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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 t h e s i s as conforming  to.the  standard r e q u i r e d from c a n d i d a t e ^ f o r the degree of MASTER OF SCIENCE  Members o f the Department o f Chemistry THE  UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1953  ABSTRACT  Two Sapotaceae  samples o f gum  supposedly o b t a i n e d from  achras and named "sapote gum"  the methods o f p a r t i t i o n chromatography. were proven t o be e n t i r e l y d i f f e r e n t c o n s t i t u t e i n f a c t two  were s t u d i e d u s i n g The two  samples  i n composition and t o  separate and d i s t i n c t gums.  The p r e v i o u s l y u n r e p o r t e d gum  was  characterized  and shown t o c o n t a i n D-xylose, L-arabinose, D-galactose, one or more g l u c u r o n i c a c i d s , i n c l u d i n g some  and  methoxy-glucuronic  acid. A new  spray reagent was  developed f o r paper  chroma-  tography of the sugars, and a n o v e l technique used f o r making permanent photographic p r i n t s o f the papergrams.  Crystalline  sugars were o b t a i n e d from h y d r o l y s a t e s i n pure form by s e p a r a t i o n on p a r t i t i o n columns o f powdered  cellulose.  ACKNOWLEDGEMENT  The author would l i k e a t t h i s time t o acknowledge h i s indebtedness t o P r o f e s s o r G. G. S. Dutton f o r the  a s s i s t a n c e and guidance so f r e e l y g i v e n d u r i n g t h e  course of t h i s work. Acknowledgement i s a l s o g r a t e f u l l y made o f the s c h o l a r s h i p p r o v i d e d by the Powell R i v e r Co. L t d . and summer r e s e a r c h funds s u p p l i e d by the N a t i o n a l Research Council.  I n a d d i t i o n , thanks are due t o Dr. E. Anderson  of U n i v e r s i t y o f A r i z o n a ; Asher, Kates and Co.; Rohm and Haas; Reeve Angel and Co.; and t h e F o r e s t Products L a b o r a t o r i e s a t Vancouver f o r v a r i o u s samples p r o v i d e d for this  project.  TABLE OF CONTENTS  Page INTRODUCTION  1  HISTORICAL  5 15  EXPERIMENTAL P u r i f i c a t i o n o f t h e Gums  16  Anderson Sapote Gum  16  Peruvian Sapote Gum  17  Peruvian Sapote Free Gum A c i d  17 18  A n a l y t i c a l Data Peruvian Sapote Gum  18"  Anderson Sapote Gum  20  Chromatographic Methods  21  Paper Chromatography  21  Partition  25  Column Chromatography  S e p a r a t i o n o f Standard sugar Mixture Graded H y d r o l y s i s o f the Gums  29 30  Peruvian Gum w i t h 0 . 4 N. A c i d  30  Anderson Gum w i t h 0.8" N. A c i d  32  Peruvian Gum With 0.8 N. A c i d  33  F u r t h e r H y d r o l y s i s ; S e p a r a t i o n and Identification  o f Products  34  Anderson Sapote Gum  34  Mesquite Gum  35  Peruvian Sapote Gum H y d r o l y s i s A  36  Column Run D  37  Peruvian Sapote Gum H y d r o l y s i s B Column Runs F t o J Column Run K DISCUSSION BIBLIOGRAPHY TABLES APPENDIX A - B i b l i o g r a p h y of r e f e r e n c e s t o chromatography o f the sugars. APPENDIX B - Diagrams showing t y p i c a l s e p a r a t i o n s obtained on paper chromatograms.  FIGURES  Page 1.  Outline of Possible mechanism for formation of pentoses from hexoses.  2.  Structure of mesquite gum according to E. V. Whitel  3.  6  Cellulose column with constant head apparatus for separation of the sugars.  5.  5  One proposed structure for mesquite gum according to F. Smith.  l+.  2  27  Rotation of 5 % Peruvian sapote gum solution on hydrolysis with 0 . 5 N. sulphuric acid.  31  \  TABLES  I. II. III.  Components of Some Water-soluble Gums. Analysis of Anderson Sapote Gum. Analysis of Barium Salts from Hydrolysis of Anderson Sapote Gum.  IV.  Color Reactions with a Number of Spray Reagents.  V.  R Values for a Number of Sugars and Their G  Methyl Derivatives.  STUDIES IN THE POLYSACCHARIDE GUMS WITH SPECIAL REFERENCE TO SAPOTE GUM  •  by  GORDON LESLIE KILGOUR  INTRODUCTION .The w a t e r - s o l u b l e  p o l y s a c c h a r i d e gums occupy a  s i n g u l a r l y i n t e r e s t i n g p o s i t i o n among carbohydrate  compounds.  They are made up o f r e l a t i v e l y s m a l l r e p e a t i n g u n i t s cont a i n i n g from 5 t o 10 molecules o f u r o n i c a c i d s , hexoses and pentoses.  In r e c e n t years., an i n c r e a s i n g amount o f a t t e n t i o n  has been p a i d t o t h e pentoses as t h e i r wide occurence i n nature  i s discovered  (10) and-to the u r o n i c a c i d s both be-  cause o f t h e i r r o l e i n p h y s i o l o g i c a l d e t o x i f i c a t i o n and because o f t h e i r suspected  r o l e i n pentose p r o d u c t i o n .  o r i g i n o f the pentoses i n nature i n t e n s e i n t e r e s t and s p e c u l a t i o n .  The  has long been a s u b j e c t o f One o f the most prominent  hypotheses i n v o l v e s the o x i d a t i o n of a hexose t o i t s c o r responding  u r o n i c a c i d f o l l o w e d by d e c a r b o x y l a t i o n t o the  pentose as shown i n f i g . 1.  2 CHO  CHO ! HCOH  CHO  !  i  HCOH  I  I  ->  HOC  I  HCOH  I  HOCH  I  HOCH ! HCOH CH 0H  HCOH ! HCOH  HCOH I HCOH CH 0H  2  I  2  COOH D-glucuronic acid  D-glucose  CHO  CHO  CHO  HCOH  HCOH ! HOCH  I  I  ! HCOH  I  I  HOCH  HOCH  I  I  I  HOCH  HOCH ! HCOH  HOCH  !  CH 0H HCOH  D-xylose  I  I  2  D-galactose  COOH D-galacturonic acid Fig. 1  While proof o f t h i s h y p o t h e s i s s t i l l  CH OH 2  L-arabinose awaits f u r t h e r work,  s u f f i c i e n t has a l r e a d y been done on the s t r u c t u r e o f t h e gums t o show t h a t such a s y n t h e s i s c o u l d not operate a t the p o l y s a c c h a r i d e l e v e l .  Any such s e r i e s o f r e a c t i o n s  would r e q u i r e p r e v i o u s degradation t o monosaccharides and l a t e r combination o f t h e p r o d u c t s . The u r o n i c a c i d s , a n d i n p a r t i c u l a r g l u c u r o n i c a c i d , which i s t h e one most commonly found i n , t h e gums,  have r e c e n t l y been found t o have important activities.  physiological  C o n s i d e r a b l e success has been r e p o r t e d i n  t r e a t i n g some types o f a r t h r i t i c a c i d or i t s l a c t o n e .  condition with glucuronic  I t i s c u r i o u s .that p l a n t s would  appear t o use the u r o n i c s f o r much the same purpose, as the gums are formed u s u a l l y i n response t o i n j u r y t o a plant.  The recent d i s c o v e r y ( 2 3 ) ( 2 4 ) ( 2 5 ) ( 6 ) ( 1 ) o f some  gums c o n t a i n i n g methoxy-uronic  a c i d s r a i s e s the q u e s t i o n  of how and why these gums d i f f e r from t h e o t h e r s i n having  one h y d r o x y l methylated.  I t i s hoped t h a t d i s c o v e r y  and i n v e s t i g a t i o n o f more such cases w i l l permit o f some g e n e r a l i z a t i o n s and i n t e r p r e t a t i o n . The w a t e r - s o l u b l e gums a l s o have another f o r the wood chemist.  interest  They p r o v i d e a type compound f o r  many o f the substances encountered i n wood chemistry and at the same time possess an ease o f h a n d l i n g f o r e i g n t o most o f them. . Thus i t i s p o s s i b l e t o work out procedures on the gums f o r l a t e r use on h e m i c e l l u l o s e and l i k e compounds i n wood. The o v e r a l l aim o f t h i s i n v e s t i g a t i o n was thus e s s e n t i a l l y two-fold. growing  I t was i n t e n d e d t o add to t h e  stock o f i n f o r m a t i o n r e g a r d i n g the gums and t h e i r  components, and a t the same time t o e s t a b l i s h methods which c o u l d l a t e r be used f o r i n v e s t i g a t i o n o f wood products In p a r t i c u l a r , i t was planned t o make use o f chromatographic methods o f a n a l y s i s and s e p a r a t i o n .  These methods  4 are o f v e r y recent i n t r o d u c t i o n and had not p r e v i o u s l y been used i n t h i s u n i v e r s i t y . The gum chosen t o c o n s t i t u t e t h e main s u b j e c t of t h i s i n v e s t i g a t i o n was sapote gum, t h e exudate of a Peruvian t r e e (supposedly Sapotaceae c h i c l e latex i s also obtained.  a c r a s ) from which  Anderson  and Ledbetter (1)  r e p o r t e d t h a t t h i s gum c o n t a i n e d o n l y D-glucuronic a c i d s , arabinose, and x y l o s e .  T h i s alone i s unusual as almost  a l l other gums thus f a r r e p o r t e d (Table I) c o n t a i n some hexose.  I n a d d i t i o n , he r e p o r t e d t h e u r o n i c a c i d possessed  one methoxyl group p e r two g l u c u r o n i c a c i d s .  One sample  o f the gum f o r t h e work d e s c r i b e d here was o b t a i n e d from Dr. Anderson,  and another l a r g e sample d i r e c t l y from  Asher, Kates and Co. o f Lima, Peru.  The Asher,  Kates  sample was used e x c l u s i v e l y i n the b e g i n n i n g o f t h e i n v e s t i g a t i o n and i t was not u n t i l some time l a t e r t h a t the two samples were found to d i f f e r w i d e l y .  I t was  planned at t h a t time t o continue work on both samples so f a r as p o s s i b l e and t o determine whether they were t r u l y d i f f e r e n t gums o r j u s t v a r i a n t samples o f sapote gum.  The  evidence thus f a r assembled would i n d i c a t e t h a t the former i s c o r r e c t and t h a t they are indeed d i f f e r e n t i n composition and probably i n source.  5 HISTORICAL As t h i s study was i n t e n d e d t o i n v e s t i g a t e new techniques, i t was considered  advisable  t o use a gum o f  known c o n s t i t u t i o n 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 o f the newer methods.  A l a r g e supply  o f the reasonably w e l l c h a r a c t e r i z e d mesquite gum being r e a d i l y a v a i l a b l e , i t was decided t o 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 p o s s i b l e l a t e r reported The  by White ( 2 3 ) ( 2 4 ) ( 2 5 )  and Smith  (believed  t o form a backbone s t r u c t u r e w i t h a l t e r n a t e branches  o f arabinose and u r o n i c to the 3 - p o s i t i o n s one  (8)(22).  proposed s t r u c t u r e o f White, as shown i n F i g . 2,  i n v o l v e s two D-galactose u n i t s l i n k e d t o g e t h e r 1-6}  structures  acid.  These branches are j o i n e d  o f the g a l a c t o s e s  and c o n s i s t i n t h e  case o f 4-methoxy-D-glucuronic a c i d (metal s a l t ) and  i n the o t h e r case o f f o u r L-arabinose u n i t s j o i n e d to each other and 1-3  t o the g a l a c t o s e  chain.  1-2  6 Smith, however, has shown t h a t the methoxyu r o n i c s are l i n k e d at t h e 4 - and 6 - p o s i t i o n s of the galactose, not  i n d i c a t i n g t h a t t h e s t r u c t u r e o f the gum i s  q u i t e as simple as o r i g i n a l l y proposed by White.  On  the b a s i s of h i s r e s u l t s , Smith has suggested a p o s s i b l e type s t r u c t u r e shown i n F i g . 3«  Fig. 3  7 At t h i s p o i n t we f i n d t h a t c o n s i d e r a b l e  dif-  f e r e n c e s e x i s t between the r e s u l t s o b t a i n e d by the  dif-  f e r e n t i n v e s t i g a t o r s 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  g a l a c t o s e : arabinose: methoxy-uronic o f 4 : 3 : 1 l a t e r changed to 4 : 2 : 1 methylated  gum.  however, now  which White  on the b a s i s of h i s work on  the  Smith's r e s u l t s and proposed s t r u c t u r e ,  require a r a t i o of 9 : 5 : 2 .  These v a r y i n g  r e s u l t s were a l l obtained before the advent o f 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 i n v o l v e d i n the c l a s s i c a l procedures. on the gum COg  Thus Anderson d i d h i s work  u s i n g the standard mucic a c i d , p h l o r o g l u c i n o l ,  type of analyses, some of which even Anderson h i m s e l f  regarded  as s u f f i c i e n t l y i n a c c u r a t e to r e q u i r e e m p i r i c a l  c o r r e c t i o n s depending on the m a t e r i a l being White's method of weighing the methylated 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 b e t t e r as there was  no accurate method of  p u r i t y of a sample of unknown sugar.  analysed.  sugar g l y c o s i d e s not a great  deal  determining  Smith's work f o l l o w e d  c l o s e l y the methods of White. Another p o i n t of d i f f i c u l t y before the d u c t i o n of chromatography was of h y d r o l y s e s . was  The  the determination  introof extent  o n l y method a v a i l a b l e i n most  cases  to t r y to c r y s t a l l i z e the component sugars out o f the  h y d r o l y s a t e q u a n t i t a t i v e l y and t o run the o l d standard analyses on the r e s i d u a l p o l y s a c c h a r i d e s .  T h i s method  8 broke down s e r i o u s l y i f two sugars h y d r o l y s e d a t the same time or i f two r e s i d u a l p o l y s a c c h a r i d e s were formed. was only with t h e r e c e n t advent  o f chromatography o f t h e  sugars t h a t i t became p o s s i b l e t o s e p a r a t e such or  even t o determine  cases.  It  mixtures  t h a t a mixture was present i n some  I t was hoped that by u s i n g mesquite gum as a  " t e s t i n g f i e l d " during the o v e r a l l a t t a c k on sapote gum, some f u r t h e r l i g h t might a l s o be shed upon the question of  the exact s t r u c t u r e o f mesquite. F i r s t i n v e s t i g a t i o n o f sapote gum was c a r r i e d  out by Anderson and L e d b e t t e r (1) u s i n g gum r e p o r t e d t o have been.obtained by Asher,  from Sapotaceae achras and s u p p l i e d  Kates and Co. o f Lima, Peru.  L-arabinose, D-xylose,  They found o n l y  and g l u c u r o n i c a c i d present and  r e p o r t e d a r a t i o o f 8.5 x y l o s e per arabinose and 7 pentoses per u r o n i c .  In a d d i t i o n , they found approximately 0.5  methoxyl per u r o n i c a c i d . of  T h i s i s the f i r s t r e p o r t e d case  a gum with both methylated  and non-methylated u r o n i c  acids. Tables I I and I I I are taken from t h e i r paper and show the a c t u a l a n a l y t i c a l  results.  On h y d r o l y s e s w i t h 4 %  s u l p h u r i c a c i d f o r two  hours at 8 0 ° C , Anderson found, a l l the arabinose was s p l i t off  along with some o f the x y l o s e .  the r e s i d u a l p o l y s a c c h a r i d e produced  Further h y d r o l y s i s of only xylose.  The  r e s i s t a n t r e s i d u a l p o l y s a c c h a r i d e i s o l a t e d a f t e r 28 hours  o at 100°C. was  found to have f o u r u r o n i c a c i d s to t h r e e  pentose u n i t s and s t i l l uronic acids.  approximately one methoxyl  Fermentable  per  two  hexoses were absent from a l l  sugar s o l u t i o n s and g a l a c t o s e i n p a r t i c u l a r was  r u l e d out  since mucic a c i d c o u l d not be o b t a i n e d from e i t h e r the  gum  or the sugar s o l u t i o n s . White ( 2 6 ) ( 2 7 ) , working w i t h a sample of sapote gum  s u p p l i e d by Anderson,  has i n v e s t i g a t e d the products  o f methanolysis of the methylated gum.  He r e p o r t e d the  presence of 2 , 3 , 4 - t r i m e t h y l and 3 , 4 - d i m e t h y l  L-arabinose  along with 2 , 3 , 4 - t r i m e t h y l and 3-methyl D-xylose.  The  presence of 2 , 3 , 4 - t r i m e t h y l and 3 , 4 - d i m e t h y l D-glucuronic a c i d was  a l s o noted.  The methyl g l y c o s i d e s o f these com-  pounds were separated u s i n g a column of Magnesol w i t h m e t h y l - e t h y l ketone/water  as s o l v e n t .  The  column  was  extruded, streaked, and the components cut out f o r e l u t i o n . White also used paper chromatography as a check on the mixtures and pure compounds o b t a i n e d .  The d i s c o v e r y of  2 , 3 , 4 - t r i m e t h y l arabinose c o n s t i t u t e s the f i r s t time t h a t arabinose has been r e p o r t e d i n nature i n the  pyranose  configuration. W i t h i n the l a s t few y e a r s , and  particularly  during the p e r i o d i n which t h i s r e s e a r c h was  being c a r r i e d  on, the uses of chromatography i n carbohydrate chemistry have i n c r e a s e d almost e x p o n e n t i a l l y w i t h time.  Thus o n l y  those papers having a d i r e c t b e a r i n g on the present p r o j e c t  10 w i l l be c o n s i d e r e d at t h i s time. of the more s i g n i f i c a n t  A complete  bibliography  and u s e f u l papers i n t h i s  field  w i l l be found i n Appendix A. Partition  chromatography was  by M a r t i n and Synge (19) i n 1941> silica-gel acids.  f i r s t introduced  using water-saturated  columns f o r the s e p a r a t i o n o f a c e t y l amino  T h i s work was  f o l l o w e d two years l a t e r by the  dis-  covery of Consden, Gordon and M a r t i n (7) t h a t f i l t e r paper c o u l d be used as a support f o r the water phase i n the s e p a r a t i o n , thus c r e a t i n g  "paper p a r t i t i o n  chromatography".  While r e d u c i n g sugars were separated by t h i s method i n 1945  by Chargaff, Levine and Green (5), i t was  the f o l l o w i n g year, when P a r t r i d g e  not u n t i l  (20) separated a l a r g e  number of sugars and sugar d e r i v a t i v e s , that the method was  r e a l l y e s t a b l i s h e d i n the carbohydrate f i e l d .  Hough, H i r s t ,  Brown,  Jones and Wadman (4) soon expanded the  scope  of the method t o i n c l u d e methylated sugars and showed  how  t h i s could be a p p l i e d t o the study of methylated p o l y saccharides.  The work of Flood, H i r s t ,  u s i n g Somogyi's micro copper reagent  and Jones ( 9 ) ,  (21) t o  determine  r e d u c i n g sugars e l u t e d from the separated spots, then e s t a b l i s h e d the u s e f u l n e s s of paper p a r t i t i o n graphy as an a c c u r a t e a n a l y t i c a l t o o l .  chromato-  T h i s was  soon  f o l l o w e d by the e q u i v a l e n t technique f o r a n a l y s i s of a number o f methylated sugar mixtures u s i n g a l k a l i n e o d i t e i n s t e a d of the Somogyi reagent  (11).  gypoi-  Results obtained  11 on methylated waxy maize s t a r c h , glycogen, and were quoted  as examples.  araban  A t h i r d a n a l y t i c a l method  d e s c r i b e d a s h o r t time l a t e r by H i r s t and Jones (13)  in-  v o l v e d the use o f sodium p e r i o d a t e with t i t r a t i o n o f the formic a c i d produced. for  T h i s method was  s a t i s f a c t o r y only  r e d u c i n g sugars and d i d not determine methylated  sugars.  The next l a r g e step forward i n paper p a r t i t i o n chromatography was Wadman (15)  the s e p a r a t i o n by Hough, Jones,  of gram amounts of sugars and t h e i r  d e r i v a t i v e s on columns of powdered c e l l u l o s e .  and  methylated Various  methods had been used p r e v i o u s l y f o r o t h e r types of compounds, u t i l i z i n g very t h i c k sheet of f i l t e r paper or "chromatopiles" i . e . s t a c k s of about 100 papers pressed t o g e t h e r .  round  filter  They had not been found very  convenient, however, and d i d not separate more than s e v e r a l hundred m i l l i g r a m s o f m a t e r i a l . c o u l d be made w i t h each one p i l e was  In a d d i t i o n , only one  run  set-up, s i n c e the sheet or  cut up to o b t a i n the separated p r o d u c t s .  Hough,  Jones and Wadman prepared t h e i r column by g r i n d i n g Whatman A c c e l e r a t o r t a b l e t s through powder dry i n t o the column. have appeared  a s i e v e , then packing the In the l a s t two years t h e r e  on the market a number of c e l l u l o s e powder  p r e p a r a t i o n s s p e c i f i c a l l y made f o r t h i s purpose.  They  a l s o used v a r i o u s s o l v e n t systems on t h e i r columns and r e p o r t e d on the degree o f s e p a r a t i o n of v a r i o u s mixtures obtained with each s o l v e n t .  H i r s t , Hough and Jones  (12)  12  then used t h i s type of column to separate the h y d r o l y s i s products of S t e r c u l i a s e t i g e r a  gum.  With a number o f methods of s e p a r a t i o n now out, a t t e n t i o n t u r n e d t o the problem a l s on the chromatograms.  of d e t e c t i o n of m a t e r i -  P a r t r i d g e 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  g e n e r a l l y accepted  i n s p i t e of i t s disadvantages up t o the end o f 1949. t h i s time P a r t r i d g e (20)  worked  At  found t h a t a n i l i n e hydrogen  p h t h a l a t e would g i v e a c o l o r r e a c t i o n w i t h both r e d u c i n g and non-reducing sugars and at the same time would d i f f e r e n t i a t e by means of c o l o r between the d i f f e r e n t of sugars.  About t h i s time a l s o , Horrocks  (14)  classes  discovered  that b e n z i d i n e could be used t o detect many sugars where other reducing substances i n t e r f e r e d with the nitrate.  silver  A number o f other sprays were found f o r s p e c i f i c  uses, but the next important c o n t r i b u t i o n was Hough, Jones and Wadman (16)  i n 1950  the work of  on improved methods  f o r s e p a r a t i o n and d e t e c t i o n o f the sugars and methylated derivatives.  At t h i s time they put forward a number of  sprays f o r both g e n e r a l d e t e c t i o n and s p e c i f i c t i o n of v a r i o u s sugars.  identifica-  They r e p o r t e d on a number of a c i d i c  aromatic, amine and phenol s o l u t i o n s g i v i n g d i f f e r e n t r e a c t i o n s with the v a r i o u s sugars and t h e i r  color  derivatives.  A n i l i n e t r i c h l o r o a c e t a t e i n g l a c i a l a c e t i c a c i d was mended f o r simple sugars and u r o n i c a c i d s and  recom-  p-anisidine  i n n-butanol f o r mixtures o f simple and methylated  sugars  13 and u r o n i c s .  Table IV shows the c o l o r s produced by  types of sugars with  various  some o f the more u s e f u l sprays.  number o f d i f f e r e n t s o l v e n t systems were a l s o  A  considered  i n t h i s paper, and the f i r s t r e p o r t i s made on r e s u l t s obtained by running room temperature.  chromatograms at 37°D<. i n s t e a d of Advantages claimed were f a s t e r running,  b e t t e r s e p a r a t i o n , and more rounded and d i s c r e t e s p o t s . Many s o l v e n t mixtures have been d e s c r i b e d 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 s e p a r a t i o n s , but few separate One  a l l the d i f f e r e n t types and  will  c l a s s e s of  sugars.  of the best f o r g e n e r a l work with both simple  methylated sugars i s n-butanol:ethanolrwater of 4 : 1 : 5 , with or without 1%  ammonia.  i n the  (17)  and t h e i r Rg v a l u e s l i s t e d .  and  The Rg value i s  obtained by u s i n g the d i s t a n c e t r a v e l l e d by t e t r a m e t h y l glucose  ratio  A l a r g e number of  sugars have been t e s t e d i n t h i s s o l v e n t by Jones Hirst  and  2,3,4?6-  as the r e f e r e n c e p o i n t i n s t e a d of the  solvent f r o n t as f o r Rf.  These values are shown i n Table  V.  T h i s solvent does not, however, separate u r o n i c a c i d s , and f a i l s to move b i u r o n i c s at a l l .  For t h i s purpose an  solvent such as n - b u t a n o l r g l a c i a l a c e t i c : w a t e r  acidic  i s required.  S e v e r a l r a t i o s of t h i s mixture have been r e p o r t e d as useful.  Hough, Jones and Wadman (1$)  P a r t r i d g e (20)  recommended 2:1:1  r e p o r t e d good r e s u l t s with 4 : 1 : 5 .  while The  l a t t e r s o l v e n t produces very much the same Rf o r Rg f o r the sugars as does the s o l v e n t c o n t a i n i n g  values  ethanol  14 referred  to above.  Thus the two  i d e n t i t y of a c i d i c components.  provide a check on Since these  two  the  solvents  are e f f i c i e n t f o r the d e s i r e d s e p a r a t i o n s , e a s i l y and r e l a t i v e l y innocuous, they were used almost  prepared, exclusively  f o r running paper chromatograms i n the work under d i s c u s sion.  For the work on c e l l u l o s e powder columns three  s o l v e n t s were used, a l l of which were recommended by Hough, Jones and Wadman (15) Those used were n-butanol ethanol: w a t e r , ' 4 : 1 : 5 ,  for different  separations.  s a t u r a t e d with water,  n-butanol:  and i s o p r o p a n o l : water, 9 : 1 .  l a s t of the t h r e e i s a one-phase s o l v e n t ; f o r the two,  only the organic phase was  used.  The other  15 EXPERIMENTAL As was has i n v o l v e d two of sapote gum,  e x p l a i n e d i n the i n t r o d u c t i o n , t h i s work separate and d i s t i n c t l y d i f f e r e n t  one from Dr. E. Anderson  samples  and the o t h e r  d i r e c t from Asher, Kates Co. i n Lima, Peru.  For  clarity,  i t has become the p r a c t i s e i n t h i s l a b o r a t o r y to r e f e r to the one as Anderson sapote gum.  sapote gum  and t o the o t h e r as Peruvian  T h i s p r a c t i s e w i l l be f o l l o w e d from here on  to prevent c o n f u s i o n .  I t 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 t h a t t h i s company b e l i e v e d them to be from the same type of t r e e . The Peruvian gum  was  o b t a i n e d as a dark c o l o r e d ,  c l e a r , hard, and very b r i t t l e m a t e r i a l .  I t c o u l d be  broken  i n t o p i e c e s with the f i n g e r s and s h a t t e r e d r e a d i l y on b e i n g s t r u c k l i g h t l y with a hammer.  In c o l d water, the  gum  d i s s o l v e d with some d i f f i c u l t y l e a v i n g as p r e c i p i t a t e p i e c e s o f bark and a s m a l l amount of an i n s o l u b l e r e s i n o u s material.  The s o l u t i o n so produced was  even at f a i r l y low c o n c e n t r a t i o n .  extremely v i s c o u s ,  A ten percent s o l u t i o n  of the Peruvian sapote was more v i s c o u s than a twenty percent s o l u t i o n of mesquite Anderson  gum,  gum.  on the other hand, was  lighter  orange  c o l o r and t r a n s l u c e n t r a t h e r than t r a n s p a r e n t .  I t appeared  t o be a much tougher s t r u c t u r e w i t h concomitant  loss i n  16 b r i t t l e n e s s , b r e a k i n g under very hard and repeated blows w i t h a hammer with a c o n c h o i d a l type f r a c t u r e .  Viscosity  o f Anderson gum s o l u t i o n was i n t e r m e d i a t e between t h a t o f mesquite gum and o f Peruvian sapote gum, w h i l e  solubility  was found t o be much t h e same as f o r t h e Peruvian v a r i e t y . P u r i f i c a t i o n o f t h e Gums The gums were p u r i f i e d by d i s s o l v i n g t h e crude m a t e r i a l i n c o l d water t o produce approximately a 1 0 % s o l u t i o n , which was then f i l t e r e d and p r e c i p i t a t e d f i v e volumes of 9 5 % e t h a n o l .  into  For further p u r i f i c a t i o n ,  the process was repeated s e v e r a l times.  To o b t a i n t h e  f r e e gum a c i d , the same procedure was used with t h e exc e p t i o n t h a t t h e a l c o h o l 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 s u l p h u r i c a c i d .  Several further  r e p r e c i p i t a t i o n s served to remove excess a c i d and s a l t s . P r e p a r a t i o n o f Pure Anderson Sapote Gum Twenty grams o f c l e a n crude Anderson sapote gum were allowed t o d i s s o l v e o v e r n i g h t i n 200 ml. c o l d water. F i l t e r i n g through a coarse s i n t e r e d f u n n e l removed a t o t a l of 1.5  gm. o f i n s o l u b l e r e s i n o u s m a t e r i a l and s m a l l b i t s  of bark.  The lemon-colored  s o l u t i o n was then taken up i n  a 100 ml. p i p e t t e and run s l o w l y i n t o S00 ml. o f 9 5 % ethanol with v i g o r o u s mechanical  stirring.  The r e s u l t a n t  white amorphous p r e c i p i t a t e was c e n t r i f u g e d down and washed twice each v/ith 9 5 % e t h a n o l , a b s o l u t e e t h a n o l ,  17 ether, and petroleum e t h e r ( 3 0 - 6 0 ° C . } , then d r i e d over Yield, 1 5 . 2  calcium c h l o r i d e and p a r a f f i n under vacuum. pure o n c e - p r e c i p i t a t e d Anderson  sapote gum  gm.  as a f i n e dry-  white powder. P r e p a r a t i o n o f Pure Peruvian Sapote  Gum  Twenty-five grams o f crude Peruvian sapote was  d i s s o l v e d over n i g h t i n 2 5 0 ml. c o l d water.  h i g h v i s c o s i t y of the s o l u t i o n , i t could not be through s i n t e r e d g l a s s and was two l a y e r s of c h e e s e c l o t h .  Due t o filtered  i n s t e a d passed twice through  Four or f i v e grams of m a t e r i a l ,  mainly bark, were removed at t h i s p o i n t .  After reprecipi-  t a t i o n as above i n t o 1 0 0 0 ml. o f 9 5 % e t h a n o l , about of the p r e c i p i t a t e was 10%  solution.  gum  half  removed and r e d i s s o l v e d to g i v e a  The remainder was  washed and d r i e d as  d e s c r i b e d above t o g i v e o n c e - p r e c i p i t a t e d Peruvian sapote gum.  The d i s s o l v e d gum  was then r e p r e c i p i t a t e d i n t o  five  volumes o f 9 5 % e t h a n o l , washed and d r i e d to g i v e t w i c e p r e c i p i t a t e d Peruvian sapote gum.  Both were o b t a i n e d as  g r a n u l a r white powders. P r e p a r a t i o n o f P e r u v i a n Sapote Free Gum T h i r t y grams o f crude gum  Acid  were s t i r r e d f o r  s e v e r a l hours w i t h 3 0 0 ml. c o l d water then f i l t e r e d twice through two l a y e r s of c h e e s e c l o t h t o remove bark and i n soluble r e s i n .  The c l e a r s o l u t i o n o b t a i n e d was  precipitated  as above i n t o 7 5 0 ml. o f 9 5 % e t h a n o l c o n t a i n i n g 1+ ml. o f  18 6 N. s u l p h u r i c a c i d . c i p i t a t e was  The r e s u l t i n g white amorphous p r e -  washed twice w i t h 9 5 %  e t h a n o l then  dissolved  i n 200 ml. water and r e p r e c i p i t a t e d u s i n g the same c o n d i tions.  T h i s time the p r e c i p i t a t e was  with 9 5 %  washed twice each  e t h a n o l and absolute ethanol before being r e -  d i s s o l v e d and p r e c i p i t a t e d i n t o nine volumes of n e u t r a l 95%  ethanol.  N e u t r a l p r e c i p i t a t i o n was  repeated t h r e e  more times, with the f i n a l product being washed twice with 95%  ethanol..and t h r e e times with a b s o l u t e e t h a n o l , then  d r i e d f i v e days i n a vacuum d e s i c c a t o r over calcium c h l o r Y i e l d , 17.5  ide.  gm.  of pure Peruvian sapote f r e e  gum  a c i d as a f i n e l i g h t - w e i g h t white powder. A n a l y t i c a l Data Peruvian Sapote  Gum  E q u i v a l e n t Weight: T i t r a t i o n s were run on two Peruvian sapote f r e e gum at the end-point was  samples o f pure  a c i d u s i n g methyl r e d .  The  pH  checked w i t h 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  ( 1 9 $ . 6 mg.)  was  d i s s o l v e d i n about  10 ml. o f 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.  I t r e q u i r e d 3.9$  ml. which  to 0.1S179 m i l l i e q u i v a l e n t s and g i v e s the gum v a l e n t weight of 1092  gm.  corresponds an e q u i -  19 Sample No.  2 (212.6 mg.)  with  the same procedure  r e q u i r e d 4«17 ml. of the base, g i v i n g an e q u i v a l e n t weight v a l u e of 1098  gm.  The mean value o f 1095  has been used f o r a l l  calculations. Ash: O n c e - p r e c i p i t a t e d gum i n a platinum  (615.05 mg.)  12.55  mg.  ignited  c r u c i b l e to constant weight, c o o l i n g a f t e r  each h e a t i n g i n a vacuum d e s i c c a t o r . was  was  Weight of r e s i d u e  g i v i n g ash of 2 . 0 4 % .  Rotation: O n c e - p r e c i p i t a t e d gum  (1.2630 gm.)  was  and made up t o 25.00 ml. with d i s t i l l e d water.  dissolved This i s  e q u i v a l e n t to a c o n c e n t r a t i o n of 5.052 gm./lOO ml. Tube l e n g t h was  one  o f 6 readings)  decimeter,  and the r e a d i n g -f-  2.$96° (mean  at 27°C. u s i n g a scdLium vapor lamp as  source. T h i s gives[a]j~ = 7  -.-57.32°  Methoxyl: Methoxyl determinations pure gum  a c i d by the  o b t a i n e d on two Periodate  were c a r r i e d out on  standard method of C l a r k .  samples were 1.94%  and 1.9#%  the  Values methoxyl.  Determination  A sample of the pure gum  acid  p l a c e d i n a 50 ml. v o l u m e t r i c f l a s k and  (168.05 mg.)  was  15.00' ml. of an  20 approximately 0.15  M.  KIO^  s o l u t i o n added.  A blank was  set  up with the same amount o f p e r i o d a t e and both s o l u t i o n s made up to the 50 ml. mark. A f t e r 22 hours, a f i v e ml. a l i q u o t was  withdrawn  from the sample f l a s k and t i t r a t e d f o r f o r m i c a c i d . r e q u i r e d 2.45 valents.  ml. of 0.0114 N. NaOH,ie. .02793 m i l l i e q u i -  A f t e r s u b t r a c t i n g .01535 m i l l i e q u i v a l e n t s f o r the  f r e e a c i d group of the gum, T h i s corresponds t o .$23 of  It  .01263 m i l l i e q u i v a l e n t s  remain.  moles f o r m i c a c i d per e q u i v a l e n t  gum. Seven days l a t e r a s i m i l a r a l i q u o t was  and t i t r a t e d , r e q u i r i n g 2.90 g i v e s a r a t i o of 1.22  withdrawn  ml. of the same base.  This  moles f o r m i c a c i d per e q u i v a l e n t o f  gum. At t h i s l a t e r date a l s o , t e n ml. a l i q u o t s of both sample and blank were withdrawn and t i t r a t e d f o r p e r i o d a t e remaining.  A value o f .13966 moles was  corresponds to 4-55  obtained which  moles p e r i o d a t e per e q u i v a l e n t of  For a l l c a l c u l a t i o n s here the v a l u e of 1095, r e p o r t e d above, was Anderson Sapote  as  used f o r e q u i v a l e n t weight.  Gum  A n a l y t i c a l data f o r t h i s gum  as o b t a i n e d by  Anderson (1) are l i s t e d i n t a b l e s I I and I I I . was  gum.  T h i s work  not repeated as the sample o b t a i n e d from him was  o f the batch on which the determinations were made.  part  21 Rotation: Once-precipitated gum  (0.5043 gm. ) was d i s s o l v e d  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 ] ^ = -5.72° 5  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. stainless  The tank was f i t t e d with  s t e e l supports holding two g l a s s troughs 8 i n .  i n length, each capable of holding two paper s t r i p s 18 cm. wide.  In p r a c t i s e ,  employed.  s t r i p s 9 x 57 cm. and 18 x 57 cm. were  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 l e s s e r 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 f o r l o a d i n g , 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 s i d e o f each o f t h e s e marks and  two m i l l i m e t e r s from i t were p l a c e d s m a l l e r l i n e s . l a r g e marks at 1.5  cm.  The  i n t e r v a l s t h e n became t h e c e n t e r s o f  t h e s p o t s and t h e s m a l l e r marks t h e b o u n d a r i e s .  With the  paper marked o u t , t h e s o l u t i o n s were t a k e n up i n f i n e l a r i e s formed by drawing out 6 mm.  capil-  soft glass tubing.  The  t i p o f each c a p i l l a r y was t h e n touched i n t u r n t o one o f the c r # s s - l i n e s and t h e s o l u t i o n a l l o w e d t o s p r e a d u n t i l i t r e a c h e d t h e s m a l l e r boundary marks.  I f necessary to  b u i l d up a s u f f i c i e n t amount o f m a t e r i a l on t h e s p o t , t h e a p p l i c a t i o n was r e p e a t e d as soon as t h e f i r s t spot d r i e d . When t h e f u l l y l o a d e d paper was t h o r o u g h l y d r y , i t was t r a n s f e r r e d t o t h e t a n k .  Some hours p r i o r t o t h i s  t i m e , t h e t a n k had been l o a d e d v/ith s o l v e n t and a l l o w e d t o reach e q u i l i b r i u m .  I n t h e case o f two-phase s o l v e n t s , t h e  o r g a n i c phase was p l a c e d i n t h e t r o u g h s a t t h e t o p o f t h e t a n k and the aqueous phase i n d i s h e s on t h e bottom.  When  r e a d y f o r r u n n i n g , t h e t o p o f t h e l o a d e d paper was p l a c e d i n one o f t h e t r o u g h s and h e l d down w i t h a heavy g l a s s r o d and the paper a l l o w e d t o hang down i n t o t h e t a n k . o f t h e t a n k was  The  s e a l e d on w i t h stopcock g r e a s e , and t h e  s o l v e n t p e r m i t t e d t o p e r c o l a t e down t h e paper.  Time o f  development ranged from 16 t o 50 hours depending on t h e room t e m p e r a t u r e and t h e degree o f s e p a r a t i o n d e s i r e d . Good s e p a r a t i o n was u s u a l l y o b t a i n e d i n about 21+ hours w i t h t h e room t e m p e r a t u r e around 20°C, w h i l e t h e same  top  23 s e p a r a t i o n could be o b t a i n e d i n about 18 hours at 25°C. The papergrams run on s h o r t e r papers at 40-50°C. were a l s o found to g i v e an e x c e l l e n t s e p a r a t i o n of the standard mixt u r e o f g a l a c t o s e , glucose, arabinose, x y l o s e , and rhamnose i n even l e s s time.  The h i g h e r temperatures  gave more  rounded and s m a l l e r spots with i n c r e a s e d Rf v a l u e s , thus a l l o w i n g good s e p a r a t i o n with s h o r t e r s o l v e n t f l o w and  con-  sequently s h o r t e r time. A f t e r removal from the tank and complete d r y i n g , the papers were sprayed with a s u i t a b l e reagent  and  then  heated two to f i v e minutes at 100°C. to b r i n g out the c o l o r s . 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 g i v e papers which  were unstable to l i g h t , soon t u r n i n g p u r p l e . attempted  but found i m p r a c t i c a l .  Washing  In a d d i t i o n , t h i s  gave spots only with r e d u c i n g sugars and d i d not between c l a s s e s of sugars at a l l . was  was  reagent  distinguish  A n i l i n e hydrogen p h t h a l a t e  a l s o 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 d e n s i t y except with l a r g e amounts of sugars. I t a l s o tended to r e a c t b e t t e r with pentoses  than hexoses  and gave poor r e s u l t s with the u r o n i c and b i u r o n i c a c i d s and rhamnose. a new was  In..an attempt  reagent was  to overcome these o b j e c t i o n s ,  c r e a t e d — a n i l i n e hydrogen malonate.  found to be d e c i d e d l y s u p e r i o r to any other reagent  This used  f o r the simple sugars, e s p e c i a l l y when l a t e r d i s s o l v e d i n g l a c i a l a c e t i c a c i d i n s t e a d of b u t a n o l .  I t not only appeared  2 4  to r e a c t e q u a l l y w i t h pentoses and hexoses, a d i s t i n c t g r e e n i s h c o l o r f o r the hexoses  but a l s o gave  and pink f o r the  pentoses which served t o d i s t i n g u i s h them b e t t e r than the brown and brownish-red o b t a i n e d w i t h a n i l i n e phthalate.  Again, however, the reagent was  hydrogen not  really  s a t i s f a c t o r y f o r the u r o n i c and b i u r o n i c a c i d s .  Aniline  t r i c h l o r o a c e t a t e i n g l a c i a l a c e t i c a c i d , as r e p o r t e d by Hough, Jones, and Wadman ( 1 6 ) ,  was  then found to meet a  m a j o r i t y of the requirements f o r t h i s work and was f o r a l l g e n e r a l purposes.  adopted  Where no o t h e r 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. should be noted, however, that t h e r e was  It  some evidence  found t h a t t h i s spray w i l l not g i v e as dark a spot with x y l o s e as with the o t h e r sugars and on the other hand overemphasises rhamnose.  Where t h i s p o i n t arose, d u p l i c a t e  papergrams were run and one was hydrogen malonate reagent.  sprayed with the new  T h i s procedure was  aniline  found o f  p a r t i c u l a r v a l u e where o n l y s m a l l amounts of x y l o s e were present. When permanent r e c o r d s of the chromatograms were d e s i r e d , p r i n t s were made on photographic paper. q u a n t i t y of Kodabromide A5 paper was a war-surplus supply company.  'Air Photo Supply Corp., New York.  A large  obtained i n r o l l s  from  These r o l l s were f o u r inches  5 5 5 E. Tremont Avenue, New  York  57,  25 wide; the exact width d e s i r e d f o r p r i n t i n g meter papergrams.  the nine  centi-  A s t r i p of the photographic paper  was  s t r e t c h e d over the curved s u r f a c e of a l a r g e f i b r e drum o f the type used i n s h i p p i n g chemicals so t h a t the d e s i r e d chromatogram c o u l d then be p l a c e d over i t and the two to the drum i n c l o s e c o n t a c t . ultraviolet  pinned  Exposure was made with an  lamp, u t i l i z i n g the a b s o r p t i o n of u l t r a v i o l e t  characteristic  of a l l the s p o t s .  Even the u r o n i c l a c t o n e  spots, d i f f i c u l t to see i n v i s i b l e but f l u o r e s c e n t i n ultraviolet  l i g h t , showed up w e l l i n the p r i n t s .  After  exposure, the s t r i p s were p r o c e s s e d as u s u a l f o r photographic prints. Partition  Column Chromatography Considerable d i f f i c u l t y was  i n attempting to s e t up c e l l u l o s e of sugars i n q u a n t i t y .  experienced at f i r s t  columns f o r s e p a r a t i o n  Attempts to pack the columns dry  as recommended by Hough, Jones and Wadman (15), met little  with  success as dye mixtures passed through the column  showed severe d i s t o r t i o n of the packing.  of the zones due t o unequal  density  Packing i n a s l u r r y of the s o l v e n t , n-  butanol s a t u r a t e d with water, was to tendency of the c e l l u l o s e t i v e l y dense medium.  then t r i e d but f a i l e d  powders to f l o a t i n t h i s  T h i s suggested the use of a  found to be q u i t e s a t i s f a c t o r y .  rela-  lighter  s o l v e n t s o l u b l e i n both water and n-butanol, and on g a t i o n , acetone was  due  investiThe  c e l l u l o s e powder was then suspended  i n t h e acetone as a  s l u r r y and poured i n t o a tube f i t t e d with a s i n t e r e d d i s c and stopcock at the bottom.  The columns used f o r t h i s work  were one and one-half and two i n c h e s i n diameter and were normally packed to a depth o f about 18 i n c h e s . tial  After par-  s e t t l i n g , s e v e r a l volumes o f acetone were run through  the  column, the l a s t two batches being saved and mixed with  the  butanol/water s o l v e n t .  T h i s mixture was then run  through under about 5 p . s . i . a i r p r e s s u r e and f o l l o w e d by pure n-butanol s a t u r a t e d w i t h water under 15 p . s . i . a i r pressure.  The same procedure was used with other s o l v e n t s ,  with the solvent to be used simply s u b s t i t u t e d at a l l p o i n t s for  butanol/water. Loading o f t h e columns was accomplished by a l l o w -  ing  the solvent l e v e l to f a l l u n t i l the t o p o f the packing  was j u s t wet.  The mixture t o be separated, d i s s o l v e d i n  water o r e t h a n o l , was then c a r e f u l l y added from a p i p e t t e in  such a way t h a t t h e speed of a d d i t i o n j u s t very s l i g h t l y  exceeded the a b s o r p t i o n by t h e column and thus a s m a l l amount o f s o l u t i o n was kept above t h e p a c k i n g .  With the  sample a l l added, the l i q u i d l e v e l was again allowed t o fall  j u s t t o the' top o f the column f i l l i n g and some o f . t h e  developing solvent added c a r e f u l l y on the top.  A constant  head apparatus ( F i g . 4) was then a t t a c h e d , and a s t e a d y f l o w of  solvent s e t up.  27  Fig. 4 .  C e l l u l o s e column with constant head apparatus f o r s e p a r a t i o n of sugars.  20 F r a c t i o n s were c o l l e c t e d i n r e g u l a r t e s t tubes u s i n g an automatic t i m e - c o n t r o l l e d c o l l e c t i n g device. mechanism used was  The  custom-built f o r the department by the  N a t i o n a l Research C o u n c i l and c o u l d be s e t to c o l l e c t  any  number of f r a c t i o n s at a time i n t e r v a l o f 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 o f sugars i n the e l u a t e f r a c t i o n s .  First,  as  a rough i n d i c a t i o n and to save time, s o l u t i o n from every f i f t h tube was  s p o t t e d on f i l t e r paper and sprayed w i t h  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. boundary or where  Those c l o s e to a  more than one pentose c o u l d be i n v o l v e d  were subjected to chromatographic a n a l y s i s .  I f necessary,  each tube over a c r i t i c a l range was t e s t e d i n t h i s way t o show composition. Two  d i f f e r e n t types o f c e l l u l o s e powder were  t e s t e d f o r use i n the columns.  At the b e g i n n i n g of t h i s  work, no g r a n u l a r type c e l l u l o s e powder was commercially i n a pure form.  There was  available  at hand, however,  a c o n s i d e r a b l e q u a n t i t y o f Griswald and Leo  Filter-Brite,  a c e l l u l o s e f i l t e r - a i d i n g r a n u l a r powder form having a l i g h t tan c o l o r .  T h i s was t e s t e d on a s m a l l column 1 1 / 2  inches i n diameter packed dry t o a depth o f about 1 2 i n c h e s . The f i r s t  few hundred m i l l i l i t e r s o f butanol/water passed  through the column came out a l i g h t y e l l o w c o l o r , but a f t e r  t h i s e x t r a c t i o n no f u r t h e r c o l o r - t h r o w or o t h e r u n d e s i r a b l e f e a t u r e s were noted.  The column was found t o separate  g a l a c t o s e and arabinose completely, and arabinose and x y l o s e almost completely.  At t h i s p o i n t , however, samples o f t h e  new Whatman " C e l l u l o s e Powder f o r Chromatography" were o b t a i n e d and found t o g i v e s i m i l a r r e s u l t s on t h e s m a l l t e s t column.  Because o f the h i g h p u r i t y o f t h i s  produce,  no e x t r a c t i o n was necessary and on t h i s b a s i s i t was adopted f o r use i n t h e l a r g e columns.  Column S e p a r a t i o n o f Standard Sugar M i x t u r e A mixture o f about 50 mg. each o f rhamnose, arabinose, and g a l a c t o s e was d i s s o l v e d i n 5 mis. o f water and a p p l i e d t o t h e 2 i n c h diameter column as d e s c r i b e d .above.  Approximately 20 ml. f r a c t i o n s were c o l l e c t e d every  10 minutes f o r a t o t a l o f 100 f r a c t i o n s .  Analysis o f the  tubes on paper showed rhamnose i n tubes 21 t o 26 i n c l u s i v e , arabinose i n 44 t o 6 3 , and g a l a c t o s e from 68 t o 9 8 . A l l o t h e r tubes were e s s e n t i a l l y f r e e from sugar. was made t o r e c o v e r t h e sugars from t h i s r u n .  No attempt  3©  • Graded H y d r o l y s i s o f the Gums with 0 . 4  Peruvian Gum  N. A c i d  A sample of o n c e - p r e c i p i t a t e d gum with 0 . 4  was  hydrolyzed  N. a c i d and the r e a c t i o n f o l l o w e d by r o t a t i o n  by chromatographic  a n a l y s i s o f time  and  samples. (5.0  O n c e - p r e c i p i t a t e d Peruvian gum  gm.)  was  dis-  s o l v e d i n 92 mi's, water and e i g h t mis. of 6 N s u l p h u r i c a c i d added.  T h i s procedure  sulphuric acid.  The  gives a 5%  s o l u t i o n was  s o l u t i o n of gum  then heated on a steam  bath and samples taken at i n t e r v a l s f o r a n a l y s i s . be seen from f i g .  5,  the r o t a t i o n was  a f t e r about 12 hours, at a v a l u e .of the end o f 19  .4 N i n  essentially  L«]Q =  hours, the s o l u t i o n was  7  As  may  constant  +77 t o 7 3 ° .  At  heated t o r e f l u x and •  maintained t h e r e f o r 12 hours while the r o t a t i o n rose to a 25  o  value of [ a ] ^ •= ±92 was  .  One  complication i n t h i s  procedure  the f a c t o r o f m u t a r o t a t i o n due to the s o l u t i o n  being a c i d w h i l e w a i t i n g to be read.  still  The l a s t r e a d i n g , f o r  i n s t a n c e , changed from +92° to +106° over a p e r i o d of twelve hours at. room  temperature.  On f o l l o w i n g the r e a c t i o n w i t h paper chromatography, i t was  found t h a t arabinose began to be s p l i t  o f f w i t h i n the  f i r s t h a l f hour whereas the g a l a c t o s e d i d not begin t o appear at a l l under 2 1/2  hours.  Xylose c o u l d not be d e t e c t e d i n  any of the f r a c t i o n s even a f t e r the r e f l u x i n g p e r i o d . sugars were found i n the o r i g i n a l gum  s o l u t i o n and  the  Ko  951  55  L  1  0  1  5  1  1—  10  ;  1  1  -i  ;  —i  15  20  1  1  25  TIME (Hours) Fig. 3 .  R o t a t i o n of 5 % Peruvian Sapote Gum S o l u t i o n on H y d r o l y s i s with 0 . 5 N S u l p h u r i c A c i d .  1  r 30  rhamnose f r a c t i o n was not encountered i n any o f t h e h y d r o l y sates. A c u r i o u s f a c t was noted i n t h e u r o n i c and b i u r o n i c s e c t i o n o f t h e papergrams run on these h y d r o l y s a t e s u s i n g the b u t a n o l / a c e t i c acid/water s o l v e n t .  Portions of  the same t e s t sample were taken and r u n s i d e by s i d e w i t h one o f the p o r t i o n s having p r e v i o u s l y been n e u t r a l i z e d w i t h I R - 4 5 and t h e o t h e r s t i l l  acidic.  the slower o f the two prominent  I t was found t h a t w h i l e  spots o b t a i n e d with both por-  t i o n s r a n t h e same d i s t a n c e , the f a s t e r spot moved f a r t h e r i n the a c i d i c p o r t i o n , and a l a c t o n e spot appeared •xylose and rhamnose.  between  I t i s probable t h a t t h i s f a s t e r spot  i s a f r e e u r o n i c a c i d w h i l e t h e slower spot corresponds t o a b i - or p o l y u r o n i c a c i d which  i s l e s s i n f l u e n c e d by change  i n pH. Anderson  Gum w i t h 0.8 N. A c i d Clean crude Anderson  sapote gum ( 2 . 0 gm) was d i s -  s o l v e d i n 1 5 ml. c o l d water and 3 ml. 6 N. s u l p h u r i c added. 4%)  acid  T h i s gave a s o l u t i o n o f 1 0 % gum i n 0.8 N. (approx.  sulphuric acid.  The s o l u t i o n was heated i n a hot water  bath at $0°C. f o r two hours, then c o o l e d and f i l t e r e d a medium s i n t e r e d f u n n e l .  The c l e a r lemon-colored  through  solution  was then run s l o w l y i n a f i n e stream i n t o 1 0 volumes o f 9 5 % and a b s o l u t e e t h a n o l . A sample o f the h y d r o l y s a t e taken b e f o r e p r e c i p i t a t i o n was examined on paper chromatograms and found t o  33 c o n t a i n c h i e f l y arabinose  and a l i t t l e x y l o s e .  In the  u r o n i c p o r t i o n o f the papergrams, i t showed t h r e e spots, a l l red-pink on s p r a y i n g with a n i l i n e acetate.  A s o l u t i o n of the washed r e s i d u a l  distinct  trichloropolysaccharide  even at h i g h c o n c e n t r a t i o n showed no moving spots whatever on the papergrams, e i t h e r f o r sugars or u r o n i c s . dissolved i n 7 . 5  The r e s i d u a l p o l y s a c c h a r i d e was ml. o f 6 N.  ml. water and 1.4  s u l p h u r i c a c i d was  again g i v i n g a s o l u t i o n of about 1 0 % 0.8  N (4%)  for  two  products  sulphuric acid.  hours without  any apparent d e p o s i t of  a f t e r c o o l i n g , was  c a l l y without  f u r t h e r treatment.  found  polysaccharide i n  T h i s s o l u t i o n was  and,  added,  refluxed decomposition  examined chromatographiOnly x y l o s e c o u l d be  on the papergrams, along with the same t h r e e u r o n i c .  spots as had been d e t e c t e d a f t e r the f i r s t  stage o f the  hydrolysis. Peruvian Gum  with 0.8  Clean  crude Peruvian  t r e a t e d i n ' e x a c t l y the Anderson gum  * sapote gum  (2.0 gm.)  Was  same manner at the same time as the  r e p o r t e d above.  the f i r s t h e a t i n g was yellow.  N. A c i d  The h y d r o l y z a t e o b t a i n e d from  c l e a r but an orange c o l o r r a t h e r than  On p r e c i p i t a t i o n i n t o a l c o h o l , the Peruvian  hydro-  l y z a t e y i e l d e d about twice as much r e s i d u a l m a t e r i a l as d i d the Anderson On  gum. chromatographic examination  o f the h y d r o l y s a t e  34  before a l c o h o l p r e c i p i t a t i o n , arabinose and a small amount of x y l o s e were the o n l y sugars found, type s p o t s .  along with f o u r u r o n i c  The t h r e e f a s t e r u r o n i c spots were found t o  have e x a c t l y t h e same  v a l u e s as those o b t a i n e d with t h e  Anderson gum but on s p r a y i n g with a n i l i n e they gave t h e olive-brown hexoses.  trichloroacetate  c o l o r u s u a l l y a s s o c i a t e d with  The f o u r t h , and slowest, spot a l s o showed t h i s  color. On the f u r t h e r h y d r o l y s i s of the washed r e s i d u a l p o l y s a c c h a r i d e , g a l a c t o s e o n l y was o b t a i n e d .  The t h r e e  f a s t e r u r o n i c spots were a l s o found t o occur, but the slow spot was completely m i s s i n g . • F u r t h e r H y d r o l y s i s ; S e p a r a t i o n and I d e n t i f i c a t i o n o f Products Anderson Sapote  Gum  One hundred grams c l e a n crude Anderson sapote were broken with d i f f i c u l t y  and d i s s o l v e d i n 1200 mis o f 2N  s u l p h u r i c a c i d with v i g o r o u s s t i r r i n g . colored l i g h t  On h e a t i n g , a brownish  f l o c c u l e n t p r e c i p i t a t e formed, p r o b a b l y due  to p r e c i p i t a t i o n o f calcium and ma'ghesium s u l p h a t e s . s o l u t i o n was r e f l u x e d 16 hours, n e u t r a l i z e d w i t h carbonate,  gum  f i l t e r e d through  The  barium  Darco and k i e s e l g u h r , and  evaporated t o 50 mis. under'reduced  pressure.  The barium  s a l t s o f the r e s i d u a l u r o n i c s and p o l y u r o n i d e s were removed by adding the. evaporated  s o l u t i o n t o 450 mis. absolute  35 ethanol.  A f t e r c e n t r i f u g i n g out the barium  remaining sugar s o l u t i o n was  salts,  evaporated t o 10-15  the  mis. o f  medium syrup. Examination o f the syrup on paper  chromatograms  showed the presence o f arabinose and x y l o s e o n l y .  No rham-  nose, g a l a c t o s e , o r u r o n i d e s c o u l d be found on any o f the papers. Mesquite  Gum Twenty grams of c l e a n crude mesquite  d i s s o l v e d i n c o l d water and f i v e ml. o f 6 N. added, g i v i n g a s o l u t i o n 1 0 % sulphuric acid. s o l u t i o n was  i n . gum  gum  were  sulphuric  and about 0.1  N. i n  A f t e r h e a t i n g i n steam f o r 22 hours, ml. 9 5 %  poured i n t o 1000  acid  the  e t h a n o l to p r e c i p i -  t a t e the r e s i d u a l p o l y s a c c h a r i d e , f i l t e r e d t h r o u g h l d e s e l g u h r , and n e u t r a l i z e d with Amberlite was  IR4B  resin.  The  solution  then evaporated under reduced p r e s s u r e t o 250ml., added  to an e q u a l volume o f 9 5 %  e t h a n o l , and  again f i l t e r e d  through k i e s e l g u h r .  At t h i s p o i n t the s o l u t i o n was  to be a c i d i c and was  again n e u t r a l i z e d w i t h IR4-B r e s i n  before e v a p o r a t i n g to about  75  found  ml.'  F i v e m i l l i l i t e r s o f t h i s sugar s o l u t i o n were then p l a c e d on the top of the two-inch  column and  fractions  c o l l e c t e d every 10 minutes at a f l o w - r a t e o f j u s t over ml. per minute.  At tube No.  63,  the time was  one  changed to  every 18 minutes t o permit running o v e r n i g h t ort the  forty-  36 f r a c t i o n t a b l e then i n use.  A n a l y s i s o f the  fractions  showed arabinose i n tubes 50 to 71,  a very f a i n t t r a c e of  g a l a c t o s e and arabinose i n tube 72,  and g a l a c t o s e o n l y  from 73 on to  105.  Contents of tubes 74-103 were combined and evapor a t e d under reduced p r e s s u r e i n a warm water bath, at about 40°C. u n t i l s o l i d m a t e r i a l began to separate out around the edges of. the l i q u i d .  On c o o l i n g and s t a n d i n g , clumps  o f c r y s t a l s formed which were f i l t e r e d off-, washed with a b s o l u t e e t h a n o l , and d r i e d i n a vacuum d e s i c c a t o r . second  crop was  A  obtained by r e t u r n i n g the mother l i q u o r t o  the f l a s k , adding a l i t t l e water to h e l p c a r r y o f f the butano1, and r e d u c i n g the volume to the same c o n d i t i o n as before.  The  c r y s t a l s gave a m e l t i n g p o i n t l 6 7 ° C . (a) and  mixed m e l t i n g p o i n t with a u t h e n t i c D-galactose of Behavior on papergrams was standard  l67°C(d).  a l s o i d e n t i c a l with t h a t of  D-galactose. Tubes 50-71  were t r e a t e d i n the same manner w i t h  the e x c e p t i o n t h a t on e v a p o r a t i o n they formed a cloudy s o l u t i o n which c r y s t a l l i z e d almost hot  (40°C.) s o l u t i o n .  immediately  M e l t i n g p o i n t and mixed m e l t i n g  p o i n t of l 6 0 ° C . as w e l l as chromatographic firmed  even i n the  behavior  con-  L-arabinose.  Peruvian Sapote Gum  Hydrolysis A  Twenty grams of c l e a n l i g h t e r p i e c e s of crude Peruvian sapote gum  were d i s s o l v e d i n 200 ml. water, f i l t e r e d  37 through two  l a y e r s o f c h e e s e c l o t h to remove a s m a l l amount  o f r e s i n , ' and a c i d i f i e d with 17 ml. o f 6 N s u l p h u r i c T h i s produced a s o l u t i o n 1 0 % acid.  gum  and 0.4  acid.  N i n sulphuric  A f t e r 10 hours on a steam bath, the s o l u t i o n  was  f i l t e r e d through a medium s i n t e r e d f u n n e l and n e u t r a l i z e d with IR4B r e s i n .  The d i l u t e d sugar syrup was  then passed  through Darco and k i e s e l g u h r to remove most of c o l o r , evaporated t o about 500 ml. about 800 ml. 9 5 %  T h i s s o l u t i o n was  e t h a n o l and the f l o c c u l e n t  polysaccharide f i l t e r e d o f f w i t h k i e s e l g u h r .  added t o residual  F u r t h e r evapo-  r a t i o n then reduced the volume o f the a l c o h o l i c t o about 50 ml.  and  solution  Paper chromatograms showed the presence  of g a l a c t o s e , a r a b i n o s e , and.xylo.se, with x y l o s e .apparently present o n l y i n s m a l l amount. speed o f rhamnose was Column Run  also  A component o f about the  indicated.  D Four ml, o f the above h y d r o l y s a t e were s e p a r a t e d  on a 1 1/2  i n c h column as w i t h t h e . h y d r o l y z e d mesquite  outlined previously.  F r a c t i o n s were again cut at 10  minute  i n t e r v a l s at a f l o w r a t e of j u s t over one ml. per minute u n t i l sugars appeared,  and then at f i v e minute  u n t i l the l a s t expected sugar zone was t o t a l of 192 tubes was  intervals  l a r g e l y through.  A  used w i t h the f a s t unknown component  found i n tubes 4 5 - 5 5 , pentoses i n 70-116, and hexose i n . 158-172.  F u r t h e r checks on the pentose f r a c t i o n showed  x y l o s e o n l y i n 70-79, a mixture o f x y l o s e and a r a b i n o s e i n  3$ 80-87,  and arabinose only i n Tubes  45-55  $£-116.  were combined and evaporated t o about  f i v e ml. but on standing showed no c r y s t a l s o r p r e c i p i t a t e . The m a t e r i a l i n s o l u t i o n behaved the same on papergrams as did an authentic sample of rhamnose both as t o Rf values and the very d i s t i n c t i v e yellow spot produced with a l l the a n i l i n e s a l t sprays on hand. Tubes 70-7$ were evaporated u n t i l small f o g - l i k e c r y s t a l s began t o appear on the sides of the f l a s k .  On  c o o l i n g and standing, c r y s t a l s were obtained with a melting point and mixed m.p. with D-xylose of 144«5°C. and the m a t e r i a l 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 c r y s t a l s . h a v i n g melting point and mixed m.p. with L^arabinose of 160°C. - so  The s p e c i f i c r o t a t i o n , [a]p^=  + 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 blockshaped c r y s t a l s with a brownish t i n g e p e r s i s t i n g even a f t e r f i l t r a t i o n through Darco and k i e s e l g u h r .  Comparison with  D-galactose on paper chromatograms i n d i c a t e d the two were the same and on o x i d a t i o n with n i t r i c a c i d an i n s o l u b l e m a t e r i a l was produced with melting-point of 213-214°C., the same as reported f o r mucic a c i d .  Complete proof was not  attempted with t h i s apparently impure m a t e r i a l , however.  39 • Peruvian Sapote Gum  Hydrolysis  B  S i x t y grams of c l e a n crtude Peruvian sapote gum d i s s o l v e d i n 500  mi.  c o l d water and  100  ml.  a c i d added.  T h i s produces a s o l u t i o n 1 0 %  i n sulphuric  acid.  a d d i t i o n of the sulphates of the t i o n was  A t u r b i d i t y was  a c i d , probably due  was  some b i u r o n i c  s o l u t i o n on .  salt.  The  not  been The  I t was  found to be  f a c t o r y f o r the purpose at hand).  ml.,  to remove  ethanol,  The  yellow c o l o r .  Haas  be-  complete  eminently  deep red  satis-  neutral kieselguhr,  A f t e r e v a p o r a t i o n to 95%  poured i n t o an equal volume o f  f i l t e r e d to remove the f l o c c u l e n t p r e c i p i t a t e ,  again evaporated under vacuum to about 50 ml.  Paper chromatographic a n a l y s i s i n d i c a t e d following:  and  of a medium  syrup.  presence of the  de-  (This r e s i n  and  then f i l t e r e d twice through Darco and  the mixture was  a com-  hydrolysate  then n e u t r a l i z e d with I R - 4 5 .  s t a b i l i t y to a c i d c o n d i t i o n s  coming out a l i g h t  solu-  successful  recommended i n p r e f e r e n c e to I R 4 B by Rohm and  syrup was  the  Paper chromatography showed  a c i d s s t i l l remained.  absence of color-throw.  about 0.8  to p r e c i p i t a t i o n of  f i l t e r e d through a coarse s i n t e r e d f u n n e l  cause o f g r e a t e r  180  and  r e f l u x e d 17 hours i n an attempt to o b t a i n  composed m a t e r i a l , was  sulphuric  noted i n the  even t h i s d r a s t i c treatment had  since  gum  c a t i o n s making up the gum  p l e t e breakdown o f the gum. that  6 N.  were  the  N.  40 a. , a component of same speed and r e a c t i o n s as rhamnose. b.  xylose  c.  arabinose  d.  Galactose  e. f , g , and h.  a s e r i e s of f o u r spots  correspond-  i n g to u r o n i c s and b i u r o n i c s , i n d i c a t i n g t h a t the treatment  with IR-45 and' a l c o h o l p r e c i p i -  t a t i o n had not removed most of the u r o n i c components- of the  hydrolysate.  Column Runs F t o J A s e r i e s o f f i v e runs was  made on the 1 1 / 2  column i n much the same manner as run D v/ith the purpose i n mind o f o b t a i n i n g a reasonable two  inch  specific  q u a n t i t y of the  components of unproven i d e n t i t y b e l i e v e d to be rhamnose  and g a l a c t o s e .  Two  of the runs had t o be abandonned,  due to the supply of s o l v e n t being i n t e r r u p t e d with  one  con-  sequent d r y i n g o f the column, and the o t h e r due to too d i l u t e a s o l u t i o n being added to the column and  resulting  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 t h r e e runs were combined so as to g i v e three s o l u t i o n s c o n t a i n i n g rhamnose ( ? ) , pentose, and hexose.  A t o t a l o f about 350 ml. o f s o l u t i o n showing  t e s t f o r rhamnose on papergrams was p r e s s u r e t o dryness.  evaporated  under reduced  Only a s m a l l amount of r e s i d u e  was  found, i n the form of very s m a l l c r y s t a l s on the bottom of.  u the f l a s k .  Chromatographic a n a l y s i s showed the m a t e r i a l  to behave e x a c t l y the same as rhamnose, but t h e r e was s u f f i c i e n t present to a l l o w d e r i v a t i v e s o r r o t a t i o n s . was  not It.  confirmed at t h i s time t h a t rhamnose g i v e s a strong  c o l o r with the sprays used even though present only i n very s m a l l amounts. The pentose  f r a c t i o n was  not t r e a t e d f u r t h e r at  the time as the components had been i d e n t i f i e d from run  column  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 s o l u t i o n j u s t began to f o g .  On s t a n d i n g ,  s m a l l , c l e a r , 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 e t h a n o l , and d r i e d thoroughly i n a vacuum desiccator. was  167  o  M e l t i n g p o i n t and mixed M.P.  C. with r o t a t i o n [ a ]  25 D  - +79.8 deg.  with n i t r i c a c i d , an i n s o l u b l e a c i d was m e l t i n g p o i n t and mixed m.p. 213-214°C.  with  D-galactose  On o x i d a t i o n  obtained with  with a u t h e n t i c mucic a c i d of  X-ray d i f f r a c t i o n p i c t u r e s o f these  crystals  and of pure D-galactose were i d e n t i c a l . Column Run  K S e p a r a t i o n of the same h y d r o l y s i s syrup as above  was  achieved u s i n g a f r e s h l y - r e p a c k e d 1 1/2  column with i s o - p r o p y l alcohol/water  (9:1)  inch  diameter  as s o l v e n t i n s t e a d  42 o f n-butanol s a t u r a t e d with water as p r e v i o u s l y .  Separation  was not quite as good but the m a t e r i a l came out i n a much s m a l l e r volume o f s o l v e n t and thus r e q u i r e d l e s s e v a p o r a t i o n and other h a n d l i n g .  Approximately 15 ml. f r a c t i o n s were  cut every 10 minutes f o r a t o t a l o f 70 tubes.  Rhamnose  i n d i c a t i o n s were found i n tubes 2 0 - 2 6 , w i t h 27 and 28 mixed. Pentose's r a n from 2 9 - 4 2 with 43-47 mixed and g a l a c t o s e i n 1+8-65. The rhamnose f r a c t i o n was evaporated t o dryness but v/ith much the same r e s u l t s as b e f o r e . C r y s t a l l i n e x y l o s e , arabinose, and g a l a c t o s e were obtained from the tubei;showing o n l y these components on papergrams.  Galactose, as an example, was o b t a i n e d by  evaporating t h e s o l u t i o n t o the f i r s t  appearance  then adding an equal volume o f pure n-butanol. the u s u a l c o l o r l e s s p l a t e s were o b t a i n e d .  of f o g On standing,  43 DISCUSSION A n a l y t i c a l data, o u t l i n e d below, p r o v i d e the f i r s t evidence t h a t the two samples are e s s e n t i a l l y The  different'gums.,  d i f f e r e n c e s i n v a l u e s o b t a i n e d f o r e q u i v a l e n t weight,  meth'oxyl, and r o t a t i o n are f a r beyond t h e small v a r i a t i o n s normally found i n d i f f e r e n t  samples o f the same gum,  Peruvian gum . Anderson gum 1095  E q u i v a l e n t weight '• Methoxyl %*  679 .  1.96  . "  2.86  2.04  Ash % * Rotation [ a ] ^  5  2.05  +57.32°  -5.72°  . 'Ash determinations and r o t a t i o n s are f o r o n c e - p r e c i p i t a t e d gum; other data are f o r pure gum a c i d s . H y d r o l y s i s o f the Peruvian sample with 0 . 4 N. 1  a c i d caused an i n c r e a s e i n r o t a t i o n o f the s o l u t i o n w i t h p o s i t i v e m u t a r o t a t i o n i n d i c a t i n g breakage o f B - g l y c o s i d i c linkages.  On papergrams, o n l y arabinose and g a l a c t o s e  c o u l d be found even a f t e r r e f l u x i n g f o r some hours. • These, r e s u l t s could- not be r e c o n c i l e d with those o f Anderson who found h i s sample o f the gum contained o n l y x y l o s e and arabinose w i t h no g a l a c t o s e at a l l . P a r a l l e l graded h y d r o l y s e s o f t h e Anderson, and sapote gums with 0 . 8 N. ( 4 % )  s u l p h u r i c a c i d showed l a r g e  d i f f e r e n c e s In the behaviour and components o f the two  44  samples.  A f t e r two  hours a t - 8 0 ° C , the Anderson gum  found by paper chromatography to have s p l i t arabinose and some x y l o s e . temperature  produced  off a l l i t s  F u r t h e r treatment  only xylose.  was  at h i g h e r  T h i s confirmed the r e s u l t s  Anderson o b t a i n e d 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 . h y d r o l y s i s syrups.  On the o t h e r hand, while  hours at 80°C. was  the.Peruvian  gum  a f t e r two  off  arabinose p l u s a . l i t t l e x y l o s e , f u r t h e r treatment  h i g h e r temperatures, produced of  was  split at  o n l y g a l a c t o s e . . From the  the spots on papergrams,-it  tose was  also, found t o have  was  apparent  sizes  t h a t the g a l a c -  present i n c o n s i d e r a b l e amounts w h i l e the x y l o s e  o n l y a minor c o n s t i t u e n t .  In the u r o n i c p o r t i o n of the  papergrams, f o u r spots were found f o r Peruvian gum  with the  f a s t e r t h r e e e x a c t l y p a r a l l e l e d by spots i n the Anderson •hydrolysate.  D i f f e r e n c e i n spray c o l o r suggests t h a t w h i l e  the s t r u c t u r e s of, these b i - or'" p o l y u r o n i c s may  be the same,  the sugars i n v o l v e d with the u r o n i c a c i d or a c i d s are ferent.  Probably i n the Anderson gum  while i n the Peruvian gum On  x y l o s e i s present  g a l a c t o s e takes  its'place.  e x t e n s i v e h y d r o l y s i s , the Peruvian gum  shown t o y i e l d about .equal amounts (as determined of  dif-  was  from  size  papergram spots) of g a l a c t o s e and arabinose with .less  than h a l f t h a t amount of x y l o s e .  When crude samples were  h y d r o l y z e d , they showed i n a d d i t i o n t r a c e amounts of rhamnose. Anderson gum  under the same c o n d i t i o n s produced  o n l y arabinose  and x y l o s e , w i t h no rhamnose i n d i c a t e d even i n the gum. h y d r o l y s a t e s .  crude  With the e x c e p t i o n o f the s m a l l amount  45 o f rhamnose i n the Peruvian gum,  the same r e s u l t s were noted  on h y d r o l y s i s of crude and o f p u r i f i e d gums. Presence  of two  u r o n i c a c i d s and/or of two methods  of u r o n i c a c i d l i n k a g e i s s t r o n g l y i n d i c a t e d f o r the Peruvian gum.  Spots i n the u r o n i c p o r t i o n of papergrams of hydro-  l y z e d Peruvian gum  were found to occur i n what appeared  be r e l a t e d p a i r s .  Each p a i r maintained  i t s own  to  relative  s e p a r a t i o n under v a r y i n g c o n d i t i o n s which changed the d i s t a n c e s moved by d i f f e r e n t p a i r s . gum  Comparison with mesquite  showed one pink spot i n common, corresponding t o a f r e e  monomethyl u r o n i c a c i d .  Two  y e l l o w spots i n the mesquite  h y d r o l y s a t e were a l s o matched i n the Peruvian syrup.  One  of these i s b e l i e v e d to be a b i u r o n i c a c i d c o n s i s t i n g o f one  D - g a l a c t o s e and one  4-methoxy-D-glucuronic  The other spot.corresponds tetrauronic acid.  acid unit.  i n a l l p r o b a b i l i t y to a t r i -  or  There were found, however, s e v e r a l a d d i -  t i o n a l spots i n the Peruvian h y d r o l y s a t e , o f which one would appear t o correspond t o a f r e e u r o n i c a c i d having no methoxyl substitution.  I t produced  a y e l l o w spot of c o r r e c t Rf or  RQ value f o r D - g l u c u r o n i c a c i d . Sugars i n d i c a t e d by paper chromatography of v a r i o u s h y d r o l y s a t e s 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 p h y s i c a l constants and d e r i v a t i v e s .  With  the e x c e p t i o n o f the g a l a c t o s e from one run, the sugars were o b t a i n e d 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 o n l y one r e c r y s t a l l i z a t i o n was r e q u i r e d  to produce pure m a t e r i a l g i v i n g a c c u r a t e p h y s i c a l c o n s t a n t s .  46 The polysaccharide gums, i n c l u d i n g sapote gum, are used commercially a s - a s i z e f o r c l o t h , as g l u e s , and •for a number of purposes i n the pharmaceutical i n d u s t r y . While the exact nature o f the gum i s not too important f o r some o f t h e uses, i t i s of v i t a l concern i n o t h e r s . Since the two samples of what i s s o l d as sapote gum have been shown t o d i f f e r two  so w i d e l y as t o c o n s t i t u t e i n r e a l i t y  d i f f e r e n t gums, the n e c e s s i t y for- a n a l y t i c a l  on gum samples used commercially i s apparent.  checks  By f a r t h e  s i m p l e s t , f a s t e s t and l e a s t expensive r o u t i n e check on samples of t h i s type would be by standard h y d r o l y s i s and examination on. paper chromatograms.  Even i n gums t h a t  c o n t a i n the same b a s i c • s u g a r components, the u r o n i c p o r t i o n of papergrams run i n - b u t a n o l / a c e t i c acid/water or a s i m i l a r solvent provides a "finger-print r e g i o n " c h a r a c t e r i s t i c of the gum.  I n any case, i t i s v e r y probable that, i f t h e  ,chromatographic - p a t t e r n s were i d e n t i c a l , d i f f e r e n c e s betwee] .the gums would be s l i g h t enough t o permit use of both f o r the same purpose.  •.  While the. terms "Anderson  sapote gum" and "Peru-  v i a n sapote gum" have been used throughout t h i s r e p o r t t o prevenp  c o n f u s i o n , they would now appear t o serve no otheh  r e a l 'purpose.  Since the gums have been-shown t o be com-  p l 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 t h a t they are obt a i n e d from the same s p e c i e s of t r e e .  Although  Anderson-  47 r e p o r t s h i s gum  as o b t a i n e d from Sapotaceae achras, the  o r i g i n of t h i s i n f o r m a t i o n i s i n doubt s i n c e Asher, Kates and Co. have no r e c o r d of the source of e i t h e r of the samples and had thought them both to have been o b t a i n e d from the same b o t a n i c a l s p e c i e s . at l e a s t two  In any case, t h e r e are  s u b - s p e c i e s ^ S. achras zapota and.S. achras  c h i c l e , both o f which produce c h i c l e l a t e x and could be sources a l s o of these gums.  The q u e s t i o n 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 b e i n g c a r r i e d out by J . L. Snyder i n order to o b t a i n i n f o r m a t i o n with regard.to t h e i r s t r u c t u r e s .  T h i s work was begun on  the P e r u v i a n sample b e f o r e the gums were found t o be  dif-  f e r e n t and i s now p r o c e e d i n g on both gums. In summary, two  samples of gum were o b t a i n e d ,  both l a b e l l e d as sapote gum.  On examination the m a t e r i a l  from Dr. Anderson was found t o c o n t a i n a r a b i n o s e , x y l o s e , and g l u c u r o n i c a c i d s only.  On h y d r o l y s i s with 0.8  N  (4%)  s u l p h u r i c a c i d , i t l o s t a l l i t s arabinose i n two hours at S0°C. and t h e r e a f t e r gave o n l y x y l o s e . d i r e c t from Asher, Kates and Co. was  The sample o b t a i n e d  found t o c o n t a i n  g a l a c t o s e , a r a b i n o s e , x y l o s e , and g l u c u r o n i c a c i d s when pure and a t r a c e amount of rhamnose when i n the crude form. On s i m i l a r h y d r o l y s i s , i t l o s t a l l arabinose and x y l o s e v  48 i n . two acid,  hours and then gave up o n l y g a l a c t o s e . x y l o s e could not be found  after refluxing. rotations  With 0.4  i n the h y d r o l y s a t e  E q u i v a l e n t weights, methoxyls,  on the two  gums d i f f e r e d  N. 1  even  and  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 scale  and  s e p a r a t i n g on a macro  the components of the. v a r i o u s h y d r o l y s a t e s .  BIBLIOGRAPHY  1.  ANDERSON, E. and-LEDBETTER, H.. D. 4 0 : 6 2 3 . 1951.  J.Am.Pharm.Assoc. •  2.  ANDERSON, E. and SANDS, L. J.Am.Chem.-Soc. 1926.  48:3172.  3. - ARBELAEZ, E. P. P l a n t a s U t i l e s de Colombia, Contral o r i a General de l a R e p u b l i c a Colombia. Bogota. 1 9 4 7 . 4.  BROWN, F., HIRST, E. L., HOUGH, L., JONES, J . K. N. and WADMAN, W. H. Nature 1 6 1 : 7 2 0 . 1 9 4 8 .  5.  CHARGAFF, E.,' LEVINE, 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.  38:224.  C. and. GREEN, C.  J.Biol.Chem.  1944.  8.  CUNEEN, J . I . and SMITH, F. J.Chem.Soc.  1948:1141.  9.  FLOOD, A., HIRST, E. and JONES, J . K. N.  Nature  1947.  J.Chem.Soc.  1948:1679.  10.  HIRST, E. L. J.Chem.Soc.  11.  HIRST, E. L., HOUGH, L..and JONES, J . K. N. Soc. 1 9 4 9 : 9 2 8 . . HIRST, E. L., HOUGH, L. and JONES, J . K. N. 163:177. 1949.  12.  160:86.  1949:522. J.Chem. Nature-  13.  HIRST, E. L. and JONES, J . K. N.  14.  HORROCKS, R. H.  15.  HOUGH, L., Jones, J . K. N. and WADMAN, W. H. Nature 1 6 2 : 4 4 3 . 1 9 4 8 . J.Chem.Soc. 1 9 4 9 : 2 5 1 1 . HOUGHS L., JONES, J . K. N., and WADMAN, W. H. J.Chem. Soc. 1 9 5 0 : 1 7 0 2 .  16. 17.  J..Chem.Soc. 1 9 4 9 : 1 6 5 9 .  Nature 1 6 4 : 4 4 4 . 1 9 4 9 .  JONES, J . K. N., and HIRST, E. L. D i s c u s s . Soc. 7:271. 1 9 4 9 .  Faraday  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 $ : 2 7 2 . 1946.  24.  WHITE, E. V. J.Am.Chem.Soc. 6 9 : 6 2 2 , 7 1 5 , 2 2 6 4 . 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  o  •rl  o  C o  •ri  Gum  C O U  -H  3 O O Ctf rH  to 1  a  o o cd ctj  rH  cd  bO  ol  CP CO  •  o 4-5 o  CU CO CO  CC  o c c cd  1  1  a3  rH  bfl  B  «  o  CO  o >.  H  !«! 1  Q  a •H .O cd <  1 h-5  CD CO  o  I  Almond Anogeissus  +  +  +  Arabic  +  +  +  Cherry  +  +  +  Cholla  +  +  +  Damson  +  +  Egg plum  +  +  Grapefruit  +*  Khaya Lemon  +  +  +  +*  +  +  Mesquite  +  +  Myrrh  +  +  Orange  +  +  Purple plum  1.  Most o f the data i n t h i s t a b l e are taken from  *  Me.thoxy-derivative,  Reference  TABLE I I * A n a l y s i s o f U n p u r i f i e d and P u r i f i e d Sapote Gum  and Gum  Unpurified Moisture, Ash,  %  %  Carbon d i o x i d e , Uronic  %  a c i d , °/o  Pentosan, °/o Methoxyl,  %  CHg c a l c u l a t e d from methoxyl, /o Total,  %  Equivalent  weight  Acid Gum  a  Gum Purified Once  Gum Acid  12,13  8.68  6.30  4.27  2.05  .06  5.92  6.44  6.27  25.84  28.11  27.51  64.7  66.5  67.9  2.59  2.41  2.86  1.17  1.09  1.30  91.71  95.70  96.71  684.  653.  679.  Pentose u n i t s per uronic a c i d  3.64  3.44  3.61  Methoxyl u n i t per uronic a c i d  0.622  0.531  0.65  'The r e s u l t s have been c o r r e c t e d f o r moisture and ash. The equivalent weights are c a l c u l a t e d from the u r o n i c a c i d . Table taken from Reference  1.  TABLE I I I * Analysis  o f Barium S a l t s from of Sapote  Gum  S a l t s from Gum Heated f o r 2 Hr. at 80° Barium,  %  Pentosan,  %  Carbon Dioxide,  %  Uronic a c i d , °/o Methoxyl, CH  2  S a l t s from Gum Heated f o r 28 Hr. in Boiling Water Bath  23.43  55.5  52.6  26.1  8.02  8.34  11.75  35-02  36.42  51.54  3.56  4.67  1.60  2.3il  %  Pentose u n i t s per uronic acid  S a l t s from Gum Heated f o r 4 Hr. at 80° 14.20  %  E q u i v a l e n t weight  ct  13.14  c a l c u l a t e d from methoxyl, /o  Total,  Hydrolysis  566. 2.31  Methoxyl u n i t s per uronic acid  104.82  103.18  553.  385.  2.1  0.74  0.61  O.564  " i n a l l cases a 4 % s o l u t i o n of s u l p h u r i c for hydrolysis. Table taken from Reference 1.  a c i d was used  TABLE IV C o l o r Reactions with a Number o f Spray Reagents Spray  AgN0 /NH 3  3  Aldohexose  A n i l i n e H Malonate  ^ n ? "  p - A n i s i d i n e HCI  greenbrn. ,  O r c i n o l or resorc i n o l with HCI  none  Diphenylamine trichloraeetate  brown  Dimethylaniline trichloraeetate  Uronic aeid  Ketose  Methyl hexose  Methyl pentose  Methyl uronic  red-brn. to maroon  cherry red  crimson  brown t o b l a c k i n a l l cases  A n i l i n e H Phthalate brn. Aniline Trichloraeetate  a-Naphthylamine t r i chloracetate.  Pentose  red  brn.  r  yellowbra.  e  d  emerald green green  none  cherry red  none  crimson lemon yellow  brn.  yellow  brn. to r e d  red  none  red  green  none  purple(arab) grey(xyl)  purple  f r e e C, purple  purple(arab) brown(xyl)  ,TABLE V . •  R  Q  VALUES  2  1  FOR A NUMBER OF SUGARS AND'  THEIR METHYL DERIVATIVES S o l v e n t : n-Butanol/ E t h a n o l / Water,  5:1:4 R  Substance  value  N  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, h e p t u l o s e  0.11  Fructose 0 12 Gulose, Arabinose, Tagatose  .  * 0.15  Xylose 4-methyl galactose  0.16  Altrose  0.17  Idose, 6-methyl g a l a c t o s e  O.lS  T a l o s e , Lyxose .  .  "  0.19  Ribose, Fucose  0.21  2-methyl  0.22  glucose  2Smethyl g a l a c t o s e  0.23  Riboketose,  0.25  3-methyl  Apiose, 2-deoxy-galactose  glucose  0 26  Xyloketose 6-methyl glucose  0.27  Quinovose  0.2S  Rhamnose  0 30  a-methyl mannoside 3:4-dimethyl  galactose  4 - methyl mannose B-methyl a r a b i n o s i d e  0.32  TABLE V.  (cont.)  Substance  R  value  N  2-deoxy a l l o s e  0.33  2-methyl {3-methyl a l t r o s i d e  0.34  Rhamnoketose, 3:6-anhydro-glucose  0.37  2-methyl x y l o s e , 2-methyl arabinose  0.38  • 2:J+-dimethyl g a l a c t o s e  0.41  4:6-dimethyl galactose  0.42  2-deoxy r i b o s e , 2 : 6 - d i m e t h y l - g a l a c t o s e  0.44  4 : 6 - d i m e t h y l glucose  O.46  2- methyl fucose  Q  ^  Q  ^  3 : 6 - d i m e t h y l glucose 3:4-dimethyl glucose 4:6-dimethyl  altrose  2:3-dimethyl mannose  0.54  2:3-dimethyl glucose 4-methyl rhamnose  0.57  4 : 6 - d i m e t h y l 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 x y l o s e  0.66  2:4:6-trimethyl galactose  O.67  2:3:6-trimethyl galactose  0.71  2:3-dimethyl x y l o s e  0.74  2 : 4 : 6 - t r i m e t h y l glucose  0.76  3 : 4 : 6 - t r i m e t h y l mannose  0.79  2 : 3 : 6 - t r i m e t h y l glucose 1:3:4-trimethyl fructose  Q  ^  TABLE V.  (cont.)  Substance  R  n  value  3:4-dimethyl rhamnose  0.84  2 : 3 : 4 - t r i m e t h y l glucose  0.85  3:4:6-trimethyl fructose  0.86  Oleandrose, 2:3:4:6-tetramethyl g a l a c t o s e  0.88  Tetramethyl f r u c t o p y r a n o s e  0.90  2:3:4-trimethyl xylose  0.94  2 : 3 : 5 - t r i m e t h y l 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 - t r i m e t h y l rhamnose 1:3:4t6-tetramethyl f r u c t o s e  1.01  APPENDIX A  A bibliography  o f t h e more i n t e r e s t i n g  and u s e f u l papers r e l a t i n g t o chromatography o f t h e carbohydrates.  1.  2.  B e l l , D. J . J.Chem.Soc. 1944:473. S e p a r a t i o n o f methyl g l u c o s e s by p a r t i t i o n on columns o f moist s i l i c a . B e l l , D. J . and Palmer, A. J.Chem.Soc. 1949:2522. Q u a n t i t a t i v e a n a l y s i s o f mixtures o f methylated f r u c t o s e s u s i n g p a r t i t i o n chromatography.  3.  B l a s s , J . , Macheboeuf, M. and Nunez, G. B u l l . Soc. Chim.Biol. 32:130. 1950. Colorimetric determination o f aldoses with a n i l i n e hydrogen p h t h a l a t e .  4.  Boissonnas, R. A. E x p e r i e n t i a 3:23S. 1947. H e l v i t i c a Chemica A c t a 30:l6#9. 1947. S e p a r a t i o n o f methylated g l u c o s e s as p-azobenzoyl d e r i v a t i v e s 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 o f 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 s p r a y i n g reagents f o r paper chromatograms.  7.  Burma, D. P. and Banerjee, B. Science and C u l t u r e 15:363. 1950. The r o l e o f 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., H i r s t , S . L., Packman, D. F., and P e r c i v a l , E. G. V. J.Chem.Soc. 1951: 34#9. S e p a r a t i o n o f products o f h y d r o l y s i s o f methylated wood s t a r c h .  9.  Cramer, F. Angew.Chem. 62:73. 1950. Review o f Paper Chromatography o f t h e Sugars.  10.  Dekker, C. A. and Long, A. G. J.Chem.Soc. 1950:3162. Determination o f g l y c o s i d e s and non-reducing carbohydrates on papergrams w i t h p e r i o d a t e .  11.  F l o o d , A. E., H i r s t , E. L., Jones, J . K. N. Nature loO:S6. 1947. S e p a r a t i o n and q u a n t i t a t i v e e s t i m a t i o n o f sugars u s i n g paper chromatography.  12.  F l o o d , A. E., H i r s t , E. L. and Jones, J . K. N. J.Chem.Soc. 1948:1679. Q u a n t i t a t i v e a n a l y s i s o f mixtures o f 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. 6 8 : 2 1 6 9 . 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. 4 4 : 2 8 9 3 . Separation of phenylosazones on calcium carbonate.  27.  Kowkabany, G. N. and Cassidy, H.. G. Anal.Chem. 22:817. 1950. A comparison o f 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 o f sugars on f i l t e r - p a p e r chromatograms by d i r e c t photometry.  29.  M o n t r e u i l , P. B u l l . S o c . C h i m . B i o l . 3 1 : 1 6 3 9 . 1949. Determination o f r e d u c i n g sugars w i t h potassium ferricyanide.  30.  Mulvany, P. K., Agar, H. D., P e n i s t o n , Q. P. and McCarthy, J . L. J.Am.Chem.Soc. 7 3 : 1 2 5 5 . 1951. A chromatographic study of s u l f i t e wate l i q u o r sugars.  31.  Nordal, A. and K l e u s t r a n d , R. Acta.Chem.Scand. 5 : 8 5 . CA. 45:5771. Paper chromatography o f f r e e sugars o f some c r e s s u l a c e o u s p l a n t s .  32.  N o v e l l i e , L. Nature 1 6 6 : 7 4 5 . 1950. A spray f o r both ketoses and a l d o s e s -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.  P a r t r i d g e , S. M. Nature 1 6 4 : 4 4 3 . 1949. Use o f a n i l i n e hydrogen p h t h a l a t e as spray reagent f o r aldoses.  35.  P o r t e r , W. L. and Fenske, C. S. J r . , J.Assoc,Agr. Chemists 32:714* 1949. Determination o f sugars separated on paper chromatograms by s p e c i f i c fermentation.  36.  Rao, P. S. and B e r i , R. M. Current S c i . (India) 20:99 C. A. 4 5 : 7 4 7 1 . A n a l y s i s o f sugars w i t h ascending paper chromatography.  37.  W h i s t l e r , R. L. and Durso, D. F. J.Am.Chem.Soc. 72:677. 1950. S e p a r a t i o n o f the sugars on c h a r c o a l columns.  38.  Wise, L. E., Green, J . W. and Rittenhouse, R. C. Tappi 32:335. 1949. Qualitative separation o f h y d r o l y s i s products o f wood substances, p u l p s , and a d d i t i v e s .  39.  Wise, L. E., Rittenhouse, R. C. and G a r c i a , C. Tappi 34:15. 1951. Paper chromatography o f pulp h y d r o l y s a t e s .  APPENDIX  B  T r a c i n g s of r e p r e s e n t a t i v e papergrams showing t y p i c a l s e p a r a t i o n s of standard mixtures and gum  hydrolysates.  sugar  S t a r t ing line  S e p a r a t i o n o f s t a n d a r d s u g a r m i x t u r e s on a paper chromatogram run 44 h o u r s a t room temperature u s i n g n-butanol/ethanol/water 5:1:4 as s o l v e n 1 0  Before heat  l i hours  3td  0  10 hours  15 hours  Arabinose  Rhamnose  H y d r o l y s i s o f P e r u v i a n sapote gum w i t h 0.4 N„ a c i d at 80°C The c e n t r e s t a n d a r d i s P e r u v i a n gum h y d r o l y s i s B s y r u p . Chroma t o g r a m run 18 h o u r s a t 4 0 ° C using n - b u t a n o l / a c e t i c a c i d / w a t e r 5:1:4. o  0  

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