit of some generalizations and int e r p r e t a t i o n . The water-soluble gums also have another i n t e r e s t f o r the wood chemist. They provide a type compound f o r many of the substances encountered i n wood chemistry and at the same time possess an ease of handling foreign to most of them. . Thus i t i s possible to work out procedures on the gums for l a t e r use on hemicellulose and l i k e com-pounds i n wood. The o v e r a l l aim of t h i s investigation was thus e s s e n t i a l l y two-fold. It was intended to add to the growing stock of information regarding the gums and t h e i r components, and at the same time to establish methods which could l a t e r be used f o r investigation of wood products In p a r t i c u l a r , i t was planned to make use of chromato-graphic methods of analysis and separation. These methods 4 are of very recent introduction and had not previously been used i n t h i s u n i v e r s i t y . The gum chosen to constitute the main subject of t h i s investigation was sapote gum, the exudate of a Peruvian tree (supposedly Sapotaceae acras) from which ch i c l e latex i s also obtained. Anderson and Ledbetter (1) reported that t h i s gum contained only D-glucuronic acids, arabinose, and xylose. This alone i s unusual as almost a l l other gums thus f a r reported (Table I) contain some hexose. In addition, he reported the uronic acid possessed one methoxyl group per two glucuronic acids. One sample of the gum for the work described here was obtained from Dr. Anderson, and another large sample d i r e c t l y from Asher, Kates and Co. of Lima, Peru. The Asher, Kates sample was used exclusively i n the beginning of the i n -vestigation and i t was not u n t i l some time l a t e r that the two samples were found to d i f f e r widely. I t was planned at that time to continue work on both samples so far as possible and to determine whether they were t r u l y d i f f e r e n t gums or just variant samples of sapote gum. The evidence thus f a r assembled would indicate that the former i s correct and that they are indeed different i n composition and probably i n source. 5 HISTORICAL As t h i s study was intended to investigate new techniques, i t was considered advisable to use a gum of known constitution as a check on the procedures and on the r e l a t i v e e f f i c i e n c y of the newer methods. A large supply of the reasonably well characterized mesquite gum being r e a d i l y available, i t was decided to use t h i s as the control. Quantitative data on t h i s gum were f i r s t pub-l i s h e d by Anderson and Sands (2) and possible structures l a t e r reported by White (23)(24)(25) and Smith ( 8 ) ( 2 2 ) . The proposed structure of White, as shown i n F i g . 2, involves two D-galactose units l i n k e d together (believed 1-6} to form a backbone structure with alternate branches of arabinose and uronic acid. These branches are joined to the 3-positions of the galactoses and consist i n the one case of 4-methoxy-D-glucuronic acid (metal sal t ) and i n the other case of four L-arabinose units joined 1-2 to each other and 1-3 to the galactose chain. 6 Smith, however, has shown that the methoxy-uronics are linked at the 4- and 6-positions of the galactose, i n d i c a t i n g that the structure of the gum i s not quite as simple as o r i g i n a l l y proposed by White. On the basis of h i s r e s u l t s , Smith has suggested a possible type structure shown i n F i g . 3« Fig. 3 7 At t h i s point we f i n d that considerable d i f -ferences exist between the r e s u l t s obtained by the d i f -ferent investigators even though each i s an acknowledged expert i n the f i e l d . Thus Anderson found a r a t i o of galactose: arabinose: methoxy-uronic of 4 : 3 : 1 which White l a t e r changed to 4 : 2 : 1 on the basis of h i s work on the methylated gum. Smith's r e s u l t s and proposed structure, however, now require a r a t i o of 9 : 5 : 2 . These varying r e s u l t s were a l l obtained before the advent of chromato- . graphy and i l l u s t r a t e some of the d i f f i c u l t i e s involved i n the c l a s s i c a l procedures. Thus Anderson did h i s work on the gum using the standard mucic acid, phloroglucinol, COg type of analyses, some of which even Anderson himself regarded as s u f f i c i e n t l y inaccurate to require empirical corrections depending on the material being analysed. White's method of weighing the methylated sugar glycosides separated by f r a c t i o n a l d i s t i l l a t i o n was not a great deal better as there was no accurate method of determining purity of a sample of unknown sugar. Smith's work followed closely the methods of White. Another point of d i f f i c u l t y before the i n t r o -duction of chromatography was the determination of extent of hydrolyses. The only method available i n most cases was to t r y to c r y s t a l l i z e the component sugars out of the hydrolysate quantitatively and to run the old standard analyses on the residual polysaccharides. This method 8 broke down seriously i f two sugars hydrolysed at the same time or i f two residual polysaccharides were formed. I t was only with the recent advent of chromatography of the sugars that i t became possible to separate such mixtures or even to determine that a mixture was present i n some cases. It was hoped that by using mesquite gum as a "testing f i e l d " during the o v e r a l l attack on sapote gum, some further l i g h t might also be shed upon the question of the exact structure of mesquite. F i r s t investigation of sapote gum was carried out by Anderson and Ledbetter (1) using gum reported to have been.obtained from Sapotaceae achras and supplied by Asher, Kates and Co. of Lima, Peru. They found only L-arabinose, D-xylose, and glucuronic acid present and reported a r a t i o of 8.5 xylose per arabinose and 7 pentoses per uronic. In addition, they found approximately 0.5 methoxyl per uronic acid. This i s the f i r s t reported case of a gum with both methylated and non-methylated uronic acids. Tables II and III are taken from t h e i r paper and show the actual a n a l y t i c a l r e s u l t s . On hydrolyses with 4 % sulphuric acid f o r two hours at 80°C, Anderson found, a l l the arabinose was s p l i t o f f along with some of the xylose. Further hydrolysis of the residual polysaccharide produced only xylose. The resistant residual polysaccharide i s o l a t e d a f t e r 28 hours o at 100°C. was found to have four uronic acids to three pentose units and s t i l l approximately one methoxyl per two uronic acids. Fermentable hexoses were absent from a l l sugar solutions and galactose i n p a r t i c u l a r was ruled out since mucic acid could not be obtained from either the gum or the sugar solutions. White ( 2 6 ) ( 2 7 ) , working with a sample of sapote gum supplied by Anderson, has investigated the products of methanolysis of the methylated gum. He reported the presence of 2 , 3 , 4-trimethyl and 3 ,4-dimethyl L-arabinose along with 2 ,3 ,4-trimethyl and 3-methyl D-xylose. The presence of 2 , 3 , 4-trimethyl and 3 ,4-dimethyl D-glucuronic acid was also noted. The methyl glycosides of these com-pounds were separated using a column of Magnesol with methyl-ethyl ketone/water as solvent. The column was extruded, streaked, and the components cut out f o r el u t i o n . White also used paper chromatography as a check on the mixtures and pure compounds obtained. The discovery of 2 , 3 , 4-trimethyl arabinose constitutes the f i r s t time that arabinose has been reported i n nature i n the pyranose configuration. Within the l a s t few years, and p a r t i c u l a r l y during the period i n which t h i s research was being car r i e d on, the uses of chromatography i n carbohydrate chemistry have increased almost exponentially with time. Thus only those papers having a direct bearing on the present project 10 w i l l be considered at t h i s time. A complete bibliography of the more s i g n i f i c a n t and useful papers i n t h i s f i e l d w i l l be found i n Appendix A. P a r t i t i o n chromatography was f i r s t introduced by Martin and Synge (19) i n 1941> using water-saturated s i l i c a - g e l columns f o r the separation of acetyl amino acids. This work was followed two years l a t e r by the d i s -covery of Consden, Gordon and Martin (7) that f i l t e r paper could be used as a support f o r the water phase i n the separation, thus creating "paper p a r t i t i o n chromatography". While reducing sugars were separated by t h i s method i n 1945 by Chargaff, Levine and Green (5), i t was not u n t i l the following year, when Partridge (20) separated a large number of sugars and sugar derivatives, that the method was r e a l l y established i n the carbohydrate f i e l d . Brown, Hough, Hirst, Jones and Wadman (4) soon expanded the scope of the method to include methylated sugars and showed how t h i s could be applied to the study of methylated poly-saccharides. The work of Flood, H i r s t , and Jones (9), using Somogyi's micro copper reagent (21) to determine reducing sugars eluted from the separated spots, then established the usefulness of paper p a r t i t i o n chromato-graphy as an accurate a n a l y t i c a l t o o l . This was soon followed by the equivalent technique f o r analysis of a number of methylated sugar mixtures using alkaline gypoi-odite instead of the Somogyi reagent (11). Results obtained 11 on methylated waxy maize starch, glycogen, and araban were quoted as examples. A t h i r d a n a l y t i c a l method described a short time l a t e r by Hi r s t and Jones (13) i n -volved the use of sodium periodate with t i t r a t i o n of the formic acid produced. This method was s a t i s f a c t o r y only for reducing sugars and did not determine methylated sugars. The next large step forward i n paper p a r t i t i o n chromatography was the separation by Hough, Jones, and Wadman (15) of gram amounts of sugars and t h e i r methylated derivatives on columns of powdered c e l l u l o s e . Various methods had been used previously f o r other types of com-pounds, u t i l i z i n g very thick sheet of f i l t e r paper or "chromatopiles" i . e . stacks of about 100 round f i l t e r papers pressed together. They had not been found very convenient, however, and did not separate more than several hundred milligrams of material. In addition, only one run could be made with each one set-up, since the sheet or p i l e was cut up to obtain the separated products. Hough, Jones and Wadman prepared t h e i r column by grinding Whatman Accelerator tablets through a sieve, then packing the powder dry into the column. In the l a s t two years there have appeared on the market a number of c e l l u l o s e powder preparations s p e c i f i c a l l y made f o r t h i s purpose. They also used various solvent systems on t h e i r columns and reported on the degree of separation of various mixtures obtained with each solvent. H i r s t , Hough and Jones (12) 12 then used t h i s type of column to separate the hydrolysis products of S t e r c u l i a setigera gum. With a number of methods of separation now worked out, attention turned to the problem of detection of materi-als on the chromatograms. Partridge had o r i g i n a l l y used ammoniacal s i l v e r n i t r a t e and t h i s was generally accepted i n spite of i t s disadvantages up to the end of 1949. At t h i s time Partridge (20) found that a n i l i n e hydrogen phthalate would give a color reaction with both reducing and non-reducing sugars and at the same time would d i f -ferentiate by means of color between the d i f f e r e n t classes of sugars. About t h i s time also, Horrocks (14) discovered that benzidine could be used to detect many sugars where other reducing substances i n t e r f e r e d with the s i l v e r n i t r a t e . A number of other sprays were found f o r s p e c i f i c uses, but the next important contribution was the work of Hough, Jones and Wadman (16) i n 1950 on improved methods for separation and detection of the sugars and methylated derivatives. At t h i s time they put forward a number of sprays f o r both general detection and s p e c i f i c i d e n t i f i c a -t i o n of various sugars. They reported on a number of a c i d i c aromatic, amine and phenol solutions giving d i f f e r e n t color reactions with the various sugars and t h e i r derivatives. Aniline trichloroacetate i n g l a c i a l acetic acid was recom-mended fo r simple sugars and uronic acids and p-anisidine i n n-butanol f o r mixtures of simple and methylated sugars 13 and uronics. Table IV shows the colors produced by various types of sugars with some of the more useful sprays. A number of d i f f e r e n t solvent systems were also considered i n t h i s paper, and the f i r s t report i s made on r e s u l t s obtained by running chromatograms at 37°D<. instead of room temperature. Advantages claimed were fas t e r running, better separation, and more rounded and discrete spots. Many solvent mixtures have been described i n the l i t e r a t u r e f o r p a r t i c u l a r separations, but few w i l l separate a l l the different types and classes of sugars. One of the best f o r general work with both simple and methylated sugars i s n-butanol:ethanolrwater i n the r a t i o of 4 : 1 : 5 , with or without 1% ammonia. A large number of sugars have been tested i n t h i s solvent by Jones and Hir s t (17) and t h e i r Rg values l i s t e d . The Rg value i s obtained by using the distance t r a v e l l e d by 2,3,4?6-tetramethyl glucose as the reference point instead of the solvent front as f o r Rf. These values are shown i n Table V. This solvent does not, however, separate uronic acids, and f a i l s to move biuronics at a l l . For t h i s purpose an a c i d i c solvent such as n-butanolrglacial acetic:water i s required. Several r a t i o s of t h i s mixture have been reported as use-f u l . Hough, Jones and Wadman (1$) recommended 2:1:1 while Partridge (20) reported good r e s u l t s with 4 : 1 : 5 . The l a t t e r solvent produces very much the same Rf or Rg values f o r the sugars as does the solvent containing ethanol 14 referred to above. Thus the two provide a check on the i d e n t i t y of a c i d i c components. Since these two solvents are e f f i c i e n t f o r the desired separations, e a s i l y prepared, and r e l a t i v e l y innocuous, they were used almost exclusively for running paper chromatograms i n the work under discus-sion. For the work on c e l l u l o s e powder columns three solvents were used, a l l of which were recommended by Hough, Jones and Wadman (15) f o r different separations. Those used were n-butanol saturated with water, n-butanol: ethanol: water, ' 4 : 1 : 5 , and isopropanol: water, 9 : 1 . The l a s t of the three i s a one-phase solvent; f o r the other two, only the organic phase was used. 15 EXPERIMENTAL As was explained i n the introduction, t h i s work has involved two separate and d i s t i n c t l y d i f f e r e n t samples of sapote gum, one from Dr. E. Anderson and the other direct from Asher, Kates Co. i n Lima, Peru. For c l a r i t y , i t has become the practise i n t h i s laboratory to re f e r to the one as Anderson sapote gum and to the other as Peruvian sapote gum. This practise w i l l be followed from here on to prevent confusion. It must be kept i n mind, however, that Asher, Kates was the o r i g i n a l source of both gums and that t h i s company believed them to be from the same type of tree. The Peruvian gum was obtained as a dark colored, clear, hard, and very b r i t t l e material. I t could be broken into pieces with the fingers and shattered r e a d i l y on being struck l i g h t l y with a hammer. In cold water, the gum dissolved with some d i f f i c u l t y leaving as p r e c i p i t a t e pieces of bark and a small amount of an insoluble resinous material. The solution so produced was extremely viscous, even at f a i r l y low concentration. A ten percent solution of the Peruvian sapote was more viscous than a twenty percent solution of mesquite gum. Anderson gum, on the other hand, was l i g h t e r orange color and translucent rather than transparent. I t appeared to be a much tougher structure with concomitant loss i n 16 b r i t t l e n e s s , breaking under very hard and repeated blows with a hammer with a conchoidal type fracture. V i s c o s i t y of Anderson gum solution was intermediate between that of mesquite gum and of Peruvian sapote gum, while s o l u b i l i t y was found to be much the same as f o r the Peruvian v a r i e t y . P u r i f i c a t i o n of the Gums The gums were p u r i f i e d by dissolving the crude material i n cold water to produce approximately a 1 0 % solution, which was then f i l t e r e d and precipitated into f i v e volumes of 9 5 % ethanol. For further p u r i f i c a t i o n , the process was repeated several times. To obtain the free gum acid, the same procedure was used with the ex-ception that the alcohol i n the f i r s t two p r e c i p i t a t i o n s was made about 0.05N i n sulphuric acid. Several further reprecipitations served to remove excess acid and s a l t s . Preparation of Pure Anderson Sapote Gum Twenty grams of clean crude Anderson sapote gum were allowed to dissolve overnight i n 200 ml. cold water. F i l t e r i n g through a coarse sintered funnel removed a t o t a l of 1.5 gm. of insoluble resinous material and small b i t s of bark. The lemon-colored solution was then taken up i n a 100 ml. pipette and run slowly into S00 ml. of 9 5 % ethanol with vigorous mechanical s t i r r i n g . The resultant white amorphous prec i p i t a t e was centrifuged down and washed twice each v/ith 9 5 % ethanol, absolute ethanol, 17 ether, and petroleum ether ( 3 0 - 6 0°C.}, then dried over calcium chloride and p a r a f f i n under vacuum. Yield, 1 5 . 2 gm. pure once-precipitated Anderson sapote gum as a fi n e dry-white powder. Preparation of Pure Peruvian Sapote Gum Twenty-five grams of crude Peruvian sapote gum was dissolved over night i n 250 ml. cold water. Due to high v i s c o s i t y of the solution, i t could not be f i l t e r e d through sintered glass and was instead passed twice through two layers of cheesecloth. Four or f i v e grams of material, mainly bark, were removed at t h i s point. After r e p r e c i p i -t a t i o n as above into 1 0 0 0 ml. of 9 5 % ethanol, about h a l f of the p r e c i p i t a t e was removed and redissolved to give a 1 0 % solution. The remainder was washed and dried as described above to give once-precipitated Peruvian sapote gum. The dissolved gum was then re p r e c i p i t a t e d into f i v e volumes of 9 5 % ethanol, washed and dried to give twice-precipitated Peruvian sapote gum. Both were obtained as granular white powders. Preparation of Peruvian Sapote Free Gum Acid Thirty grams of crude gum were s t i r r e d f o r several hours with 300 ml. cold water then f i l t e r e d twice through two layers of cheesecloth to remove bark and i n -soluble r e s i n . The clear solution obtained was precipitated as above into 7 5 0 ml. of 9 5 % ethanol containing 1+ ml. of 18 6 N. sulphuric acid. The r e s u l t i n g white amorphous pre-c i p i t a t e was washed twice with 9 5 % ethanol then dissolved i n 200 ml. water and reprecipitated using the same condi-ti o n s . This time the p r e c i p i t a t e was washed twice each with 9 5 % ethanol and absolute ethanol before being re-dissolved and pre c i p i t a t e d into nine volumes of neutral 9 5 % ethanol. Neutral p r e c i p i t a t i o n was repeated three more times, with the f i n a l product being washed twice with 9 5 % ethanol..and three times with absolute ethanol, then dried f i v e days i n a vacuum desiccator over calcium chlor-ide. Yi e l d , 17.5 gm. of pure Peruvian sapote free gum acid as a f i n e light-weight white powder. An a l y t i c a l Data Peruvian Sapote Gum Equivalent Weight: T i t r a t i o n s were run on two samples of pure Peruvian sapote free gum acid using methyl red. The pH at the end-point was checked with a Beckmann Model N pH meter and found to be 6 .0 f o r one sample and 5 .$ f o r the other. Sample No. 1 (19$.6 mg.) was dissolved i n about 10 ml. of d i s t i l l e d water and t i t r a t e d v/ith 0.04643 N. sodium hydroxide. It required 3.9$ ml. which corresponds to 0.1S179 m i l l i e q u i v a l e n t s and gives the gum an equi-valent weight of 1092 gm. 19 Sample No. 2 (212.6 mg.) with the same procedure required 4«17 ml. of the base, giving an equivalent weight value of 1098 gm. The mean value of 1095 has been used f o r a l l calculations. Ash: Once-precipitated gum (615.05 mg.) was i g n i t e d i n a platinum crucible to constant weight, cooling a f t e r each heating i n a vacuum desiccator. Weight of residue was 12.55 mg. giving ash of 2.04%. Rotation: Once-precipitated gum (1.2630 gm.) was dissolved and made up to 25.00 ml. with d i s t i l l e d water. This i s equivalent to a concentration of 5.052 gm./lOO ml. Tube length was one decimeter, and the reading -f- 2.$96° (mean of 6 readings) at 27°C. using a scdLium vapor lamp as source. This gives[a]j~ 7= -.-57.32° Methoxyl: Methoxyl determinations were carr i e d out on the pure gum acid by the standard method of Clark. Values obtained on two samples were 1.94% and 1.9#% methoxyl. Periodate Determination A sample of the pure gum acid (168.05 mg.) was placed i n a 50 ml. volumetric f l a s k and 15.00' ml. of an 20 approximately 0.15 M. KIO^ solution added. A blank was set up with the same amount of periodate and both solutions made up to the 50 ml. mark. After 22 hours, a f i v e ml. aliquot was withdrawn from the sample f l a s k and t i t r a t e d f o r formic acid. It required 2.45 ml. of 0.0114 N. NaOH,ie. .02793 m i l l i e q u i -valents. After subtracting .01535 m i l l i e q u i v a l e n t s f o r the free acid group of the gum, .01263 m i l l i e q u i v a l e n t s remain. This corresponds to .$23 moles formic acid per equivalent of gum. Seven days l a t e r a similar aliquot was withdrawn and t i t r a t e d , requiring 2.90 ml. of the same base. This gives a r a t i o of 1.22 moles formic acid per equivalent of gum. At t h i s l a t e r date also, ten ml. aliquots of both sample and blank were withdrawn and t i t r a t e d f o r periodate remaining. A value of .13966 moles was obtained which corresponds to 4-55 moles periodate per equivalent of gum. For a l l calculations here the value of 1095, as reported above, was used f o r equivalent weight. Anderson Sapote Gum An a l y t i c a l data f o r t h i s gum as obtained by Anderson (1) are l i s t e d i n tables I I and I I I . This work was not repeated as the sample obtained from him was part of the batch on which the determinations were made. 21 Rotation: Once-precipitated gum (0.5043 gm. ) was dissolved and made up to 25 ml. with d i s t i l l e d water. This i s equi-valent to a concentration of 2.0172 gm./lOO ml. Tube length was 2 decimeters and the reading -0.231° (mean of 8 readings) at 25°C. using a sodium vapor lamp as source. This gives [a]^ 5= -5.72° Chromatographic Methods Paper Chromatography Most of the papergrams (paper chromatograms) were run by the descending technique i n a tank 12 i n . i n diameter and 24 i n . i n depth. The tank was f i t t e d with stainless steel supports holding two glass troughs 8 i n . i n length, each capable of holding two paper s t r i p s 18 cm. wide. In practise, s t r i p s 9 x 57 cm. and 18 x 57 cm. were employed. The paper used i n t h i s tank was from Whatman Mo. 1 f i l t e r paper sheets and the chromatograms were run at room temperature. A lesser number of'papergrams were run i n a s i m i l a r but smaller tank i n an oven at temperatures between 30°C. and 50°C. using both Whatman No. 1 and No. 4« In preparing the papers for loading, a l i n e was drawn 9 cm. from one end and marks made at 1 .5 c.m„ i n t e r v a l s 22 across t h i s l i n e . On each side of each of these marks and two m i l l i m e t e r s from i t were placed smaller l i n e s . The l a r g e marks at 1.5 cm. i n t e r v a l s then became the centers of the spots and the smaller marks the boundaries. With the paper marked out, the s o l u t i o n s were taken up i n f i n e c a p i l -l a r i e s formed by drawing out 6 mm. s o f t g l a s s t u b i n g . The t i p of each c a p i l l a r y was then touched i n t u r n to one of the c r # s s - l i n e s and the s o l u t i o n allowed to spread u n t i l i t reached the smaller boundary marks. I f necessary t o b u i l d up a s u f f i c i e n t amount of m a t e r i a l on the spot, the a p p l i c a t i o n was repeated as soon as the f i r s t spot d r i e d . When the f u l l y loaded paper was thoroughly dry, i t was t r a n s f e r r e d to the tank. Some hours p r i o r to t h i s time, the tank had been loaded v/ith solvent and allowed to reach e q u i l i b r i u m . In the case of two-phase s o l v e n t s , the organic phase was placed i n the troughs at the top of the tank and the aqueous phase i n dishes on the bottom. When ready f o r running, the top of the loaded paper was placed i n one of the troughs and h e l d down with a heavy g l a s s rod and the paper allowed to hang down i n t o the tank. The top of the tank was sealed on with stopcock grease, and the solvent permitted to p e r c o l a t e down the paper. Time of development ranged from 16 t o 50 hours depending on the room temperature and the degree of separation d e s i r e d . Good separation was u s u a l l y obtained i n about 21+ hours w i t h the room temperature around 20°C, while the same 23 separation could be obtained i n about 18 hours at 25°C. The papergrams run on shorter papers at 40-50°C. were also found to give an excellent separation of the standard mix-ture of galactose, glucose, arabinose, xylose, and rhamnose i n even less time. The higher temperatures gave more rounded and smaller spots with increased Rf values, thus allowing good separation with shorter solvent flow and con-sequently shorter time. After removal from the tank and complete drying, the papers were sprayed with a suitable reagent and then heated two to f i v e minutes at 100°C. to bring out the colors. The f i r s t few standard papers were sprayed with ammoniacal s i l v e r n i t r a t e , but t h i s was found to give papers which were unstable to l i g h t , soon turning purple. Washing was attempted but found impractical. In addition, t h i s reagent gave spots only with reducing sugars and did not distinguish between classes of sugars at a l l . Aniline hydrogen phthalate was also t r i e d , but could not be made to produce spots of s a t i s f a c t o r y density except with large amounts of sugars. It also tended to react better with pentoses than hexoses and gave poor r e s u l t s with the uronic and biuronic acids and rhamnose. In..an attempt to overcome these objections, a new reagent was c r e a t e d — a n i l i n e hydrogen malonate. This was found to be decidedly superior to any other reagent used fo r the simple sugars, e s p e c i a l l y when l a t e r dissolved i n g l a c i a l acetic acid instead of butanol. It not only appeared 2 4 to react equally with pentoses and hexoses, but also gave a d i s t i n c t greenish color f o r the hexoses and pink f o r the pentoses which served to distinguish them better than the brown and brownish-red obtained with a n i l i n e hydrogen phthalate. Again, however, the reagent was not r e a l l y s a t i s f a c t o r y for the uronic and biuronic acids. Aniline trichloroacetate i n g l a c i a l acetic acid, as reported by Hough, Jones, and Wadman ( 16) , was then found to meet a majority of the requirements f o r t h i s work and was adopted for a l l general purposes. Where no other spray i s s p e c i f i e d i n t h i s report, a n i l i n e t r i c h l o r o a c e t a t e i s implied. I t should be noted, however, that there was some evidence found that t h i s spray w i l l not give as dark a spot with xylose as with the other sugars and on the other hand over-emphasises rhamnose. Where t h i s point arose, duplicate papergrams were run and one was sprayed with the new a n i l i n e hydrogen malonate reagent. This procedure was found of p a r t i c u l a r value where only small amounts of xylose were present. When permanent records of the chromatograms were desired, p r i n t s were made on photographic paper. A large quantity of Kodabromide A5 paper was obtained i n r o l l s from a war-surplus supply company. These r o l l s were four inches 'Air Photo Supply Corp., 555 E. Tremont Avenue, New York 5 7 , New York. 25 wide; the exact width desired for printing the nine c e n t i -meter papergrams. A s t r i p of the photographic paper was stretched over the curved surface of a large f i b r e drum of the type used i n shipping chemicals so that the desired chromatogram could then be placed over i t and the two pinned to the drum i n close contact. Exposure was made with an u l t r a v i o l e t lamp, u t i l i z i n g the absorption of u l t r a v i o l e t c h a r a c t e r i s t i c of a l l the spots. Even the uronic lactone spots, d i f f i c u l t to see i n v i s i b l e but fluorescent i n u l t r a v i o l e t l i g h t , showed up well i n the p r i n t s . After exposure, the s t r i p s were processed as usual f o r photographic p r i n t s . P a r t i t i o n Column Chromatography Considerable d i f f i c u l t y was experienced at f i r s t i n attempting to set up c e l l u l o s e columns f o r separation of sugars i n quantity. Attempts to pack the columns dry as recommended by Hough, Jones and Wadman (15), met with l i t t l e success as dye mixtures passed through the column showed severe d i s t o r t i o n of the zones due to unequal density of the packing. Packing i n a s l u r r y of the solvent, n-butanol saturated with water, was then t r i e d but f a i l e d due to tendency of the c e l l u l o s e powders to f l o a t i n t h i s r e l a -t i v e l y dense medium. This suggested the use of a l i g h t e r solvent soluble i n both water and n-butanol, and on i n v e s t i -gation, acetone was found to be quite s a t i s f a c t o r y . The c e l l u l o s e powder was then suspended i n the acetone as a slurry and poured into a tube f i t t e d with a sintered disc and stopcock at the bottom. The columns used f o r t h i s work were one and one-half and two inches i n diameter and were normally packed to a depth of about 18 inches. After par-t i a l s e t t l i n g , several volumes of acetone were run through the column, the l a s t two batches being saved and mixed with the butanol/water solvent. This mixture was then run through under about 5 p . s . i . a i r pressure and followed by pure n-butanol saturated with water under 15 p . s . i . a i r pressure. The same procedure was used with other solvents, with the solvent to be used simply substituted at a l l points for butanol/water. Loading of the columns was accomplished by allow-ing the solvent l e v e l to f a l l u n t i l the top of the packing was just wet. The mixture to be separated, dissolved i n water or ethanol, was then c a r e f u l l y added from a pipette i n such a way that the speed of addition just very s l i g h t l y exceeded the absorption by the column and thus a small amount of solution was kept above the packing. With the sample a l l added, the l i q u i d l e v e l was again allowed to f a l l just to the' top of the column f i l l i n g and some of.the developing solvent added c a r e f u l l y on the top. A constant head apparatus (Fig. 4) was then attached, and a steadyflow of solvent set up. 2 7 F i g . 4 . Cellulose column with constant head apparatus f o r separation of sugars. 2 0 Fractions were co l l e c t e d i n regular test tubes using an automatic time-controlled c o l l e c t i n g device. The mechanism used was custom-built for the department by the National Research Council and could be set to c o l l e c t any number of fracti o n s at a time i n t e r v a l of one to 4 0 0 minutes. Paper chromatography was used to determine the d i s t r i b u t i o n of sugars i n the eluate f r a c t i o n s . F i r s t , as a rough in d i c a t i o n and to save time, solution from every f i f t h tube was spotted on f i l t e r paper and sprayed with a n i l i n e t r i c h l o r a c e t a t e and i n some cases a n i l i n e hydrogen malonate to show the presence of sugars and to d i f f e r e n t i a t e between rhamnose, pentoses, and hexoses. Those close to a boundary or where more than one pentose could be involved were subjected to chromatographic analysis. I f necessary, each tube over a c r i t i c a l range was tested i n t h i s way to show composition. Two d i f f e r e n t types of c e l l u l o s e powder were tested for use i n the columns. At the beginning of t h i s work, no granular type c e l l u l o s e powder was available commercially i n a pure form. There was at hand, however, a considerable quantity of Griswald and Leo F i l t e r - B r i t e , a c e l l u l o s e f i l t e r - a i d i n granular powder form having a l i g h t tan color. This was tested on a small column 1 1 / 2 inches i n diameter packed dry to a depth of about 1 2 inches. The f i r s t few hundred m i l l i l i t e r s of butanol/water passed through the column came out a l i g h t yellow color, but a f t e r t h i s extraction no further color-throw or other undesirable features were noted. The column was found to separate galactose and arabinose completely, and arabinose and xylose almost completely. At t h i s point, however, samples of the new Whatman "Cellulose Powder for Chromatography" were obtained and found to give s i m i l a r r e s u l t s on the small test column. Because of the high p u r i t y of t h i s produce, no extraction was necessary and on t h i s basis i t was adopted f o r use i n the large columns. Column Separation of Standard Sugar Mixture A mixture of about 50 mg. each of rhamnose, arabinose, and galactose was dissolved i n 5 mis. of water and applied to the 2 inch diameter column as described .above. Approximately 20 ml. f r a c t i o n s were co l l e c t e d every 10 minutes f o r a t o t a l of 100 f r a c t i o n s . Analysis of the tubes on paper showed rhamnose i n tubes 21 to 26 i n c l u s i v e , arabinose i n 44 to 63 , and galactose from 68 to 98. A l l other tubes were e s s e n t i a l l y free from sugar. No attempt was made to recover the sugars from t h i s run. 3© • Graded Hydrolysis of the Gums  Peruvian Gum with 0 . 4 N. Acid A sample of once-precipitated gum was hydrolyzed with 0 . 4 N. acid and the reaction followed by r o t a t i o n and by chromatographic analysis of time samples. Once-precipitated Peruvian gum (5 .0 gm.) was d i s -solved i n 92 mi's, water and eight mis. of 6 N sulphuric acid added. This procedure gives a 5 % solution of gum . 4 N i n sulphuric acid. The solu t i o n was then heated on a steam bath and samples taken at i n t e r v a l s f o r analysis. As may be seen from f i g . 5, the rotation was e s s e n t i a l l y constant a f t e r about 12 hours, at a value .of L « ] Q 7 = +77 to 7 3 ° . At the end of 19 hours, the solution was heated to r e f l u x and • maintained there f o r 12 hours while the rotation rose to a 25 o value of [ a ] ^ •= ±92 . One complication i n t h i s procedure was the factor of mutarotation due to the solution s t i l l being acid while waiting to be read. The l a s t reading, f o r instance, changed from +92° to +106° over a period of twelve hours at. room temperature. On following the reaction with paper chromatography, i t was found that arabinose began to be s p l i t o f f within the f i r s t h a l f hour whereas the galactose did not begin to appear at a l l under 2 1/2 hours. Xylose could not be detected i n any of the f r a c t i o n s even afte r the r e f l u x i n g period. Ko sugars were found i n the o r i g i n a l gum solution and the 951 55 L 1 1 1 1— ; 1 1 -i ;—i 1 1 1 r 0 5 10 15 20 25 30 TIME (Hours) Fig. 3 . Rotation of 5 % Peruvian Sapote Gum Solution on Hydrolysis with 0 .5 N Sulphuric Acid. rhamnose f r a c t i o n was not encountered i n any of the hydroly-sates. A curious fact was noted i n the uronic and b i -uronic section of the papergrams run on these hydrolysates using the butanol/acetic acid/water solvent. Portions of the same tes t sample were taken and run side by side with one of the portions having previously been neutralized with IR - 4 5 and the other s t i l l a c i d i c . I t was found that while the slower of the two prominent spots obtained with both por-tions ran the same distance, the fa s t e r spot moved farther i n the ac i d i c portion, and a lactone spot appeared between •xylose and rhamnose. It i s probable that t h i s f a s t e r spot i s a free uronic acid while the slower spot corresponds to a b i - or polyuronic acid which i s l e s s influenced by change i n pH. Anderson Gum with 0.8 N. Acid Clean crude Anderson sapote gum ( 2 . 0 gm) was d i s -solved i n 15 ml. cold water and 3 ml. 6 N. sulphuric acid added. This gave a solution of 1 0 % gum i n 0.8 N. (approx. 4%) sulphuric acid. The solution was heated i n a hot water bath at $0°C. for two hours, then cooled and f i l t e r e d through a medium sintered funnel. The clear lemon-colored solution was then run slowly i n a f i n e stream into 10 volumes of 9 5 % and absolute ethanol. A sample of the hydrolysate taken before p r e c i p i -t a t i o n was examined on paper chromatograms and found to 33 contain c h i e f l y arabinose and a l i t t l e xylose. In the uronic portion of the papergrams, i t showed three d i s t i n c t spots, a l l red-pink on spraying with a n i l i n e t r i c h l o r o -acetate. A solution of the washed residual polysaccharide even at high concentration showed no moving spots whatever on the papergrams, either f o r sugars or uronics. The residual polysaccharide was dissolved i n 7 . 5 ml. water and 1.4 ml. of 6 N. sulphuric acid was added, again giving a solution of about 1 0 % polysaccharide i n 0.8 N ( 4 % ) sulphuric acid. This solution was refluxed fo r two hours without any apparent deposit of decomposition products and, after cooling, was examined chromatographi-c a l l y without further treatment. Only xylose could be found on the papergrams, along with the same three uronic. spots as had been detected af t e r the f i r s t stage of the hydrolysis. Peruvian Gum with 0.8 N. Acid * Clean crude Peruvian sapote gum (2.0 gm.) W a s treated in'exactly the same manner at the same time as the Anderson gum reported above. The hydrolyzate obtained from the f i r s t heating was clear but an orange color rather than yellow. On p r e c i p i t a t i o n into alcohol, the Peruvian hydro-lyzate yielded about twice as much residual material as did the Anderson gum. On chromatographic examination of the hydrolysate 3 4 before alcohol p r e c i p i t a t i o n , arabinose and a small amount of xylose were the only sugars found, along with four uronic type spots. The three f a s t e r uronic spots were found to have exactly the same values as those obtained with the Anderson gum but on spraying with a n i l i n e trichloroacetate they gave the olive-brown color usually associated with hexoses. The fourth, and slowest, spot also showed t h i s color. On the further hydrolysis of the washed residual polysaccharide, galactose only was obtained. The three f a s t e r uronic spots were also found to occur, but the slow spot was completely missing. • Further Hydrolysis; Separation and  I d e n t i f i c a t i o n of Products Anderson Sapote Gum One hundred grams clean crude Anderson sapote gum were broken with d i f f i c u l t y and dissolved i n 1200 mis of 2N sulphuric acid with vigorous s t i r r i n g . On heating, a brownish colored l i g h t flocculent p r e c i p i t a t e formed, probably due to p r e c i p i t a t i o n of calcium and ma'ghesium sulphates. The solution was refluxed 16 hours, neutralized with barium carbonate, f i l t e r e d through Darco and kieselguhr, and evaporated to 50 mis. under'reduced pressure. The barium s a l t s of the residual uronics and polyuronides were removed by adding the. evaporated solution to 450 mis. absolute 35 ethanol. After centrifuging out the barium s a l t s , the remaining sugar solution was evaporated to 10-15 mis. of medium syrup. Examination of the syrup on paper chromatograms showed the presence of arabinose and xylose only. No rham-nose, galactose, or uronides could be found on any of the papers. Mesquite Gum Twenty grams of clean crude mesquite gum were dissolved i n cold water and f i v e ml. of 6 N. sulphuric acid added, giving a solution 1 0 % in. gum and about 0.1 N. i n sulphuric acid. After heating i n steam f o r 22 hours, the solution was poured into 1000 ml. 9 5 % ethanol to p r e c i p i -tate the r e s i d u a l polysaccharide, f i l t e r e d throughldeselguhr, and neutralized with Amberlite I R 4 B r e s i n . The solution was then evaporated under reduced pressure to 250ml., added to an equal volume of 9 5 % ethanol, and again f i l t e r e d through kieselguhr. At t h i s point the solution was found to be a c i d i c and was again neutralized with IR4-B r e s i n before evaporating to about 75 ml.' Five m i l l i l i t e r s of t h i s sugar solution were then placed on the top of the two-inch column and f r a c t i o n s c o l l e c t e d every 10 minutes at a flow-rate of just over one ml. per minute. At tube No. 63, the time was changed to every 18 minutes to permit running overnight ort the f o r t y -36 f r a c t i o n table then i n use. Analysis of the f r a c t i o n s showed arabinose i n tubes 50 to 71, a very f a i n t trace of galactose and arabinose i n tube 72, and galactose only from 73 on to 105. Contents of tubes 74-103 were combined and evapo-rated under reduced pressure i n a warm water bath, at about 40°C. u n t i l s o l i d material began to separate out around the edges of. the l i q u i d . On cooling and standing, clumps of cr y s t a l s formed which were f i l t e r e d off-, washed with absolute ethanol, and dried i n a vacuum desiccator. A second crop was obtained by returning the mother l i q u o r to the f l a s k , adding a l i t t l e water to help carry o f f the butano1, and reducing the volume to the same condition as before. The crystals gave a melting point l67°C. (a) and mixed melting point with authentic D-galactose of l 6 7°C(d). Behavior on papergrams was also i d e n t i c a l with that of standard D-galactose. Tubes 50-71 were treated i n the same manner with the exception that on evaporation they formed a cloudy solution which c r y s t a l l i z e d almost immediately even i n the hot (40°C.) solution. Melting point and mixed melting point of l60°C. as well as chromatographic behavior con-firmed L-arabinose. Peruvian Sapote Gum Hydrolysis A Twenty grams of clean l i g h t e r pieces of crude Peruvian sapote gum were dissolved i n 200 ml. water, f i l t e r e d 37 through two layers of cheesecloth to remove a small amount of resin,' and a c i d i f i e d with 17 ml. of 6 N sulphuric acid. This produced a solution 1 0 % gum and 0.4 N i n sulphuric acid. After 10 hours on a steam bath, the solution was f i l t e r e d through a medium sintered funnel and neutralized with IR4B r e s i n . The diluted sugar syrup was then passed through Darco and kieselguhr to remove most of color, and evaporated to about 500 ml. This solution was added to about 800 ml. 9 5 % ethanol and the f l o c c u l e n t residual polysaccharide f i l t e r e d o f f with kieselguhr. Further evapo-ra t i o n then reduced the volume of the alc o h o l i c solution to about 50 ml. Paper chromatograms showed the presence of galactose, arabinose, and.xylo.se, with xylose .apparently present only i n small amount. A component of about the speed of rhamnose was also indicated. Column Run D Four ml, of the above hydrolysate were separated on a 1 1/2 inch column as with the.hydrolyzed mesquite outlined previously. Fractions were again cut at 10 minute i n t e r v a l s at a flow rate of just over one ml. per minute u n t i l sugars appeared, and then at f i v e minute i n t e r v a l s u n t i l the l a s t expected sugar zone was l a r g e l y through. A t o t a l of 192 tubes was used with the fas t unknown component found i n tubes 4 5 - 5 5 , pentoses i n 70-116, and hexose in. 158-172. Further checks on the pentose f r a c t i o n showed xylose only i n 70-79, a mixture of xylose and arabinose i n 3 $ 80-87, and arabinose only i n $ £ - 1 1 6 . Tubes 4 5 - 5 5 were combined and evaporated to about f i v e ml. but on standing showed no cry s t a l s or p r e c i p i t a t e . The material i n solution behaved the same on papergrams as did an authentic sample of rhamnose both as to Rf values and the very d i s t i n c t i v e yellow spot produced with a l l the a n i l i n e s a l t sprays on hand. Tubes 70-7$ were evaporated u n t i l small f o g - l i k e c r y s t a l s began to appear on the sides of the f l a s k . On cooling and standing, crystals were obtained with a melting point and mixed m.p. with D-xylose of 144«5°C. and the material behaved the same chromatdgraphically as an authentic sample of D-xylose. Evaporation and c r y s t a l l i z a t i o n of tubes 90-110 produced small crystals.having melting point and mixed m.p. with L^arabinose of 160°C. The s p e c i f i c r o t a t i o n , [a]p^= - s o + 102.6 , and chromatographic behavior also helped confirm i d e n t i f i c a t i o n as L-arabinose. Evaporation o f tubes 160-170 gave small block-shaped crystals with a brownish tinge p e r s i s t i n g even aft e r f i l t r a t i o n through Darco and kieselguhr. Comparison with D-galactose on paper chromatograms indicated the two were the same and on oxidation with n i t r i c acid an insoluble material was produced with melting-point of 213-214°C., the same as reported f o r mucic acid. Complete proof was not attempted with t h i s apparently impure material, however. 39 • Peruvian Sapote Gum Hydrolysis B Sixty grams of clean crtude Peruvian sapote gum were dissolved i n 500 mi. cold water and 100 ml. 6 N. sulphuric acid added. This produces a solution 1 0 % gum and about 0.8 N. i n sulphuric acid. A t u r b i d i t y was noted i n the solution on . addition of the acid, probably due to p r e c i p i t a t i o n of the sulphates of the cations making up the gum s a l t . The solu-t i o n was refluxed 17 hours i n an attempt to obtain a com-plete breakdown of the gum. Paper chromatography showed that even t h i s drastic treatment had not been successful since some biuronic acids s t i l l remained. The hydrolysate was f i l t e r e d through a coarse sintered funnel to remove de-composed material, then neutralized with IR - 4 5 . (This r e s i n was recommended i n preference to IR4B by Rohm and Haas be-cause of greater s t a b i l i t y to acid conditions and complete absence of color-throw. It was found to be eminently s a t i s -factory for the purpose at hand). The deep red neutral syrup was then f i l t e r e d twice through Darco and kieselguhr, coming out a l i g h t yellow color. After evaporation to 180 ml., the mixture was poured into an equal volume of 9 5 % ethanol, f i l t e r e d to remove the flocculent p r e c i p i t a t e , and again evaporated under vacuum to about 50 ml. of a medium syrup. Paper chromatographic analysis indicated the presence of the following: 40 a. , a component of same speed and reactions as rhamnose. b. xylose c. arabinose d. Galactose e. f,g, and h. a series of four spots correspond-ing to uronics and biuronics, i n d i c a t i n g that the treatment with IR-45 and' alcohol p r e c i p i -t a t i o n had not removed most of the uronic components- of the hydrolysate. Column Runs F to J A series of f i v e runs was made on the 1 1 / 2 inch column i n much the same manner as run D v/ith the s p e c i f i c purpose i n mind of obtaining a reasonable quantity of the two components of unproven i d e n t i t y believed to be rhamnose and galactose. Two of the runs had to be abandonned, one due to the supply of solvent being interrupted with con-sequent drying of the column, and the other due to too di l u t e a solution being added to the column and r e s u l t i n g i n water-phase band t r a v e l l i n g r i g h t through the column. The f r a c t i o n s from the other three runs were combined so as to give three solutions containing rhamnose (?), pentose, and hexose. A t o t a l of about 350 ml. of solution showing tes t f o r rhamnose on papergrams was evaporated under reduced pressure to dryness. Only a small amount of residue was found, i n the form of very small c r y s t a l s on the bottom of. u the f l a s k . Chromatographic analysis showed the material to behave exactly the same as rhamnose, but there was not s u f f i c i e n t present to allow derivatives or rotations. It. was confirmed at t h i s time that rhamnose gives a strong color with the sprays used even though present only i n very small amounts. The pentose f r a c t i o n was not treated further at the time as the components had been i d e n t i f i e d from column run D. L Approximately 800 ml. of the hexose f r a c t i o n was evaporated under reduced pressure i n a hot water bath at 50°C. u n t i l the solution just began to fog. On standing, small, clear, c o l o r l e s s , l e a f - l i k e c r y s t a l s were found on the bottom of the f l a s k and were then f i l t e r e d o f f , washed with absolute ethanol, and dried thoroughly i n a vacuum desiccator. Melting point and mixed M.P. with D-galactose o 25 was 167 C. with rotation [ a ] D - +79.8 deg. On oxidation with n i t r i c acid, an insoluble acid was obtained with melting point and mixed m.p. with authentic mucic acid of 213-214°C. X-ray d i f f r a c t i o n pictures of these c r y s t a l s and of pure D-galactose were i d e n t i c a l . Column Run K Separation of the same hydrolysis syrup as above was achieved using a freshly-repacked 1 1/2 inch diameter column with iso-propyl alcohol/water (9:1) as solvent instead 42 of n-butanol saturated with water as previously. Separation was not quite as good but the material came out i n a much smaller volume of solvent and thus required less evaporation and other handling. Approximately 15 ml. fra c t i o n s were cut every 10 minutes for a t o t a l of 70 tubes. Rhamnose indications were found i n tubes 2 0 - 2 6 , with 27 and 28 mixed. Pentose's ran from 2 9 - 4 2 with 43-47 mixed and galactose i n 1+8-65. The rhamnose f r a c t i o n was evaporated to dryness but v/ith much the same res u l t s as before. C r y s t a l l i n e xylose, arabinose, and galactose were obtained from the tubei;showing only these components on papergrams. Galactose, as an example, was obtained by evaporating the solution to the f i r s t appearance of fog then adding an equal volume of pure n-butanol. On standing, the usual colorless plates were obtained. 43 DISCUSSION Ana l y t i c a l data, outlined below, provide the f i r s t evidence that the two samples are e s s e n t i a l l y different'gums., The differences i n values obtained f o r equivalent weight, meth'oxyl, and rotation are f a r beyond the small variations normally found i n di f f e r e n t samples of the same gum, Peruvian gum . Anderson gum Equivalent weight 1095 679 . '• Methoxyl %* . " 1 . 9 6 2 . 8 6 Ash % * 2 . 0 4 2 . 0 5 Rotation [ a ] ^ 5 +57 .32° - 5 . 7 2 ° . 'Ash determinations and rotations are f o r once-precipitated gum; other data are f o r pure gum acids. Hydrolysis of the Peruvian sample1 with 0 . 4 N. acid caused an increase i n rotation of the solution with posit i v e mutarotation i n d i c a t i n g breakage of B-glycosidic linkages. On papergrams, only arabinose and galactose could be found even a f t e r refluxing for some hours. • These, res u l t s could- not be reconciled with those of Anderson who found h i s sample of the gum contained only xylose and arabinose with no galactose at a l l . P a r a l l e l graded hydrolyses of the Anderson, and sapote gums with 0 . 8 N. ( 4 % ) sulphuric acid showed large differences In the behaviour and components of the two 4 4 samples. After two hours at-80°C, the Anderson gum was found by paper chromatography to have s p l i t o f f a l l i t s arabinose and some xylose. Further treatment at higher temperature produced only xylose. This confirmed the r e s u l t s Anderson obtained by f r a c t i o n a l c r y s t a l l i z a t i o n of the . hydrolysis syrups. On the other hand, while the.Peruvian gum aft e r two hours at 80°C. was also, found to have s p l i t o f f arabinose plus a . l i t t l e xylose, further treatment at higher temperatures, produced only galactose. . From the sizes of the spots on papergrams,-it was apparent that the galac-tose was present i n considerable amounts while the xylose was only a minor constituent. In the uronic portion of the papergrams, four spots were found f o r Peruvian gum with the faster three exactly p a r a l l e l e d by spots i n the Anderson •hydrolysate. Difference i n spray color suggests that while the structures of, these b i - or'" polyuronics may be the same, the sugars involved with the uronic acid or acids are d i f -ferent. Probably i n the Anderson gum xylose i s present while i n the Peruvian gum galactose takes i t s ' p l a c e . On extensive hydrolysis, the Peruvian gum was shown to y i e l d about .equal amounts (as determined from size of papergram spots) of galactose and arabinose with .less than h a l f that amount of xylose. When crude samples were hydrolyzed, they showed i n addition trace amounts of rhamnose. Anderson gum under the same conditions produced only arabinose and xylose, with no rhamnose indicated even i n the crude gum. hydrolysates. With the exception of the small amount 45 of rhamnose i n the Peruvian gum, the same re s u l t s were noted on hydrolysis of crude and of p u r i f i e d gums. Presence of two uronic acids and/or of two methods of uronic acid linkage i s strongly indicated f o r the Peruvian gum. Spots i n the uronic portion of papergrams of hydro-lyzed Peruvian gum were found to occur i n what appeared to be related p a i r s . Each pair maintained i t s own r e l a t i v e separation under varying conditions which changed the distances moved by di f f e r e n t pairs. Comparison with mesquite gum showed one pink spot i n common, corresponding to a free monomethyl uronic acid. Two yellow spots i n the mesquite hydrolysate were also matched i n the Peruvian syrup. One of these i s believed to be a biuronic acid consisting of one D-galactose and one 4-methoxy-D-glucuronic acid unit. The other spot.corresponds i n a l l p r o b a b i l i t y to a t r i - or tetrauronic acid. There were found, however, several addi-t i o n a l spots i n the Peruvian hydrolysate, of which one would appear to correspond to a free uronic acid having no methoxyl substitution. It produced a yellow spot of correct Rf or RQ value f o r D-glucuronic acid. Sugars indicated by paper chromatography of various hydrolysates were separated on c e l l u l o s e columns and. i d e n t i f i e beyond doubt by physical constants and derivatives. With the exception of the galactose from one run, the sugars were obtained i n a r e l a t i v e l y pure form d i r e c t l y from the column. In almost a l l cases only one r e c r y s t a l l i z a t i o n was required to produce pure material giving accurate physical constants. 46 The polysaccharide gums, including sapote gum, are used commercially as-a size f o r cloth, as glues, and •for a number of purposes i n the pharmaceutical industry. While the exact nature of the gum i s not too important for some of the uses, i t i s of v i t a l concern i n others. Since the two samples of what i s sold as sapote gum have been shown to d i f f e r so widely as to constitute i n r e a l i t y two di f f e r e n t gums, the necessity for- a n a l y t i c a l checks on gum samples used commercially i s apparent. By f a r the simplest, fastest and least expensive routine check on samples of t h i s type would be by standard hydrolysis and examination on. paper chromatograms. Even i n gums that contain the same basic•sugar components, the uronic portion of papergrams run in-butanol/acetic acid/water or a sim i l a r solvent provides a "finger-print region" c h a r a c t e r i s t i c of the gum. In any case, i t i s very probable that, i f the ,chromatographic - patterns were i d e n t i c a l , differences betwee] .the gums would be s l i g h t enough to permit use of both f o r the same purpose. •. While the. terms "Anderson sapote gum" and "Peru-vian sapote gum" have been used throughout t h i s report to prevenp confusion, they would now appear to serve no otheh r e a l 'purpose. Since the gums have been-shown to be com-pl e t e l y d i f f e r e n t , i t i s most u n l i k e l y that they are ob-tained from the same species of tree. Although Anderson-47 reports h i s gum as obtained from Sapotaceae achras, the o r i g i n of t h i s information i s i n doubt since Asher, Kates and Co. have no record of the source of either of the samples and had thought them both to have been obtained from the same botanical species. In any case, there are at least two sub-species^ S. achras zapota and.S. achras  c h i c l e , both of which produce ch i c l e latex and could be sources also of these gums. The question of sources and proper nomenclature f o r both gums w i l l only be cleared up by examination of samples of known o r i g i n . An i n v e s t i g a t i o n of the methylated gums i s being ca r r i e d out by J. L. Snyder i n order to obtain information with regard.to t h e i r structures. This work was begun on the Peruvian sample before the gums were found to be d i f -ferent and i s now proceeding on both gums. In summary, two samples of gum were obtained, both l a b e l l e d as sapote gum. On examination the material from Dr. Anderson was found to contain arabinose, xylose, and glucuronic acids only. On hydrolysis with 0.8 N (4%) sulphuric acid, i t l o s t a l l i t s arabinose i n two hours at S0°C. and thereafter gave only xylose. The sample obtained direct from Asher, Kates and Co. was found to contain galactose, arabinose, xylose, and glucuronic acids when pure and a trace amount of rhamnose when i n the crude form. On similar hydrolysis, i t l o s t a l l arabinose and xylose v 48 in. two hours and then gave up only galactose. With 0.4 N1. acid, xylose could not be found i n the hydrolysate even after r e f l u x i n g . Equivalent weights, methoxyls, and rotations on the two gums d i f f e r e d widely. Methods of p a r t i t i o n chromatography were estab-' l i s h e d and used i n determining and separating on a macro scale the components of the. various hydrolysates. BIBLIOGRAPHY 1. ANDERSON, E. and-LEDBETTER, H.. D. J.Am.Pharm.Assoc. • 40:623. 1951. 2. ANDERSON, E. and SANDS, L. J.Am.Chem.-Soc. 48:3172. 1926. 3. - ARBELAEZ, E. P. Plantas U t i l e s de Colombia, Contra-l o r i a General de l a Republica Colombia. Bogota. 1947. 4. BROWN, F., HIRST, E. L., HOUGH, L., JONES, J. K. N. and WADMAN, W. H. Nature 161:720. 1948. 5. CHARGAFF, E.,' LEVINE, C. and. GREEN, C. J.Biol.Chem. 175:67. 1948. 6. CONNELL,- J. J., HAINSWORTH, R.. M., HIRST, E. L. and JONES, J. K. N. J.Chem.Soc. 1950:1696. 7. CONSDEN, R., GORDON, A. H. and MARTIN, A. J. P. Bioch.J. 3 8 : 2 2 4 . 1944. 8. CUNEEN, J. I. and SMITH, F. J.Chem.Soc. 1948:1141. 9. FLOOD, A., HIRST, E. and JONES, J. K. N. Nature 160:86. 1947. J.Chem.Soc. 1948:1679. 10. HIRST, E. L. J.Chem.Soc. 1949:522. 11. HIRST, E. L., HOUGH, L..and JONES, J. K. N. J.Chem. Soc. 1949:928. . 12. HIRST, E. L., HOUGH, L. and JONES, J. K. N. Nature-163:177. 1949. 13. HIRST, E. L. and JONES, J. K. N. J..Chem.Soc. 1949:1659. 14. HORROCKS, R. H. Nature 164:444. 1949. 15. HOUGH, L., Jones, J. K. N. and WADMAN, W. H. Nature 162:443. 1948. J.Chem.Soc. 1949:2511. 16. HOUGHS L., JONES, J. K. N., and WADMAN, W. H. J.Chem. Soc. 1950:1702. 17. JONES, J . K. N., and HIRST, E. L. Discuss. Faraday Soc. 7:271. 1949. 18. JONES,' J. K. N . , HOUGHy E. L . and WADMAN, W. H. J . . Chem. Soc. 1950:704.' 19. MARTIN, A. J. P. and SYNGE, R. L . M. Bioch.J. 3 5 : 9 1 . 1941. 20. PARTRIDGE, S. M. Nature 158:270. 1946. Bioch.J. 42:538 1948. 21. S0M0GYI,"I. J.Biol.Chem. l 6 0 : 6 l . 1945. 22. SMITH, F. J.Chem.Soc. 1951:2646. 23. WHITE, E. V. J.Am.Chem.Soc. 6$:272. 1946. 24. WHITE, E. V. J.Am.Chem.Soc. 69:622,715,2264. 1947. 25. WHITE, E. V. J.Am.Chem.Soc. 70:367. 194$. 26. WHITE, E. V. J.Am.Chem.Soc. 75:257- 1953. 27". WHITE, E. V. Personal Communication. TABLE I.' COMPONENTS OF SOME WATER-SOLUBLE GUMS Gum Almond Anogeissus Arabic Cherry Cholla Damson Egg plum Grapefruit Khaya Lemon Mesquite Myrrh Orange Purple plum o • r l o C •ri o CP CU C CO CO O o o U - H 4-5 CO a 3 O o o o o CO •H O Ctf cd ctj • a3 c o .O r H r H c H cd r H cd CC cd >. to bO bfl B !«! < 1 1 l 1 1 1 a o « Q h-5 + + + + + * + * + + + + + + + + + + + + + + + + + + + + + + + CD CO o I 1 . Most of the data i n t h i s table are taken from Reference * Me.thoxy-derivative, TABLE I I * Analysis of Unpurified and P u r i f i e d Sapote Gum and Gum A c i d a Unpurified Gum Gum P u r i f i e d Once Gum Acid Moisture, % 12,13 8.68 6.30 Ash, % 4.27 2.05 .06 Carbon dioxide, % 5.92 6.44 6.27 Uronic acid, °/o 25.84 28.11 27.51 Pentosan, °/o 64.7 66.5 67.9 Methoxyl, % 2.59 2.41 2.86 CHg calculated from methoxyl, /o 1.17 1.09 1.30 Total, % 91.71 95.70 96.71 Equivalent weight 684. 653. 679. Pentose units per uronic acid 3.64 3.44 3.61 Methoxyl unit per uronic acid 0.622 0.531 0.65 'The re s u l t s have been corrected f o r moisture and ash. The equivalent weights are calculated from the uronic acid. Table taken from Reference 1. TABLE I I I * Analysis of Barium Salts from Hydrolysis ct of Sapote Gum Barium, % Pentosan, % Carbon Dioxide, % Uronic acid, °/o Methoxyl, % CH 2 calculated from methoxyl, /o Total, % Equivalent weight Pentose units per uronic acid Methoxyl units per uronic acid Salts from Gum Heated for 2 Hr. at 80° 13.14 55.5 8 .02 35-02 Salts from Salts from Gum Heated Gum Heated 566. 2.31 f o r 4 Hr. at 80° 14.20 52.6 8 .34 36.42 3.56 1.60 104.82 553. 2 .1 0.61 for 28 Hr. i n B o i l i n g Water Bath 23.43 26.1 11.75 51.54 4.67 2.3il 103.18 385. 0 .74 O.564 " i n a l l cases a 4 % solution of sulphuric acid was used f o r hydrolysis. Table taken from Reference 1. TABLE IV Color Reactions with a Number of Spray Reagents Spray Aldo- Uronic Methyl Methyl Methyl hexose Pentose aeid Ketose hexose pentose uronic AgN03/NH3 A n i l i n e H Phthalate A n i l i n e Trichloraeetate brown to black i n a l l cases brn. red brn. none red-brn. cherry to maroon red crimson A n i l i n e H Malonate ^ n ? " r e d yellow-bra. crimson p-Anisidine HCI green-brn. emerald green cherry red lemon yellow brn. red a-Naphthylamine t r i - , chloracetate. green yellow brn. to red green Orcinol or resor-c i n o l with HCI none none red none none Diphenylamine tr i c h l o r a e e t a t e brown purple purple(arab) grey(xyl) Dimethylaniline t r i c h l o r a e e t a t e free C, purple purple(arab) brown(xyl) ,TABLE V. 1 • R Q VALUES2 FOR A NUMBER OF SUGARS AND' THEIR METHYL DERIVATIVES Solvent: n-Butanol/ Ethanol/ Water, 5:1:4 Substance RN value Raffinose . O.D01 Lactose 0.016 Maltose 0.021 Sucrose 0.03 •Turanose 0.060 Galactose 0.070 Glucose 0.090 Sorbose 0.3i0 Mannose, heptulose 0.11 Fructose 0 12 Gulose, Arabinose, Tagatose . * Xylose 0 . 1 5 4-methyl galactose 0.16 Altrose 0.17 Idose, 6-methyl galactose O.lS Talose, Lyxose . . " 0.19 Ribose, Fucose 0.21 2- methyl glucose 0.22 2Smethyl galactose 0 . 2 3 Riboketose, Apiose, 2-deoxy-galactose 0 . 2 5 3- methyl glucose 0 26 Xyloketose 6-methyl glucose 0 . 2 7 Quinovose 0.2S Rhamnose 0 30 a-methyl mannoside 3:4-dimethyl galactose 4 - methyl mannose 0.32 B-methyl arabinoside TABLE V. (cont.) Substance RN value 2-deoxy al l o s e 0.33 2-methyl {3-methyl al t r o s i d e 0 .34 Rhamnoketose, 3:6-anhydro-glucose 0 .37 2-methyl xylose, 2-methyl arabinose 0 .38 • 2:J+-dimethyl galactose 0 .41 4:6-dimethyl galactose 0 .42 2-deoxy ribose, 2 :6-dimethyl-galactose 0 .44 4:6-dimethyl glucose O.46 2- methyl fucose Q ^ 3:6-dimethyl glucose 3:4-dimethyl glucose Q ^ 4:6-dimethyl altrose 2:3-dimethyl mannose 0 .54 2:3-dimethyl glucose 4-methyl rhamnose 0.57 4:6-dimethyl mannose 3:4-dimethyl mannose 0.58 3 - methyl quinovose 0 .60 3:4-dimethyl fructose 0 ^ 2-deoxy rhamnose 2:3-dimethyl arabinose Q 2 : 3 : 4-trimethyl galactose 2:4-dimethyl xylose 0.66 2 : 4 : 6-trimethyl galactose O.67 2 : 3 : 6-trimethyl galactose 0.71 2:3-dimethyl xylose 0 .74 2 : 4 : 6-trimethyl glucose 0.76 3 : 4 : 6-trimethyl mannose 0.79 2 : 3 : 6-trimethyl glucose Q ^ 1 : 3 : 4-trimethyl fructose TABLE V. (cont.) Substance Rn value 3:4-dimethyl rhamnose 0.84 2 :3 :4-trimethyl glucose 0.85 3 :4 :6-trimethyl fructose 0.86 Oleandrose, 2 :3:4:6-tetramethyl galactose 0.88 Tetramethyl fructopyranose 0.90 2 :3 :4-trimethyl xylose 0.94 2 :3 :5-trimethyl arabinose 0.95 2:3:4:6-tetramethyl mannose 0.96 2:3:4:6-tetramethyl glucose 1.00 2:3:5:6-tetramethyl glucose 2:3 :4-trimethyl rhamnose 1.01 1:3:4t6-tetramethyl fructose APPENDIX A A bibliography of the more in t e r e s t i n g and useful papers r e l a t i n g to chromatography of the carbohydrates. 1. B e l l , D. J . J.Chem.Soc. 1944:473. Separation of methyl glucoses by p a r t i t i o n on columns of moist s i l i c a . 2. B e l l , D. J. and Palmer, A. J.Chem.Soc. 1949:2522. Quantitative analysis of mixtures of methylated fructoses using p a r t i t i o n chromatography. 3. Blass, J ., Macheboeuf, M. and Nunez, G. B u l l . Soc. Chim.Biol. 32:130. 1950. Colorimetric determination of aldoses with a n i l i n e hydrogen phthalate. 4. Boissonnas, R. A. Experientia 3:23S. 1947. H e l v i t i c a Chemica Acta 30:l6#9. 1947. Separation of methylated glucoses as p-azobenzoyl derivatives on alumina columns. 5. Brown, F., H i r s t , E. L., Hough, L., Jones, J. K. N., and Wadman, W. H. Nature 161:720. 1948. Separation of methyl sugars on paper chromatograms. 6. Bryson, J . L. and M i t c h e l l , T. J . Nature 167:$64. 1951. Improved spraying reagents f o r paper chromatograms. 7. Burma, D. P. and Banerjee, B. Science and Culture 15:363. 1950. The r o l e of c e l l u l o s e i n f i l t e r -paper chromatography. 8. Campbell, W. G., Frahn, J . L., Hi r s t , S. L., Packman, D. F., and Perc i v a l , E. G. V. J.Chem.Soc. 1951: 34#9. Separation of products of hydrolysis of methylated wood starch. 9. Cramer, F. Angew.Chem. 62:73. 1950. Review of Paper Chromatography of the Sugars. 10. Dekker, C. A. and Long, A. G. J.Chem.Soc. 1950:3162. Determination of glycosides and non-reducing carbohydrates on papergrams with periodate. 11. Flood, A. E., Hi r s t , E. L., Jones, J . K. N. Nature loO:S6. 1947. Separation and quantitative estimation of sugars using paper chromatography. 12. Flood, A. E., Hirs t , E. L. and Jones, J . K. N. J.Chem.Soc. 1948:1679. Quantitative analysis of mixtures of sugars by p a r t i t i o n chromatography. 13. Forsyth, W. G. G. Nature 161:239. 1948. Use of resorcinol and napharesorcinol as spray reagents for reducing and non-reducing sugars. 14. Georges, L. W. and Bower, R. S. J.A.C.S. 68:2169. 194o. Separation of sugars and derivatives on Silene EF columns. 15. Gustafsson, C., Sundman, J. and Lindh, T. Paper and Timber 3 3 : 1 . 1951. Photometric determination of sugars on papergrams of pulp hydrolysates. 16. Haskins, J. F. and Hogsed, M. J. J.Org.Chem. 15:1275. 1950. Chromatography of acetylated carbohydrate acids. 17. Hirst, E. L., Hough, L. and Jones, J. K. N. Nature 163:177. 1949. Separation of polysaccharide hydrolysis products on powdered cellulose columns. 18. Hirst, E. L., Hough, L. and Jones, J. K. N. J.Chem. Soc. 1949:928 Separation of 62 sugars and methyl derivatives on paper, chromatograms using butanol/ ethanol/water solvent. 19. Hirst, E. L. and Jones, J. K. N. J.Chem.Soc. 1949: 1659. Oxidation with periodate as a method for quantitative determinations of sugars. 20. Horrocks, R. H. Nature 164:444. 1949. Use of benzidine as a spray for reducing sugars. 21. Hough, L., Jones, J. K. N. and Wadman, W. H. J.Chem. Soc. 1949, 2511. Separation of sugars and methyl derivatives on cellulose columns. 22. Isherwood, F. A. and Jermyn, M. A. Biochem.J. 48:515« 1951. Relationship between structure of the simple sugars and their behavior on the paper chromatogram. 23. Jeanes, A., Wise, C. S. and Dimler, R. J. Anal.Chem. 23:415. 1951. Improved techniques in paper chromatography of carbohydrates. 24. Jermyn, M. A. and Isherwood, F. A. Bioch.J. 44:402. 1949. Filter-paper chromatography of the sugars. 25. Jones, J. K. N. J.Chem.Soc. 1944:333. Separation of methylated methyl glycosides by adsorption on alumina. 26. Jorgensen, P. F. Dansk.Tids,Farm. 2 4 : 1 . 1950. CA. 44:2893. Separation of phenylosazones on calcium carbonate. 27. Kowkabany, G. N. and Cassidy, H.. G. Anal.Chem. 22:817. 1950. A comparison of f i l t e r papers f o r chromatography. 28. McFarren, E. F., Brand, K. and Rutkowski, H. R. Anal.Chem. 23:1146. 1951. Determination of sugars on f i l t e r - p a p e r chromatograms by di r e c t photometry. 29. Montreuil, P. Bull.Soc.Chim.Biol. 31:1639. 1949. Determination of reducing sugars with potassium fer r i c y a n i d e . 30. Mulvany, P. K., Agar, H. D., Peniston, Q. P. and McCarthy, J . L. J.Am.Chem.Soc. 73:1255. 1951. A chromatographic study of s u l f i t e wate liquor sugars. 31. Nordal, A. and Kleustrand, R. Acta.Chem.Scand. 5 : 8 5 . CA. 45:5771. Paper chromatography of free sugars of some cressulaceous plants. 32. Novellie, L. Nature 166:745. 1950. A spray f o r both ketoses and aldoses -3-napthylamine with f e r r i c sulphate. 33. Pacsu, E., Mora, T. P. and Kent, P. W. Science 110:446. 1949. Permanganate spray f o r sugars. 34. Partridge, S. M. Nature 164:443. 1949. Use of ani l i n e hydrogen phthalate as spray reagent f o r aldoses. 35. Porter, W. L. and Fenske, C. S. J r . , J.Assoc,Agr. Chemists 32:714* 1949. Determination of sugars separated on paper chromatograms by s p e c i f i c fermentation. 36. Rao, P. S. and Beri, R. M. Current S c i . (India) 20:99 C. A. 45:7471. Analysis of sugars with ascending paper chromatography. 37. Whistler, R. L. and Durso, D. F. J.Am.Chem.Soc. 72:677. 1950. Separation of the sugars on charcoal columns. 38. Wise, L. E., Green, J . W. and Rittenhouse, R. C. Tappi 32:335. 1949. Qualitative separation of hydrolysis products of wood substances, pulps, and additives. 39. Wise, L. E., Rittenhouse, R. C. and Garcia, C. Tappi 34:15. 1951. Paper chromatography of pulp hydrolysates. A P P E N D I X B Tracings of representative papergrams showing t y p i c a l separations of standard sugar mixtures and gum hydrolysates. S t a r t ing l i n e S e paration of standard sugar mixtures on a paper chromatogram run 44 hours at room temperature using n-butanol/ethanol/water 5:1:4 as s o l v e n 1 0 Before l i 10 15 heat hours 3 t d 0 hours hours Arabinose Rhamnose H y d r o l y s i s of Peruvian sapote gum with 0.4 N„ a c i d at 80°C o The centre standard i s Peruvian gum h y d r o l y s i s B syrup. Chroma togram run 18 hours at 40°C 0 using n - b u t a n o l / a c e t i c acid/water 5:1:4. 

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