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Structural polysaccharide chemistry with analytical applications to lemon gum Gibney, Kelly Blair 1971

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STRUCTURAL POLYSACCHARIDE CHEMISTRY WITH ANALYTICAL APPLICATIONS TO LEMON GUM by KELLY BLAIR GIBNEY B.Sc.; University of British Columbia, 1963 M.Sc, University of British Columbia, 1967  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Chemistry  We accept this thesis as conforming to xhe required standard  THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1971  In presenting this thesis in partial  fulfilment  of the requirements for  an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make it  freely available for reference and study.  I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives.  It  is understood that copying or publication  of this thesis for financial gain shall not be allowed without my written permission.  Department of  CMB*\I*TM  The University of B r i t i s h Columbia Vancouver 8, Canada  Date  AfglL  3 1 , Mf\  ABSTRACT Chairman:  Professor G.G.S. Dutton  A gas chromatographic method has been developed whereby the following compounds may be determined quantitatively and simultaneously:  ethylene  glycol, glycerol, erythritol, threitol, arabinose, xylose, monnose, galactose and glucose.  These products correspond to the constituent parts of periodate  oxidized, borohydride reduced polysaccharides. Quantitative analysis of the fractions obtained on total and partial hydrolysis of such polyalcohols provides a new and powerful analytical tool in polysaccharide chemistry.  Optimum  conditions for the Smith degradation of a polysaccharide have been illustrated by a detailed analysis of lemon gum. A new method for the reduction of uronic acids in poly- and oligosaccharides has been developed. the uronic acids.  Reduction can be carried out after only esterification of Reaction at this stage avoids possible degradation or  fractionation during esterification and subsequent saponification of polysaccharide hydroxyl groups.  Good levels of reduction [75$] have been achieved  with reduction of highly branched lemon gum. Silylated polyhydroxy compounds and galacturonic acid were examined by proton magnetic resonance spectroscopy.  The trimethylsilyl [TMS] protons of  such ethers and esters show good separation from one another.  Analysis of the  trimethylsilyl proton peaks provides information concerning the number and degree of subsitution of hydroxyl and acid functions present in the parent molecule.  Signal strength due to the nine protons on each trimethyl s i l y l  group provides a chemical means of signal enhancement. Analysis was carried out on. 2 mg. of a minor TMS glucofuranose derivative.  [Ill] Lemon gum has been analysed by the method of Lindberg et a l [ 8 l ] . Analysis of lemon gum is the f i r s t example of the application of partially methylated a l d i t o l acetate mass spectroscopy to a plant gum.  The results  of this analysis are in good agreement with previous analysis of this gum.  [iv] T A B L E OF  CONTENTS  Pap.e N o .  'INTRODUCTION  . . . .'  1  P E R I O D A T E O X I D A T I O N OF CARBOHYDRATES PRODUCTS BY GAS  AND  EVALUATION OF REACTION  L I Q U I D CHROMATOGRAPHY  2  P E R I O D A T E O X I D A T I O N I N STRUCTURAL S T U D I E S O F POLYSACCHARIDES  2  SIMULTANEOUS  5  E S T I M A T I O N O F POLYHYDRIC ALCOHOLS AND  CONTROL DURING  SUGARS  .  T H E S M I T H DEGRADATION  l6  T H E OPTIMUM HYDROLYSIS O F O X I D I Z E D LEMON GUM  19  SMITH FRAGMENTS  28  REDUCTION  OF URONIC A C I D S  JO  M E T A L HYDRIDE REDUCING AGENTS E V A L U A T I O N O F NEW  .  ROUTE TO REDUCED POLYSACCHARIDES  56 59  S I G N A L ENHANCEMENT U S I N G T R I M E T H Y L S I L Y L [ T M S ] D E R I V A T I V E S AND U T I L I Z A T I O N OF TMS AS A PROBE FOR DETERMINATION  O F NUMBER  OF  HYDROXYLS I N A COMPOUND  1+5  S I G N A L ENHANCEMENT  k-9  T R I M E T H Y L S I L Y L A T I O N O F GALACTURONIC A C I D METHYLATION  51  O F LEMON GUM  MASS SPECTROMETRY  OF METHYLATED  62 POLYOL A C E T A T E S  67  METHYLATION A N A L Y S I S O F LEMON GUM  71  METHYLATED  7k  POLYOLS FROM LEMON GUM  METHYLATED DEGRADED LEMON GUM METHYLATION A N A L Y S I S O F LEMON GUM AND  BOROHYDRIDE REDUCTION  80 AFTER PERIODATE OXIDATION. .  82  Cv] TABLE OF CONTENTS [Cont'd] Pafte No. METMLATION OF LEMON GUM  [Cont'd] 85  METHYLATION OF LEMON GUM SI EXPERIMENTAL  . ..  88  •  88  GENERAL METHODS  95  I] HAKOMORI METHOD II] III]  95  PURDIE METHOD KUHN METHOD  .  96  METHYLATION OF OLIGO-AND MONOSACCHARIDES  96  PURDIE AND KUHN METHODS  96  .  HAKOMORI METHOD . . . . . . . . .  97  PERIODATE OXIDATION  97  ESTIMATION OF PERIODATE  98  SIMULTANEOUS ESTIMATION OF POLYHYDRIC ALCOHOLS AND SUGARS .  98  PURIFICATION OF LEMON GUM . . . .  101  OPTIMUM HYDROLYSIS OF LEMON GUM - PERIODATE DEGRADED . . .  102  SMITH FRAGMENTS  105  • THE REDUCTION OF ACIDIC POLYSACCHARIDES . . . .  '105  ESTERIFICATION  105  REDUCTION OF ETHYLENE GLYCOL ESTER OF LEMON GUM . . .  106 107  SIGNAL ENHANCEMENT TRIMETHYL SILYLATION OF GALACTURONIC ACID METHYLATION ANALYSIS  .........  107 108  [vi] TABLE OP CONTENTS. [Cont'd] To,f\e No.  110  APPENDIX I . . . . ESTIMATION OF DEGREE OF POLYMERIZATION  110  EXPERIMENTAL  112  * APPENDIX II  . . . . . . . . . .  •. i  Ilk  ENZYMATIC HYDROLYSIS OF LEMON GUM  Ilk  EXPERIMENTAL  117  ENZYMATIC HYDROLYSIS OF LEMON GUM  117  FRACTIONATION OF PECTINASE, CELLULASE AND HP-150 . . .  118  PECTINOL kl-V CONCENTRATE CELLULASE 36 . . . . . .  . . . . . .  Il8  .  119 120  HP-150  121  APPENDIX III . .• CARBON-CARBON BOND CLEAVAGE, DIALYSIS AND DESTRUCTIVE . PURIFICATION, AN UNDERGRADUATE ORGANIC EXPERIMENT  125  CHARACTERIZATION  126  APPENDIX IV LEMON GUM:  121  DEGRADATION WITH 0.1U SULFURIC ACID  126  EXPERIMENTAL  126  AUTOHYDROLYSIS OF LEMON GUM  126  EXPERIMENTAL  128  . .  128  OLIGOSACCHARIDES  130  APPENDIX V A TECHNIQUE FOR HANDLING MICRO SAMPLES COLLECTED FROM THE GAS CHROMATOGRAPH  , .,  130  [vii] TABLE OF CONTENTS [Cont'd] Pape No. 131+  APPENDIX VI COMPUTER SEARCHING THE CURRENT CARBOHYDRATE LITERATURE . . PROFILE NUMBER 2, EVALUATION  139  '  PROFILE NUMBER 3  1^1  APPENDIX VII LEMON GUM 1/lON H2S0 LEMON GUM  LEMON GUM SI BIBLIOGRAPHY  . . .  150  ,. .  150 160  4  SMITH POLYALCOHOL LEMON GUM  1J'+  . . . . . .  163 167 172  [viii] LIST OF TABLES Table No. TABLE I  Pa.qe Wo. Possible Reaction Products from Periodate Oxidized Polysaccharides  6  TABLE II  Analysis of Reaction Products from an Arabinoxylan  8  TABLE III  Ana,lysis of Reaction Products of a Glucomannan . .  10  TABLE TV  Analysis of Reaction Products of a Galactoglucomannan  10  TABLE V  Analysis of Threitol and Erythritol Mixtures . . .  10  TABLE VI  Analysis of Simulated Crude Hemicellulose Hydrolysates  10  TABLE VII  Molar Response Factors of Polyols and Sugars . . .  12  TABLE VIII  Composition of Peaks in Gas Pha.se Chroma, to gram of a Mixture of Arabinose, Xylose, Galactose, Glucose and Mannose  TABLE IX  15  Number of Sugar Anomers Detected by Gas-Liquid Chromatography and the Percentage Composition of Equilibrium Solutions  TABLE X  Analysis of Lemon Gum Smith Polyalcohol and i t s Hydrolysis and Oxidation Products  TABLE XII  55  Carbohydrate Analysis of Lemon Gum and i t s Reduced Product  TABLE XTV  26  Analysis of the Polysaccharides Obtained from Alginic Acid by Different Reduction Procedures . .  TABLE XIII  15  K2  Yields and Degrees of Methylation of Various Polysaccharides  65  [ix] LIST OF TABLES [Cont'd] Table No.  Paffe No, ....  63  TABLE XV  Recently Developed Methylation Procedures  TABLE XVI  Methylation Analysis of Lemon Gum  79  TABLE XVII  Methylation Analysis of Degraded Lemon Gum . . . .  8l  TABLE XVIII  Methylation Analysis of Lemon Gum Smith Polyalcohol  83  TABLE XIX  Methylation Analysis of Lemon Gum SI  86  TABLE XX  Anomeric Ratios for BTMSA Silylation of 108  Galacturonic Acid in Different Solvents TABLE XXI  Enzyme Preparations Examined for Activity on Lemon Gum  Ilk  TABLE XXII  Variable Steps i n Preparation of Erythritol  . . .  TABLE XXIII  Oligosaccharides Isolated on Hydrolysis of Lemon 127  Gum TABLE XXIV  122  Neutral and Acidic Components Obtained on Hydrolysis .  129  TABLE XXV  Computer Print Out of Profile One  136  TABLE XXVI  Computer Print Out of Profile Two  138  TABLE XXVII  Computer Print Out of Profile Three  IkO  TABLE XXVIII  Computer Print Out of Profile Four  Ikl  TABLE XXIX  Evaluation of Search Profiles  ikk  of Lemon Gum Oligosaccharides  [x] LIST OF FIGURES Figure No. FIGURE 1  Pap;e No. Model Periodate Oxidation Products Before and After Reduction  FIGURE 2  Separation of Products as Trimethylsilyl Derivatives from an Arabinogalactan  FIGURE 3  k  . . . . . . . .  Separation of Products as Trimethylsilyl Derivatives from an Arabinoxylan  FIGURE k  9  Separation of Products as Trimethylsilyl Derivatives from Crude Hemicellulose  FIGURE 7  9  Separation of Products as Trimethylsilyl Derivatives from Galactoglucomannan  FIGURE 6  8  Separation of Products as Trimethylsilyl Derivatives, from a Glucomannan  FIGURE 5  8  9  Gas Chroma to grams of Trimethylsilyl Derivatives from Lemon Gum Smith Polyalcohol Hydrolysed With Various Sulfuric Acid Strengths for 2k Hours at 20°C  FIGURE 8  21  Gas Chromatograros of Trimethylsilyl Derivatives from Lemon Gum Smith Polyalcohol Hydrolysed for Different Periods of Time With 0.5N Sulfuric Acid at 20°C  FIGURE 9  Relative G.L.C. Peak Areas for Components 2,3,4 from Lemon Gum Smith Polyalcohol  FIGURE 10  23  2k  Relative G.L.C. Peak Areas for Components 1 [Glycerol] and 5 [Arabinose] from Lemon Gum Smith Polyalcohol  2k  Cxi] . LIST OF FIGURES [Cont'd] Figure No. FIGURE 11  Page No. Gas Chromatogram of Lemon Gum Smith Polyalcohol Hydrolysed for 32 Hours With 0.5N Sulfuric Acid at 20°C After Dialysis  FIGURE 12  25  Gas Chromatogram of the Supernatant From the Isolation of Lemon Gum SI Before and After 30  Total Hydrolysis FIGURE 13  The Reduction of Esters to Ethers with an Excess 32  of Diborane FIGURE 14  The /S -Elimination Mechanisms for Uronic Acids  FIGURE 15  100 MHz P.M.R. Spectrum for the E r y r t h r i t o l  .  Protons i n Trimethylsilylated Erythritol FIGURE 16  56  P.M.R. Spectrum of Trimethylsilylated Galacturonic Acid [G.L.C. Peak 4]  FIGURE 21  55  P.M.R. Spectrum of Trimethylsilylated Galacturonic Acid [G.L.C. Peak 2]  FIGURE 20  54  P.M.R. Spectrum of Trimethylsilylated Galacturonic Acid [G.L.C. Peak 1]  FIGURE 19  47  Proton Magnetic Resonance Spectra for the T r i methylsilyl Protons of Silylated Galacturonic Acid  FIGURE 18  k6  Compounds Subjected to P.M.R. [100 MHz] Analysis of Trimethylsilyl Protons  FIGURE 17  kl  57  Mass Spectrum of the Trimethylsilylation Product of Galacturonic Acid [G.L.C. Peak 1]  58  [xii] LIST OF FIGURES [Cont'd] Figure No. FIGURE 22  Page No. Mass Spectrum of the Trimethylsilylation Product of Galacturonic Acid [G.L.C. Peak 2]  FIGURE 23  59  Mass Spectrum of the Trimethylsilylation Product of Galacturonic Acid [G.L.C. Peak 3]  FIGURE 24  60  Mass Spectrum of the Trimethylsilylation Product 6l  of Galacturonic Acid [G.L.C. Peak k] FIGURE 25  Gas Chroma to gram of Lemon Gum After Methylation, Lithium Aluminum Hydride Reduction, Hydrolysis, 72  Borohydride Reduction and Acetylation FIGURE 26  Thin Layer Chromatogram of the Ten Fractions of 72  Lemon Gum Methylated Polyol Acetates FIGURE 27  Gas Chromatogram of Degraded Lemon Gum 80  Methylated Polyol Acetates FIGURE 28.  Gas Chromatogram of Lemon Gum Smith Polyalcohol Methylated Polyol Acetates  FIGURE 29  ...  Gas Chromatogram of Lemon Gum SI Methylated Polyol Acetates  FIGURE 30  '.  111  4  Gas Chromatogram of Glycitol Showing Undersilylation After 10 Min. of Reaction  FIGURE 32  86  Gas Chromatogram of Glucitol Hydrolysed with 1.0N H2S0 for 2k Hours  FIGURE 31  83  .  112  Collection of Gas Chromatography Samples i n Glass Capillaries and Oven Design for Their Micromanipulation  132  [xiii] LIST OF FIGURES [Cont'd] Fip<u.re No.  Pap;e No.  FIGURE 33  Oven and Gas Supply for Micromanipulation . . . .  133  FIGURE 34  Lemon Gum Peak 3  150  FIGURE 35  Lemon Gum Peak 4  151  FIGURE 36  Lemon Gum Peak 5  152  FIGURE 37  Lemon Gum Peak 6B  . . . . . . . . . .  153  FIGURE 38  Lemon Gum Peak 6T . . . . . .  .  154  FIGURE 39  Lemon Gum Peak 7B  155  FIGURE 40  Lemon Gum Peak 7T .  156  FIGURE 41  Lemon Gum Peak 8  157  FIGURE 42  Lemon Gum Peak 9  158  FIGURE 43  Lemon l/lON Smith Lemon  .-  Gum Peak 10 H2S0 Lemon Gum Peak 4 Polyalcohol Lemon Gum Peak 6 Gum SI Peak 6 4  159  FIGURE 44  l/lON HsSO* Lemon Gum Peak 1  FIGURE 45  l/lON HeS0 Lemon Gum Peak 2  FIGURE 46  l/lON H£S0 Lemon Gum Peak 3  FIGURE 47  Smith Polyalcohol Lemon Gum Peak 2  FIGURE 48  Smith Polyalcohol Lemon Gum Peak 3  164  FIGURE 49  Smith Polyalcohol Lemon Gum Peak 4  165  FIGURE 50  Smith Polyalcohol Lemon Gum Peak 5  166  FIGURE 51  Lemon Gum SI Peak 1  167  FIGURE 52  Lemon Gum SI Peak 2 . . . . . . . .  FIGURE 53  Lemon Gum SI Peak 5 . . . . . '  169  FIGURE 54  Lemon Gum SI Peak 4  170 •  FIGURE 55  Lemon Gum SI Peak 5 . . .  171  4  160 l6l  . . .  162  4  .  .  163  168  ACKNOWLEDGEMENTS The author wishes to express his grateful appreciation for the help, encouragement and advice given him by Professor G.G.S. Dutton, who directed the work described i n this thesis. He wishes to thank Dr. P.E. Reid, Dr. J.J.M. Rowe, Dr. K. Hunt, Dr. L.D. Hall and Dr. J.N.C. Whyte for their valued counsel, the University of British Columbia for the award of the MacMillan Bloedel Graduate Scholarship and Miss N. Housenga for her diligence during the preparation of this manuscript.  [xv]  To my wife.  1  INTRODUCTION Lemon gum is a highly branched polysaccharide exudate.  During the  course of structural investigations of this polymer, a detailed structure was published by Jones and Stoddart [ l j . Although work continued to confirm the structure proposed, the direction of investigation was altered. Methods were designed to improve the quality of polysaccharide structural determinations while decreasing the quantities of material required. The isolation of pure polysaccharides has been greatly stimulated by several important improvements in methods and techniques.  Investigators  can now isolate small quantities of extremely pure polysaccharides using preparative moving boundary electrophoresis, starch block electrophoresis, ultracentrifugation using density gradients, gel permiation chromatography, and various types of ion exchange chromatography. The general drawback of these methods i s an inability to isolate large quantities of polymer. problem can be further complicated by a lack of available sample.  This  Biological  materials may contain polysaccharides in low concentrations or as very complex mixtures. nature.  Time can be a c r i t i c a l factor since a glyco protein may  de-  However, while the techniques of polymer isolation have improved so  have techniques for the investigation of small quantities of carbohydrates. In the ensuing sections new methods detailing improvements in techniques already applied to carbohydrates and analytical procedures not previously described w i l l  be presented.  Their, application to  model  compounds  and lemon gum [as an example of a complex polysaccharide] w i l l be developed.  PERIODATE OXIDATION OF CARBOHYDRATES AND EVALUATION OF REACTION PRODUCTS BY GAS LIQUID CHROMATOGRAPHY PERIODATE OXIDATION IN STRUCTURAL STUDIES OF POLYSACCHARIDES  Periodate oxidation results In a specific attack on a carbohydrate polymer. Periodic acid, or functions, the cleaved  i t s salts w i l l oxidize v i c i n a l hydroxy 1 glycols forming a dialdehyde. The existence of  three contiguous hydroxyls v i l l result i n the formation of a mole of formic acid from the central carbon atom. Periodate oxidation i s complicated by steric effects, electronic effects and over oxidation.  Since  this f i e l d has been v e i l outlined i n several reviews [ 2 , 3 ] i t v i l l be sufficient to say that the dialdehyde that results from an oxidation forms an extremely complex system. a l l y impossible to interpret.  Direct hydrolysis of the dialdehyde i s v i r t u Severe degradation of the small component  units formed in the oxidation occurs. [4],  In previous work in this laboratory  experiments aimed at preparation of partially methylated tetroses by  oxidation between C-2 and C-3 of two hexoses were unsuccessful because of side reactions, lack of hydrolysis and degradation. I f this simple system does not yield clear results, the concept of analysis of complex polysaccharides at the polyaldehyde stage becomes impossible. The Barry degradation [ 5 ] was developed to analyse the polyaldehyde oxidation product.  The polyaldehyde was treated with phenylhydrazine i n  dilute acetic acid solution.  An insoluble product formed, containing ap-  proximately one molecule of phenylhydrazine condensed with each dialdehyde group.  This complex broke down when heated i n an aqueous or ethanolic solution  3 of phenylhydrazine and acetic acid.  The products were phenylosazones and  unoxidized parts of the polysaccharide a l l of which may be Identified. were, however, two areas of d i f f i c u l t y with this procedure.  There  The products of  the oxidized portion of the molecule were osazones and hence structural asymmetry of a portion of the molecule was l o s t .  Secondly i t was relatively  d i f f i c u l t to purify and quantitatively estimate the osazones produced. A method was developed by Smith [ 6 ] to simplify the interpretation of results from periodate oxidation of polysaccharides.  The procedure involved  reduction with metal borohydrides and subsequent controlled hydrolysis of the polyalcohol which was formed. Dialdehydes formed on periodate oxidation do not exist as free aldehydes but like sugars themselves, undergo cyclization and hydration [ 7 ] *  Some examples of cyclized model compounds [ I , I I , III]  which have been analysed are shown i n Figure 1.  Alcohols such as IV are  readily hydrolysed with dilute mineral acid at room temperature. acetals such as I, I I , and III are relatively stable.  Cyclic  Smith realized that  i n s t a b i l i t y of alcohols such as IV toward dilute acid at room temperature, compared with glycosides, provided a new and more powerful tool for controlled degradation of polysaccharides.  When a sugar moeity of a polysaccharide i s  cleaved by periodate and reduced, the resulting alcoholic derivative, being a true acetal, i s sensitive to acid.  Sugar units which are not cleaved and  are attached glycosidicly to cleaved or unattached units remain intact being relatively stable to acid.  Using the marked differences i n s t a b i l i t y o f  glycosides over true acetals a vide variety of oligosaccharides have been obtained.  The structures o f the stable units provide an indication of the  polysaccharide subunits.  FIGURE 1 MODEL PERIODATE OXIDATION PRODUCTS BEFORE AND AFTER REDUCTION  IE  W  Named the Smith degradation this procedure has been reviewed and u t i l i z e d extensively [ 8, 9, 10, 1 1 ] .  Periodate oxidation, reduction and t o t a l hy-  drolysis of a polysaccharide, often incorrectly called a Smith degradation, also provides a considerable source o f information regarding fine structure in the polymer. Two limitations exist which detract from the use of t o t a l or p a r t i a l hydrolysis of the Smith polyalcohol.  One of these problems i s the i n a b i l i t y  of standard analytical techniques to determine quantitatively the totally reduced products o f the oxidation, that i s to say ethylene glycol, glycerol, erythritol and t h r e i t o l .  Ethylene glycol i s especially d i f f i c u l t to qual-  i t a t i v e l y identify on paper Cihrona to grams because of diffusion i n chromatographic solvents, streaking and lack o f reaction. The second problem i s the  5 wide variety of rates of hydrolysis of different glycosidic linkages and different tx-ue acetals. p a r t i a l hydrolysis.  These factors cause d i f f i c u l t i e s i n carrying out  The solutions to these problems w i l l be outlined i n the  following two subsections.  They w i l l demonstrate a whole new approach and  usefulness of Smith oxidations involving p a r t i a l and total hydrolysis.  SIMULTANEOUS ESTIMATION OF POLYHYDRIC ALCOHOLS AND SUGARS As mentioned before, one method of investigating the structure of a polysaccharide  involves periodate oxidation, borohydride reduction and com-  plete hydrolysis [12].  For example, in the case of a linear 1 —5-4 linked  pentosan, the products w i l l be ethylene glycol from the non reducing end and glycerol from the interior units. When any of the interior units carry side chains these units are immune to periodate oxidation and thus subsequent hydrolysis gives monosaccharides as well as polyhydric alcohols. Much useful information can be obtained from a knowledge of the ratio between non reducing end groups, interior units and branch points.  Two different analytical methods  have normally been used i n this connection after separation of the components by paper or thin-layer chromatography. Thus, polyhydric alcohols have been estimated by chronotropic acid [13] and monosaccharides by phenol sulfuric [14] or other colorimetric method. The necessity of using two distinct analytical procedures in addition to the separations involved is tedious and introduces possibilities of error.  The present work reports a method using  gas-liquid chromatography [G.L.C] whereby mixtures of polyhydric alcohols and monosaccharides may be analysed simultaneously and accurately.  6  TABLE I POSSIBLE REACTION PRODUCTS FROM PERIODATE OXIDIZED POLYSACCHARIDES  Polysaccharide  Possible Products  A rab inoxylan  Ethylene glycol, glycerol, xylose, arabinose  Glucomannan  Glycerol, erythritol, glucose, mannose  Galactoglucomannan  Glycerol, erythritol, threitol, glucose, mannose, galactose  Arab ino galactan  Ethylene glycol, glycerol, threitol, arabinose, galactose  This investigation arose from the need to have a simple method for the analysis of polysaccharides occurring i n wood and plant gums. Table I illustrates the possible products from four such typical polysaccharides. The method involves transformation of the mixture of sugars and polyhydric alcohols resulting from the periodate oxidation of a polysaccharide into derivatives sufficiently volatile for separation by G.L.C.  In order to  test the v i a b i l i t y of the method, several test mixtures were prepared corresponding to the possible products shown i n Table I. For each system studied the relative proportions of the components were varied over wide limits thus covering the different situations encountered i n various naturally occurring polysaccharides.  The components were separated as their t r i -  methylsilyl [TMS] derivatives [15] on a column employing SF-96 as the l i q u i d phase.  7 The separation of the products arising from an arabinogalactan i s shown in Figure  2 and the data obtainable from an arabinoxylan are given in Table  II with the separation shown i n Figure  3.  Similarly the simulated gluco-  mannan i s represented by Table III and Figure k.  In a l i k e manner the simu-  lated galactoglucomannan system i s shown i n Table IV and Figure 5. results need l i t t l e explanation except in the last case.  These  According to the  current view [16 ] of the structure of a wood galactoglucomannan one would not expect galactose to survive the periodate oxidation. However, galactose was included as a check on hexose analysis. The model experiments reported here were an opportunity to test the v a l i d i t y of the proposed method and therefore the most general cases were studied.  I t i s clearly seen from the figures that the separations are ex-  cellent and the agreement between calculated and found values i s good.  The  worst separation in the present study i s that between t h r e i t o l and erythritol in the galactoglucomannan system.  But even here i t i s possible to calculate  the relative amounts [17] i n f a i r agreement with theory [Table V].  This  problem w i l l only commonly arise where a polysaccharide contains galactose units linked 1 - 4  together with glucose and/or mannose similarly linked.  Examination of Figure 6 w i l l show that the pentoses have a lower retention time [R^,] than the hexoses.  This means that the same column and  procedure may be used to separate a l l of the polyhydric alcohols and sugars mentioned in Table I. More importantly i t means that this same system may be used to separate and estimate a l l five of the sugars commonly occurring in wood polysaccharides.  This separation has now been described by others  whose results appeared while our work was in progress [ 18-21 ].  Particularly  FIGURE 2: SEPARATION OF PRODUCTS AS TRIMETHYLSILYL DERIVATIVES FROM AN ARABINOGALACTAN  ETHYLENE GLYCOL  ARABINOGALACTAN BUTANE1,4-OIOL THREITOL I  u  -//•  r-YA-r—ih  10  16  —i—  28  I  34  44  52  FIGURE 3: SEPARATION OF PRODUCTS AS TRIMETHYLSILYL DERIVATIVES FROM AN ARABINOXYLAN  ARABINOXYLAN  TABLE II ANALYSIS OF REACTION PRODUCTS FROM A N ARABINOXYLAN  • % composition by weight  Found Calculated  .  Found Calculated 1  Found Calculated  Ethylene glycol  Glycerol  15-6 15-8  18.8 19.0 ..  . 3°-5 30.8 42.9 42.8  :  38.1 37-7 5-2° 5-14  , Arabinose  29-3 29.9  ,  29.2 " ' ' 28.9 3-31 4.07  Xylose  36.0 35-5 3-15 3-44 48.5 47.8  FIGURE k: SEPARATION OF PRODUCTS AS TRIMETHYLSILYL DERIVATIVES FROM A GLUCOMANNAN  GLUCOMANNAN  GLYCERCI. ERYTHRITOL  J -If—i  16  it  1  //-  26  v.  43  60  •56  FIGURE 5: SEPARATION OF PRODUCTS AS TRIMETHYLSILYL DERIVATIVES FROM GALACTOGLUCOMANNAN  GALACTOGLUCOMANNAN  43  46  50  FIGURE 6: SEPARATION OF PRODUCTS AS TRIMETHYLSILYL DERIVATIVES FROM CRUDE HEMICELLULOSE ETHYLENE GLYCOL GLYCEROL BUTANE1,4-DIOL I  ARABINOSE  THRE ERYTH  ll  I vJl 0  J u l  j\—-J  1—it—r-"-i—o—i  4  10 16  26  u  1  34  1  38  '  43  1  46  r—  50  56  10 TABLE I I I " ANALYSIS OF REACTION PRODUCTS OF A GLUCOMANNAN  % composition by weight Glycerol  Erythritol  Mannose  Glucose 36-4 3<>.4  Found Calculated  19.2 19.2  9.20 8.70  35-5 36.0  Found Calculated  3i-4 33-2  1.48 1.46  65.1 62.8  Found Calculated  6.78  6.20 5.85  29.6 28.6  6-34  2.22 3.11 . 57-5 59*  TABLE IV ANALYSIS OF REACTION PRODUCTS OF A GALACTOGLUCOMANNAN  % composition by weight Glycerol  Galactose  Mannose  Glucose  Found Calculated  17.6 •• '53  25-7 27.8  29.38 28.4  28.2 28.5  Found Calculated  4.12 4.02  , 53-2 54-8  39.1 37-4  Found Calculated  4.08 3-94  2.85 302  38.6 36.8  Found Calculated  —  54-7 55-7  2.61 4.81  47-5 47-2  —-*  3-71 3-74  50.2 48.4  -  TABLE V ANALYSIS O F THREITOL AND ERYTHRITOL  MIXTURES  % composition by weight Thrcitol  Erythritol  Found Calculated  54-5 5i-5  455 48.5  Found Calculated  83.2 84.1  16.8 15-9  Found Calculated  20.8 17-5  79.2' 82.5  •  TABLE VI ANALYSIS OF SIMULATED C R U D E H E M I C E L L U L O S E  HYDROLYSATES  % composition by weight Arabinose Found Calculated  17.9 18.4  Found Calculated  17-75 18.2  Found Calculated Found Calculated  3-15 3.28 33-8 33-6  .  Xylose  Galactose  . 23.1 ... 21.6  , 20.4 21.1  22.6 21.7  ' 21.6 21.3  39.8 38.7 4.07 3-85  .  3.03 3-8i 38.5 38.9  Mannose •;  16.95 17-25 34-° 34-4  ' >  -'  16.62 15-3 3-64 3-94  Glucose ' 22.0 21.7 4.07 4-37 39.0 39-0 ' 20.0 19.92  11 notable Is the excellent Swedish paper to which further reference w i l l be made [22J. The results presented in Table VI are further confirmation of the accuracy of the method used in this laboratory. The results quoted i n Tables I I - VI were obtained using an instrument with a thermal conductivity detector and systems containing free sugars. Each of these factors requires brief comment. F i r s t l y , different substances cause such a detector to respond to a varying degree, thus for identical molar amounts of different compounds the areas under the peaks are not the same. I t i s therefore necessary to determine the Molar Response Factor [M.R.F.] for each compound to be determined.  This i s done relative to an internal  standard, which in the present work was butane -1,4-diol.  This compound i s  readily available, easily purified and has a convenient retention time, as Figure  6 shows. The view has recently been expressed that an inert com-  pound i s perferable [23] and terphenyl has been suggested.  Although this  compound has certain advantages, solubility i n carbohydrate hydrolysis mixtures can limit i t s use as a quantitative internal standard. When working with compounds of similar molecular complexity i t may be acceptable to assume that a l l the response factors are identical. when the compounds examined range from Ca to C [24], as shown i n Table VTI.  6  However,  this assumption i s incorrect  These values are presented here to show the vari  ation which may be expected. I t i s our experience that the exact M.R.F. applic able to any analysis depends on the precise experimental conditions used. The value i s influenced by such factors as the, rate o f temperature programming. A contrary view has been expressed [25] but at this time i t would seem advisable for each worker to determine suitable response factors using exactly  12 the same experimental conditions as in the analytical determinations. I t would be most unwise to assume without verification that the figures quoted in Table VII are valid for other systems.  TABLE VII MOLAR RESPONSE FACTORS OF POLYOLS AND SUGARS  Compound  Molar Response Factor  Ethylene glycol  O.832 + 0.033  Butane -1,4-diol  1.00  + 0.000  Glycerol  1.26  + 0.014  Threitol  1.78  + 0.088  Erythritol  1.72  + 0.061  Arabinose  1.74  + 0.061  Xylose  + 0.064  Galactose  1.77 2.08  Glucose  2.02  + 0.091  Mannose  2.02  + 0.059  + 0.126  Secondly, in solution any one sugar exists as an equilibrium mixture of the anomars of the furanose and pyranose forms. When certain peaks overlap i t w i l l be necessary to use a peak corresponding to one anomeric form as a measure of the total amount of that particular sugar. This fact has been clearly discussed in the Swedish paper cited previously [22].  Since the r e l -  ative equilibrium concentrations of the different forms are highly dependent on the solvent used [26 ] i t follows that the figures quoted i n that paper [22 ] are only valid for that particular system.  For accurate results i t i s thus  necessary for each worker to adopt a standardized procedure for the treatment  13 of polysaccharide hydrolysates. The composition of the sugar peaks obtained in the present work i s given i n Table VIII and shown i n Figure  6. The  equilibrium composition of the solutions i s shown i n Table XX.  TABLE VIII COMPOSITION OF PEAKS IN GAS PHASE CHROMATOGRAM OF A MIXTURE OF ARABINOSE, XYLOSE, GALACTOSE, GLUCOSE AND MANNOSE  Major Component Arabinose 1, 2, 3  Minor Component[s] Xylose 1 + 2  Xylose 3 + 4 Mannose 1  Galactose 1  Galactose 2 Galactose 3  Mannose 2 Glucose 1  Glucose 2  TABLE IX NUMBER OF SUGAR ANOMERS DETECTED BY GAS-LIQUID CHROMATOGRAPHY AND THE PERCENTAGE COMPOSITION OF EQUILIBRIUM SOLUTIONS  Sugar  Anonym  Arabinose  3  Xylose  4  1 +2 3 + 4  3-58 + 0.28 96.4 + 0.28  Galactose  3  l 2 3  8.19 + O.80 29.3 + 1.27 62.6 + 1.02  Mannose  2  1 2  7^5 25.5  + 1.21 + 1.21  Glucose  2  1 2  38.3 61.7  + 0.33 + O.36  a b  b  Percentage Compos i t i o n  a  Anomers numbered i n order o f elution. I t i s possible that glucose shows a third anomer present i n very small concentration.  Ik The analysis of a periodate oxidized glucomannan by gas-liquid chromatography of the derived erythritol and glycerol acetates was reported i n I960 by Bishop and Cooper [ 2 7 ] .  More recently Zinbo and Timell [28] have  used the s i l y l derivatives for the analysis of a xylan.  In neither case has  the fate of glycol aldehyde, obtainable from carbons one and two, been studied, although the former authors noted the existence of an unidentified peak with a greater retention time than glycerol triacetate and suggested this might be due to glycol aldehyde diacetate. In an attempt to discover what happens to the glycol aldehyde a solution of this compound i n pyridine was silylated and examined using the same chromatographic conditions as before. There resulted a single peak indistinguishable from glycerol and attributed to the dimer [ 2 9 ] .  When an aqueous solution  of glycol aldehyde was concentrated to dryness and silylated, two peaks were obtained. One o f these had the same retention time as glycerol and the other a shorter [presumably the monomer]. I f an aqueous solution of glycol aldehyde was f i r s t reduced with sodium borohydride only one peak corresponding to ethylene glycol was obtained. This suggested one way of dealing with the problem.  When, however, synthetic mixtures of glycol aldehyde and the neutral  products l i s t e d i n Table I were analysed, several new and unexpected peaks were obtained. These peaks were assumed to be due to acetals formed between glycol aldehyde and the polyhydric alcohols [ 2 9 ] .  These observations did not  accord with those made on periodate oxidized amylose, where the amount o f glycol aldehyde should equal the sum of glycerol and erythritol.  Furthermore,  the gas chromatographic analysis of the products from mesquite gum gave results in good agreement with those obtained by paper chromatographic separations [50].  15 The difference i n treatment of the model systems and the polysaccharides was an acid hydrolysis step. When the model systems containing glycol aldehyde -were heated at 100° for 2k hours with IN sulfuric acid and then analysed* no unidentifiable peaks were obtained. There was no change i n the ratio of the sugars present. precipitate.  The hydrolysates were dark brown and contained a brown  There was a small increase i n the glycerol concentration and a  small decrease i n ethylene glycol.  The former i s no doubt due to traces of  glycol aldehyde dimer since in a separate experiment i n which glycol aldehyde alone was treated with aoid a small peak occurred in the glycerol region. The apparent loss of ethylene glycol cannot at present be explained since i t was verified that this compound was not l o s t on ion exchange resins nor on concentration of i t s aqueous solutions. I t i s concluded that the glycol aldehyde i s almost entirely destroyed i n the total hydrolysis of the polyalcohol and i s thus not a serious factor in such analyses. Where a highly accurate value for glycerol i s necessary this may be obtained by reduction of the hydrolysate with borohydride before analysis to convert any remaining glycol aldehyde dimer to ethylene glycol. The f i r s t applications of gas-liquid chromatography to polysaccharide chemistry were reported for methylated sugars [31] because of their v o l a t i l i t y . This technique i s now widely applied and has also been used in conjunction with periodate degradation to f a c i l i t a t e the resolution of a complex mixture of isomeric sugars [32-34].  With the introduction of trimethylsilylation by  Sweeley et a l . [15] this technique has been extended to the separation of sugars [18-22 ] and glycosylalditols obtained by Smith degradation [ 3 ^ and references therein].  Although the work reported here arose out of our interests  16 in wood polysaccharides and plant gums I t i s clear that the procedure i s a general one and may be used for many other types of polysaccharides.  I t has  already been applied with success to a study of sapote gum [35] and mesquite gum [30].  Work has been carried out to extend the method to systems con-  taining deoxy and amino sugars [36] thus permitting the study of a wider variety of polysaccharides.  (  ,.  CONTROL DURING THE SMITH DEGRADATION In addition to using gas-liquid chromatography for the examination of t o t a l hydrolysis of a Smith polyalcohol, this analytical method has provided a new and most powerful tool i n the structural elucidation of polysaccharides by Smith degradation. As mentioned before, Smith degradations involve u t i l i zation of differences i n the ease of hydrolysis of various acetal structures generated during the periodate oxidation and subsequent reduction. Unfortunately the Smith degradation i s not as simple as would f i r s t appear. The problem arises from the different rates at which different glycosides and acetals hydrolyse. Two extreme examples may be used to illustrate this point. Pyranosidic linkages are considerably more stable than corresponding furanosidic linkages. I t has been possible to u t i l i z e this property to isolate L-arabinose from mesquite gum [ 3 7 ] .  L-arabinose i n polysaccharides often occurs as  furanoside and hence may be readily hydrolysed by O.OIN sulfuric acid at 95°C. Hydrolysis of mesquite gum for 36 hours under these conditions produced a residual polysaccharide containing no L-arabinose. At the other extreme, 4-0methyl-D-glucuronic acid when attached glycosidically to other carbohydrate residues i s extremely d i f f i c u l t to hydrolyse. Under conditions of hydrolysis sufficiently drastic to hydrolyse a l l other pyranosidic linkages, two thirds  17 of the 2-0-[4-0-methyl-D-glucopyranosyluronic acid]- o< -g-xylose of a softwood xylsn w i l l remain unhydrolysed [ 3 8 ] . This resistance to hydrolysis can probably be extended to the acetal resulting from cleavage between carbons two and three of the uronic acid. The a b i l i t y to choose correct hydrolysis conditions, acid strength and duration, without an adequate analytical technique for a Smith polyalcohol^ i s severely hampered. One must contend with glycosidic linkages which are easily hydrolysed as well as stable acetals. No set conditions appear to be employed i n preparing Smith degraded residual polysaccharides.  An oat /3 -D-  glucan, oxidized and reduced, was hydrolysed with O.5N hydrochloric acid for 8 hours at room temperature [6  ]. Since there are no uronic acids in the  glucan the acid treatment might seem rather severe. on the other hand i s known to contain uronic acid. hydrolysed with 0.1N  Brome grass hemicellulose Its Smith polyalcohol was  hydrochloric acid at room temperature for 6 hours [6  ].  Although the isolation of small glycosylalditols was the object of the latter experiment, these conditions might not be expected to completely hydrolyse a l l acetals generated by the oxidation and reduction.  A further example where  possibly too much acid was used was. in the hydrolysis reported for lemon gum [1  ]. - Experimental data shox-js that optimum acid concentration i s 0.5N  than 1.0N.  rather  As well," optimum duration of acetal hydrolysis with minimum loss  of arabinose is 16 hours rather than the reported kQ hours. A normal acetal resulting from a periodate oxidized and borohydride reduced monosaccharide glycoside can be hydrolysed completely by 0.1N  sulfuric  Smith polyalcohol w i l l imply a periodate oxidized and sodium borohydride reduced polysaccharide.  18 acid at room temperature in eight hours.  These conditions indicate the  minimum acid strength required for analysis of Smith polyalcohols. s i d i c linkages v i l l be preserved.  Glyco-  This property v i l l allow isolation of  intact glycosylaiditoIs released during hydrolysis vhile retaining as much as possible of the unoxidized polysaccharide.  The degree of polymerization [D.P.]  v i l l remain high preserving important structural features for subsequent examination. Linkages other than the above may not be hydrolysed under the specified conditions.  Methyl 4-0-methyl- c< -D-glucuronate methyl ester vas oxidized  v i t h periodate and reduced with borohydride.  The resulting polyol, although  easier to hydrolyse than the related neutral parent glycoside, required hydrol y s i s with 0.5K sulfuric acid for 16 hours at room temperature.  This i s more  than sufficient acid strength to hydrolyse a considerable proportion of L-arabinofuranosyl linkages. well.  Some pyranosidic linkages w i l l be broken as  A problem exists therefore i n the structural analysis of some polymers.  Mild acid hydrolysis w i l l not cleave a l l oxidized and reduced uronic acids. Subunits which would be potential sites of periodate oxidation or methylation w i l l s t i l l be blocked.  Stronger acid on the other hand w i l l cleave glycosidic  bonds which have formed a constituent part of the overall structure of the polysaccharides.  There are two potential solutions available for this problem.  The f i r s t involves use of optimum hydrolysis conditions.  These conditions  involve control of hydrolysis parameters to achieve maximum acetal cleavage with minimal glycosidic hydrolysis. The second solution requires the reduction of uronic acids i n the polymer. This reduction eliminates those acetal structures which were d i f f i c u l t to hydrolyse.  Thus as much as possible  of the. intact original structure v i l l be retained by mild hydro l y t i c conditions.  19 The analysis of polyols and sugars outlined in the f i r s t portion of this thesis provides the f i r s t practical method for carrying out detailed analysis of any Smith polymer for optimum hydrolysis conditions. be varied; time, temperature, acid strength. Smith polysaccharide  A l l parameters may  Since only small amounts of  are required for hydrolysis, in normal practice only 5  milligrams or less, the l i m i t i s set by the a b i l i t y to handle precipitation of the residual polysaccharide.  Precipitating the residual polysaccharide as  hydrolysis proceeds, produces a fraction containing only high molecular weight products.  This fraction may then be dialysed and freeze dried i f desired. The  resultant residual polysaccharide  can be subjected to total hydrolysis, thus  avoiding complications which result from glycol aldehyde acetal formation. This technique has been successfully applied to lemon gum. Much milder conditions than those previously used are necessary for maximum y i e l d of residual polysaccharide with a minimum amount of unnecessary degradation.  THE OPTIMUM HYDROLYSIS OF OXIDIZED LEMON GUM Determination of optimum hydrolysis conditions for periodate oxidized and borohydride reduced lemon gum involves examination of parameters affecting the degree of hydrolysis.  Lemon gum polyalcohol was prepared as outlined in the  experimental section.  Small samples of the polyalcohol were dissolved in  water.  The normality was adjusted to a series from 0.05N to 1.0N by the .  addition of concentrated acid.  Each sample was allowed to hydrolyse for  twenty-four hours a t room temperature. The samples were neutralized and  20 residual polysaccharide  was isolated. Analyses were carried out to deter-  mine remaining unhydrolysed acetal linkages.  The sample hydrolysed with  0-5N  sulfuric acid showed almost total hydrolysis of linkages which could be residual acetal l i n k s .  1  Figure  7  strength was increased from 0.05N to  shows the reduction in glycerol as acid O.5K.  Removal of a l l glycerol can be seen to require a 0-5N  acid strength.  lower acid strength would have required a prolonged hydrolysis.  A  The acid  strength having been chosen i t was necessary to determine optimum hydrolysis time. Optimum hydrolysis time w i l l minimize loss of L-arabinose with respect to elimination of glycerol.  Lemon gum polyalcohol was dissolved in 0.5N  sulfuric acid and samples were taken at intervals to follow removal of components x-)ith respect to time. to accurately assess.  To a certain extent this i s a d i f f i c u l t thing  Fragments may be lost during hydrolysis which contain  unoxidized carbohydrates.  These fragments result from two forms of hydrolysis.  Hydrolysis occurring at acetal linkages may liberate Smith fragments, for example, arabinosyl glycerol or galactosyl glycerol. In addition there w i l l be hydrolysis which xtfill break normal glycosidic bonds such as arabinofuranose linked units.  The assay of how well acetals have been cleaved w i l l be i n -  dicated by residual glycerol since only glycosidic glycerol should be stable.  New gas-liquid chromatography columns were prepared in an identical manner •to those used i n i t i a l l y . Peaks eluted prior to glycerol were obscured by a pyridine t a i l i n g peak. The peak resulted from some form of adsorption or reaction between copper and pyridine. I t is at this time s t i l l not - clear what difference exists between the two tubing samples. Results with the second tubing were only slightly affected since the major peak produced by the polyol on hydrolysis and attributable to an acetal was the glycerol peak. It was possible to eliminate the t a i l i n g peak by switching to stainless steel columns. Since only ethylene glycol was affected extensive use was s t i l l made of copper tubing columns. I t has been subsequently discovered that copper tubing may be made passive by silvering the inner side with a mirror silvering solution.  FIGURE 7: GAS CHROMATOGRAMS OF TRIMETHYLSILYL DERIVATIVES FROM LEMON GUM SMITH POLYALCOHOL HYDROLYSED WITH VARIOUS SULFURIC.ACID '. STRENGTHS FOR 2k HOURS AT 20°C . . .  22  Additional end groups w i l l be formed i f large fragments are released by the action of acetal and glycoside hydrolysis. Under these conditions an increase in stable glycerol w i l l occur.  There exists however a large difference in  rate of hydrolysis between acetal and glycosidicly bound glycerol. By plotting the degree of hydrolysis against time, i t i s possible to follow the decrease in components as hydrolysis proceeds.  Examination of the plot w i l l indicate  the optimum hydrolysis time for the polymer. Figure  8  shows  gas chromato-  grams indicating progressive loss of glycerol. Figures 9 and 10 show loss of components plotted with respect to a response for galactose. loss of galactose.  There was, however,  This fact was clearly seen on hydrolysis of the soluble  fraction from the precipitation solvent.  The graphs indicate the time for  optimum yield of residual polysaccharide with a minimum of contaminating acetal structures. During the course of these hydrolysis studies i t was confirmed that residual glycerol present in the precipitated polysaccharide was chemically bound and not mechanically entrained or hydrogen bonded.  This confirmation was  de-  termined in the following manner. The polysaccharide obtained after 32 hours of hydrolysis was dissolved in water and dialysed against running water for 2k hours.  The polymer isolated by freeze drying was shown to contain the same  amount of residual glycerol. Clearly the glycerol was covalently bonded or i t would have passed freely from the dialysis tubing. Analysis of the graphs which plot decrease in components, indicated that removal of a l l but a residual amount of glycerol required 8 - 16 hours.  Hy-  drolysis for that period formed a polysaccharide containing minimal glycerol. These conditions are considerably milder than those reported by Jones and  23  0 HR. (Log often.)  GLYCEROL(l) GALACTOSE (6) ETHYLENE GLYCOL  -i—  v  |  1  i  1  1  r  2 HR.  FIGURE 8: GAS CHROMATOGRAMS OF TRIMETILYLSILYL DERIVATIVES FROM LEMON GUM SMITH POLYALCOHOL HYDROLYSED FOR DIFFERENT PERIODS OF TIME WITH 0.5N SULFURIC ACTJD AT 20°C  20r  0 12  4  8  16  TIME (HR)  FIGURE 9: RELATIVE G.L.C. PEAK AREAS FOR COMPONENTS 2,3,4 FROM LEMON GUM SMITH POLYALCOHOL  FIGURE 10: RELATIVE G.L.C. PEAK AREAS FOR COMPONENTS 1 [GLYCEROL] AND 5 [ARABINOSE] FROM LEMON GUM SMITH POLYALCOHOL  25 Stoddart [ l ] .  Their hydrolysis of borohydride reduced - periodate oxidized  lemon gum involved treatment at room temperature with one normal sulfuric acid for forty-eight hours* FIGURE 11 GAS CHROMATOGRAM OF LEMON GUM SMITH POLYALCOHOL HYDROLYSED FOR 32 HOURS WITH 0.5N SULFURIC ACID AT 20°C AFTER DIALYSIS 601 50-  40 30 20-  10 0  4  8  12  16  20  24  28  32  36  40  44  48  52  56 MIN  Evaluation of data presented indicates that even at the milder conditions used i n our work degradation of the polysaccharide has occurred. Only trace quantities of galactose were present i n the supernatant confirming that the galactan portion of the polysaccharide had been subjected to only mild degradation.  I f this were not the case one would have expected signif-  icantly larger losses of free galactose from the polymer. The galactose which was lost has been i n the form of low molecular weight Smith fragments such as galactosyl glycerol* supernatant.  Arabinose was found to a considerable extent i n the  Since arabinose i s furanosidicly linked, one would conclude  that i t had been produced from the polymer by two mechanisms*  Some free  26 arabinose resulted from direct hydrolysis of araban portions of lemon gum. The second portion of arabinose resulted from hydrolysis of Smith fragments since subunits such as arabinofuranosyl glycerol are susceptible to mild acid hydrolysis.  TABLE  A N A L Y S I S O F LEMON GUM AND  X  SMITH POLYALCOHOL AND  Glycerol  Lemon gum S m i t h Lemon  [16  Lemon after  polyalcohol  gum S I  hr.  - 0.5N  H  2  S O a -  20°C]  gum S m i t h p o l y a l c o h o l s u p e r n a t a n t J2 h r s . [ T O T A L H Y D R O L Y S I S ]  Lemon  gum S m i t h p o l y a l c o h o l  after  32.hrs.  Lemon  gum S I  [NO  FURTHER  supernatant HYDROLYSIS]  polyalcohol  Lemon gum S I I  [16 h r s . - 0.5N H S0 . - 20°C] 2  I T S HYDROLYSIS  O X I D A T I O N PRODUCTS  Mole  $  Arabinose  Galactose  27.1  14.0  8.8  2.8  14.6  82.7  , 21.1  21.8  57-1  75-3  20.8  4.0  32.8  11.1  49.2  3.2  13-7  83.I  4  A sample of lemon gum was oxidized, reduced and hydrolysed under optimum conditions to produce lemon gum SI. dicated above in Table X.  The composition of the polymer was as i n -  . Lemon gum SI was oxidized, reduced and hydrolysed  in a similar manner to that used i n i t s own preparation. designated lemon gum SII.  This product was  Analysis of the polyalcohol SII indicated a lower  percentage of arabinose than had been observed by Jones and Stoddart [ l ] .  The  27 retention of some arabinose is to be expected from reported substitution patterns.  The large difference observed may be related to increased acid  strength during their hydrolysis. More side chains v i l l have been removed from the galactan framework, exposing additional sites for oxidation by periodate.  With more acetals being formed during the oxidation and reduction  the polymer's galactan framework v i l l undergo greater depolymerization. Additional losses vere encountered during both the i n i t i a l and second oxidation-reduction operations.  Each of the two operations resulted in ap-  proximately forty percent weight losses*  These decreases cannot be attributed  to oxidation weight loss due to formic acid or other minor constituents. They must reflect a degree of autohydrolysis and base catalysed depolymerization occurring during the periodate oxidation and borohydride reduction.  Polyalde-  hydes are known to be susceptible to basic hydrolysis and depolymerization [ 3 9 ] . The polyalcohol can be degraded by two acid sources.  Acetic acid was added  to destroy the excess borohydride while oxidized uronic acids vere present in the polymer during reduction and dialysis.  28 SMITH FRAGMENTS The optimum yield of residual polysaccharide after Smith oxidation w i l l not necessarily produce an optimum yield of Smith fragments*  Degradation of  substituent units during hydrolysis i s reflected in the appearance of free sugars.  For example, any Smith fragment containing arabinofuranosyl  linkages  w i l l undergo hydrolysis under mildly acidic conditions. Examination of the alcohol-water soluble fraction from mild acid hydrolysis to lemon gum SI gave results presented in Table X.  The alcohol-water soluble  fraction obtained on hydrolysis to lemon gum SI contained large quantities of glycerol. This fragment arises from cleavage of arabinofuranose, arabinopyranose [blocked at position four] and galactopyranose [blocked at position two and/or s i x ] . I t i s known from our own and previous work that galactose i n lemon gum i s not substituted at C-2  hydroxyl.  The amount of glycerol present i n intact lemon gum SI alcohol-water solubles may be divided into two distinct classes. glycerol results from multiple oxidations.  The largest portion of  For example, when side chains  terminated in k-Q[4-0-methyl- d-D-glucopyranosyluronic acid]-L-arabinose  are  oxidized, both sugars w i l l be cleaved and free glycerol [from arabinose] w i l l be released on mild hydrolysis.  The second source of free glycerol on mild  hydrolysis arises from glycosidic hydrolysis of labile furanosidic linkages. Two  complications affect the yield of glycerol. Stable uronic acid de-  rived subunits may effect liberation of glycerol while s t i l l forming part of the soluble fraction. Secondly, glycolaldehyde  formed by periodate cleavage  can readily complex with free hydroxyIs to form an acetal blocking group.  29 The complexity of this problem was mentioned earlier in discussion of the need for t o t a l hydrolysis during analysis of periodate oxidized polymers* Substitution such as this results i n chromatographic complexity. These acetals, as new components, have unique retention times and molar responses. A possible but untried solution to this problem may be to hydrolyse i n the presence of a large amount of v i c i n a l d i o l [i.e. butane -2,3-diol].  It is  possible that added d i o l w i l l act as a scavenger for available glycol aldehyde. Glycerol also exists i n the alcohol-water soluble fraction from lemon gum SI i n the form of glycosidicly bound glycerol. may be regarded as structurally significant. from studies of this material. in acetone-methanol [1:1].  I t i s this material which  Two conclusions may be drawn  A portion of the soluble fraction was dissolved  This soluble material contained low molecular  weight monosaccharide fragments i n a free and glycol aldehyde combined form as well as small amounts of arabinose, galactose and a single glycerol containing Smith fragment.  The Smith fragment was a galactopyranosyl glycerol.  No other Smith fragments could be positively identified among the other trace components. The remainder of the original SI soluble fraction [insoluble in acetonemethanol] was shown by GLC to contain no low molecular weight Smith fragments. It contained small amounts of low molecular weight arabinose and galactose oligosaccharides but the bulk of the sample was higher molecular weight oligosaccharides terminated by a glycerol moeity*  This was clearly seen  when samples of acetone-methanol insolubles were hydrolysed and checked for reducing power.  20 The presence of high molecular weight galactan oligosaccharides terminated by glycerol provides further proof that the internal galactan framework o f lemon gum contains some galactose units substituted at positions six and one only. FIGURE 12: GAS CHROMATOGRAM OF THE SUPERNATANT FROM THE ISOLATION OF LEMON GUM SI BEFORE AND AFTER TOTAL HYDROLYSIS [at 52 min. the temperature was raised at 3°/min. to a f i n a l temperature of 250°C] SOLUBLE TOTAL HYDROLYSIS COMPONENT (2) THREITOL AltABINOSE  GALACTOSE  (4)  AJ 24  12  36  44  48  52  56  SOLUBLE FROM MILD HYDROLYSIS (2)  GLYC  GAL-GLYC ARAB THRE GAL  12  16  —i  20  i  24  28  I  I  —  32  i  36  40  52  56  • i  60  • // • 64 90  REDUCTION OF URONIC ACIDS As indicated earlier uronic acids were a potential source of error i n evaluation of polysaccharide structure by mild acid hydrolysis of the Smith polyalcohol.  There exists a solution to the problem of resistance to hydrolysis  of uronic acids or acid containing subunits.  Reduction of the uronic acids  prior to oxidation w i l l result i n an acetal which i s easily hydrolysed.  31 Two basic approaches exist for the reduction of an acidic polysaccharide. They involve reduction of either the free uronic acid or an ester of the acid. The most common reduction of a polysaccharide i s performed on the f u l l y methylated product.  This i s readily accomplished with lithium aluminum hydride [LAH]  in tetrahydrofuran [THF] or similar dry ether-type solvents.  I t does not how-  ever provide a route to free unsubstituted polysaccharides. One use that has been made of an ether blocking group i s contained i n a paper by Rees and Samuel [40].  Alginic acid was esterified with diazomethane,  trimethylsilylated with hexamethyldisilazane and trimethylchlorosilane [15] and then treated with LAH in cold THF.  The product separated slowly as re-  duction proceeded. Unfortunately LAH i s a poor reagent to use with sugar derivatives containing or forming unblocked hydroxyl groups. strong aluminate complexes with these free bydroxyls.  LAH forms  I t i s often necessary  to esterify the product to free the substrate from residual reducing agent. Some workers have used diborane as their reducing agent.  I t was noted  by Smith and Stephen [hi] that metal hydrides failed to reduce acylated acidic polysaccharides.  Ester groups were attacked f i r s t and as a result acidic  polysaccharide was precipitated from the reaction mixture.  Brown [42] however  has shown that diborane reduces carboxyl groups i n preference to ester groups. Smith and Stephen [41] f i r s t showed that diborane could be an effective reducing agent for acylated acidic polysaccharides.  They also noted that the  propionate derivative of alginate was more soluble i n diglyme and hence reduced more easily. Other workers have used this procedure to reduce acidic polysaccharides. Hirst et a l [43] reduced alginic acid by Smith's method of in situ generation of diborane.  This reagent reduced 90f> o f the uronic acids and produced $.2$  32 residual n-propoxy group.  The degree of reduction was slightly higher than  that which Smith and Stephen found.  Ross and Thompson [44] treated an acetyl-  ated 4-0-methyl-glucuronoxylan with externally generated diborane, reducing 90$ of the carboxyl groups. Manning and Green [45] compared the reduction of alginic acid by d i f ferent methods. One of these methods was the use of diborane generated externally on di-O-propionyl alginic acid.  A large excess of diborane [6.5  moles per mole of carboxyl group] was used.  The same polymer was also re-  duced by in situ generation of diborane from the addition of boron trifluoride to sodium borohydride.  Thus any difference between these two reduced polymers  could be attributed to the effect of sodium borohydride and boron trifluoride in the polymer solution.  This use of externally produced diborane showed  for the f i r s t time that an ester could be reduced to an ether by diborane alone.  They explained this reduction by a modification of the mechanism  suggested by P e t t i t and Kasturi [46 ] to account for the reduction of esters to ethers with diborane and boron t r i f l u o r i d e .  The proposed mechanism ex-  plained why more n-propionyl esters were reduced to n-propyl ethers with diborane generated i n s i t u than externally.  As the sodium borohydride con-  centration was reduced, boron trifluoride was f e l t to catalyse the following reaction: FIGURE 13:  THE REDUCTION OF ESTERS TO ETHERS WITH AN EXCESS OF DIBORANE [adapted from Pettit and Kasturi [46]]  BH3  9  R-C-OR  DH3  5==i  R-C-OR •  BH3  BH,  \  n  1  2  ck,:H__ R-CH-OR  0H2  O ,.H R-C-OR  R-C-OR •  •BH  RCHgOR *  BH2  b  !' BH3 S^*-  BH2  4 . R-CH-OR  r  h  c  TABLE XII ANALYSIS 0? THE POLYSACCHARIDES OBTAINED FROM ALGINIC ACID BY DIFFEREHT REDUCTION PROCEDURES  Functional Group Reduction Procedure  Polymer  Anhydrouronic acid [<f>f  b n-Propionyl ester [$]  Total propoxygroup [$>]  0.00  o.oo  0.00  Negative  c  Alkali-labile n-propoxygroup [#]  Esterified uronic^ acids  Degree of polymsrisatii  Methyl di-O-propionyl alginate with LiBH^  Bl  Dl with LiBH  B2  10.4  0.00  3.9  0.25  Negative .  Di-O-propionyl alginic acid with B2HS  Dl  11.0  7.6  3-5  2.3  Negative  111  Di-O-propionyl alginic acid with NaBHj + BF  D2  I6.3  3.2  7.5  2.7  Negative  66  4  3  Determined by CO2 evolution. e  Determined by a Zeisel method. Contained  Q.kQfja  6.3  e  Determined by the hydroxylamine-ferric perchlorate colour test. cl. Determined by osn»metry of the triacetate in 1,1,2-trichloroethane.  methoxy-group from diazomethane methylation of di-O-propionyl alginic acid.  103 83  a  34 Less uronic acid i s reduced when diborane i s generated in s i t u .  Since  boron trifluoride catalyses the reduction o f n-propionyl esters to ethers, i t may also catalyse reductive cleavage of n-propionyl esters* An increased rate of reductive cleavage would have caused the polymer to precipitate sooner thereby lowering the reactivity of the carboxyl group. Sodium ions present i n the i n situ diborane generation may form sodium carboxylate which i s not reduced by diborane. Manning and Green [45] suggested that the differences in D.P. were a result of the strong Lewis acid, boron t r i f l u o r i d e .  The degree o f depolymer-  ization was dependent on how fast and i n what quantity boron trifluoride was added. The use of diborane for the reduction of acidic polysaccharides prior to periodate oxidation has several serious drawbacks. Even when diborane was generated externally the D.P. of di-O-propionyl alginic acid was reduced from 158 to H I , a drop of 30 percent. Although data are not available, a corresponding drop i n D.P. might be expected to occur on reduction o f other acidic polysaccharides, neutral linkages being more susceptable to acidic hydrolysis. The introduction of ether blocking groups i s the most serious drawback to u t i l i z a t i o n of this method of reduction. In the best diborane case there was 3*5$ pro poxy ether substitution.  This represents i n i t s lowest case an  conversion of original n-propionyl ester to n-propyl ether. a linear 1 — • 4 linked polyuronide.  iQjo  Alginic acid i s  Its di-O-propionyl derivative w i l l have  two out of ten esters reduced to ethers.  For every five uronic acid groups  oxidizable by periodate i n the original polymer only slightly greater than three of the five w i l l be oxidizable after reduction with diborane. In  addition esterification must be employed to produce a soluble polymer and saponification i s necessary for removal of the ester groups. Some fractionation may occur on esterification, altering slightly the composition of the polymer.  Saponification may produce a small amount of alkaline degradation.  It i s important to stress again that diborane suffers severely as a potential reagent for preparing neutral polysaccharide for periodate oxidation. The most serious drawback is the introduction of ether linked n-propyl groups. The second method employed commonly for conversion of acidic into neutral polysaccharides involves reduction of uronic acid esters \*ith a metal hydride.  Two main esters are used, methyl and hydroxyethyl.  Methyl esters are generally formed in one of two ways. A polysaccharide can be esterified by dispersion in methanol containing concentrated sulfuric acid or hydrogen chloride. Bishop and Zitko [47] in an analysis of sunflower pectic acid esterified the galacturonan in methanol containing sufficient sulfuric acid to make the concentration 2N. The mixture was kept at 0-2°C for nineteen days before isolation of the esterified product.  This pro-  cedure resulted in a significant reduction in D.P. of the polysaccharide. A procedure such as this would be extremely detrimental to a polymer containing labile furanosidic linkages. The second method for forming the methyl ester of an acidic polysaccharide involves the use of diazomethane in an ethereal suspension of the polymer. Direct esterification with diazomethane i s not a practical route in preparation of a neutral polysaccharide.  Methylation w i l l  occur at a number of alcoholic functions in the polymer, interfering with subsequent periodate oxidation of the reduced polysaccharide.  Esterification with  diazomethane i s best carried out on the acetylated or propionylated polysaccharide.  The esterified polymers have very limited numbers of free hydroxyl  groups capable of methylation.  56 Hydroxyethylation  is an alternate method of esterifying uronic acids.  This reaction involves esterification of the uronic acid v i t h ethylene oxide. -which reacts directly v i t h the polysaccharide acid in aqueous solution. There i s reported to be no evidence for hydroxyalkylation of alcohol groups by ethylene oxide.  An investigation on a model compound would however be  a useful undertaking since isolation of polyethylene glycol esters has been reported in the literature [48],  METAL HYDRIDE REDUCING AGENTS Sodium borohydride has been reported to give good reductions of ethylene oxide esters [49 ]•  Attempts to u t i l i z e this reagent have generally been  unsuccessful in this laboratory.  Sodium borohydride saponifies the esters  and the acids and salts produced are not readily reducible. Potassium borohydride has also been used to reduce esterifled uronic acids [ 5 0 ] . Bishop and Zitko [47] in their paper on the reduction of galacturonan form sunflower pectic acid analysed in d e t a i l the effect of solvent on potassium borohydride efficiency.  They examined water, Q0% aqueous  methanol, and &0$ aqueous dimethyl sulfoxide. Their results indicated that reductions in 80# aqueous methanol were most efficient. De-esterification was suppressed, a reasonably high proportion of galacturonic acid was reduced and recoveries were good. In water, a high proportion of galacturonic acid was reduced but there was considerable de-esterification and recoveries were low.  The low recovery was f e l t to result from degradation of esterifled gal-  acturonan in alkaline media [ 5 1 ] . In  BOJ»  aqueous dimethyl sulfoxide [DMSO],  de-esterification was greatly suppressed. The greatest reduction was obtained  37 when a partially reduced product was reacted. offset by low recovery.  These advantages however were  Indeed i t i?as reported that when this solvent was  used for the f i r s t reduction of a f u l l y esterified galacturonan, complete degradation occurred.  No product precipitable by methanol could be obtained.  Reductions were found to be only slightly more efficient with methyl than hydroxyethyl esters. Bishop and Zitko [47] examined the efficiency of esterification.  As  galacturonan was reduced heterogeneous esterification with diazomethane became less efficient.  At a galacturonic acid content of 11$, no more than 45$  of the carboxyl groups could be esterified. dried to provide a large surface area.  The substrate was even freeze  Esterification with ethylene or pro-  pylene oxide gave good results but the products when precipitated by organic solvents, tended to form gels which were d i f f i c u l t to handle. however to esterify residual carboxyl groups i n highly reduced  I t was possible galacturonans  with diazomethane i n dimethyl sulfoxide. In this procedure the esterified product did not need to be isolated before reduction since the same solvent could be used. However, the main drawback to using the method proposed by Bishop and Zitko was the accumulated decrease in degree of polymerization [from 270 to 21]. Lithium borohydride has also been used to reduce acidic polysaccharides. This reagent was f i r s t used by Rees and Samuel [40 3 for the reduction of a l ginic acid.  A heterogeneous reaction in boiling THF was carried out on both  2-acetoxy-ethyl di-O-acetylalginate and methyL-di-0-acetylalginate.  Special  precautions were taken to ensure that the polysaccharides were completely accessible.  The authors report that reduction yielded a polysaccharide contain-  ing 4.7$ anhydro uronic acid.  38 Manning and Green [45] i n their examination of the reduction of alginic acid, reduced methyl di-O-propionyl alginate by the method of Rees and Samuel. Methyl di-O-propionyl alginate [89$ of i t s uronic acids esterified] was however soluble in tetrahydrofuran.  A l l n-propionyl and methyl esters were reductively  cleaved in the reduced polysaccharide. converted to glycitols.  A l l reducible terminal sugar units were  In addition the authors reported a kyf> reduction of  uronic acid carboxyl group to primary alcohols* Rees and Samuel suggested that reduction of functional groups on polysaccharides under heterogeneous conditions i s incomplete because molecules of the solid phase are inaccesible. Manning and Green [45] however showed that 94$ of the hemiacetal end group were reduced under heterogeneous conditions. It was f e l t that lowered reactivity was not due to lack of accessibility. Iheir alternate explanation i s based on activation energies.  Addition of  solvation energy for heterogeneous reaction to the activation energy s t i l l makes the total energy for reaction of aldehydes lower than that for carboxylic acids.  Hence aldehydes react more rapidly than carboxylic acids under hetero-  geneous conditions. I t should be noted that there may be vast differences i n accessibility.  The reducing end group of a linear molecule such as alginate  should, i f well dispersed, be accessible to reduction. Uronic acids on the other hand can be strongly associated with hydroxyl functions of other polymer chains.  Areas of polysaccharide may thus be rendered inaccessible.  Aspinall and McKenna [52] esterified Araucaria b i d w i l l l gum with ethylene oxide.  Acetylation and subsequent reduction by the method of Rees and Samuel  gave a product polysaccharide with less than 1$ uronic acid.  39 EVALUATION OF NEW ROUTE TO REDUCED POLYSACCHARIDES The method of Rees and Samuel [kO ] for reducing acidic polysaccharides is obviously effective.  I t does however involve several reactions; poly-  saccharide esterification, uronic acid esterification. reduction and saponification.  Each reaction may have to be repeated and possible fractionation  or degradation of the polysaccharide can occur.  Removal of any of these steps  might increase the chance of obtaining a neutral polysaccharide which retains potential periodate oxidation sites and i s closely related to the original polymer. To prevent unnecessary degradation, esterification should not be carried out in methanol with an acid catalyst because of D.P. reduction. Diazomethane i s unsuitable on the unsubstituted polymer due to ether formation. oxide has shown none of these detrimental effects.  Ethylene  I t has certain advantages;  water soluble, volatile, i f hydrolysed to ethylene glycol, i t may be readily removed by dialysis, and i t also inhibits any bacterial degradation.  Its two  disadvantages are long reaction time for esterification as mentioned previously and possible ether  formation.  The latter point requires further evaluation,  however no etherification has thusfar been reported. Certain reducing agents are unacceptable i n the reduction of acidic polysaccharides. Diborane to be effective requires a homogeneous reaction. But, soluble ester derivatives are reduced in part to ethers which block subsequent periodate oxidation. S i l y l blocking groups which also make a soluble derivative, bydrolyse as the reaction proceeds producing a heterogeneous reaction.  Lithium aluminum hydride complexes free hydroxyls, the result being  a heterogeneous reaction. The complex i s d i f f i c u l t to break down, introducing  ko problems in isolating the reduced polysaccharide. ducing agents i s limited to the borohydrides.  Thus the choice of re-  Their a b i l i t y to reduce esters  and acids is increased with increasing covalent character.  Lithium boro-  hydride best f i t s this requirement [53]. In the work conducted in this laboratory, choice of solvent was a departure from those previously employed.  I t was shown by Bishop and Zitko [47] that  8C$ aqueous DMSO greatly suppressed de-esterification and a high proportion of reduction occurred.  Since only the hydrolysis of reduction complex involves  water in a borohydride reduction i t was decided to use pure DMSO. Addition of water after complex formation w i l l complete the reaction. A large number of polysaccharides are soluble in this reagent and tests showed only a slow reaction with lithium borohydride.  The only disadvantage in the use of  DMSO was reported decomposition of polysaccharide [47]-  An explanation as to  why depolymerization occurs was not indicated, however there are two possibilities.  Alkaline cleavage by /3 -alkoxycarbonyl elimination could lead to  depolymerization.  DMSO is effective in preserving methyl ester necessary for  this elimination. However, i t should not greatly increase the a b i l i t y to co-ordinate transition states or leaving groups in comparison to water or methanol. When methyl 4-0-methyl c< -D-glucuronic acid methyl ester was reduced with lithium borohydride in DMSO there was gas chromatographic evidence for the presence of small amounts of by-product.  The by-product was tentatively  assigned the structure of the 4,5-dehydro-D-glucuronate [54]. this structure w i l l require further analysis.  Confirmation of  The proposed mechanisms for the  elimination in uronic acids are outlined in Figure 14.  in  FIGURE Ik THE /3 -ELIMINATION MECHANISMS FOR URONIC ACIDS [It has been recently suggested that the eliminations proceed via an ElcB mechanism [ 5 5 3 3  EtO  * H  E2:  — OPh  EtO ElcB:  X-  X-  H  b  OPh  ;=  X-  OPh  The second possible explanation i s that DMSO altered the precipitation characteristics of pectin. Work in our laboratory on isolation of methylated polysaccharides from DMSO solutions indicated changes i n ease of precipitation. Methylated polysaccharides in DMSO occasionally failed to precipitate on addition to water. Methylation mixtures poured directly into dialysis tubing with no addition of water to the DMSO resulted i n cases of almost complete loss o f partially methylated polysaccharides.  Dilution of methylation mixture with  water, followed by dialysis to remove DMSO resulted in easily isolated methylated polysaccharide i n excellent y i e l d . Lemon gum was u t i l i z e d as a model substance to evaluate reduction of esterified polysaccharides dissolved in DMSO. Lithium borohydride was the reducing agent employed. Polysaccharide reduction was monitored by G.L.C.  42 Inclusion of erythritol during polysaccharide hydrolysis gave quantitative results on retrieval of constituent monomers. The results indicated additional d i f f i c u l t i e s in obtaining a neutral polysaccharide by reduction.  Esterification  with ethylene oxide requires reaction for a period of ten days [4-7 3 • However, loss of arabinose occurred during the esterification of lemon gum. TABLE XIII CARBOHYDRATE ANALYSIS OF LEMON GUM AND ITS REDUCED PRODUCT  Arabinose  Rhamnose *  Glucose  Neutral Sugar Recovery  63.9  —  66.6  66.8  —  4-0-Methyl Galactose Glucose  Lemon Gum  32.7  3.4  —  Lemon Gum OCH2CH2OH  29.9  3-3  —  Lemon Gum OCH CH OH 1 x LiBH  25.0  1.5  15-9  51.3  5.3  84.0  Lemon Gum 2 x OCH^CHaOH 2 x LiBH  26.25  1.7  18.8  48.6  4.8  89.5  2  2  —  4  4  Calculations based on aldobiouronic acid survival during hydrolysis i n dicated quantitative levels of reduction.  These calculations were based on  survival of aldobiouronic acids during hydrolysis as well as degradation of individual sugars.  The results accounted for a l l sugars present prior to hy-  drolysis and correlated well with recovery of neutral sugar i n hydrolysate. The loss of some arabinose during esterification was not unexpected. The polysaccharide was deionized hence a l l uronic acids were free to cause hydrolysis.  Although the large amount of ethylene oxide present provided a  substrate for reaction of ionized acids, there was s t i l l water present and cleavage of labile arabinose linkages could occur.  As the reaction proceeded  the pH decreased to 7 and hydrolytic action was reduced.  Hydrolysis could be  suppressed by substitution of DMSO for water during esterification.  I t was  necessary however to isolate the polymer before reduction as residual ethylene oxide reacted with lithium borohydride. Lithium borohydride was added as a DMSO solution to lemon gum 2-hydroxy ethyl ester i n DMSO. Hydrogen was released during the i n i t i a l vigorous reaction through a mercury gas trap to exclude moisture.  By the 15th hour of  reduction a thick gel had formed. This gel could be partially broken down by s t i r r i n g or gentle shaking. several days.  A slow evolution of hydrogen continued for  The solution was then treated with small portions of dilute  acetic acid to destroy excess lithium borohydride. with water and dialysed.  The solution was diluted  Saponification occurred during isolation making re-  es t e r i f ication necessary for continued reduction. The i n i t i a l ethylene oxide esterification did not f u l l y block a l l uronic acids.  The lack of complete reduction with an excess of lithium borohydride  suggested either a problem of accessibility or hydrolysis of esters during reduction.  Similar results i n subsequent reductions could be attributed to effects  of either of these factors. dialysis.  Some losses occurred as a result of work-up and  However yields were generally good.  The mild nature of the reaction was evident from the yields of L-arabinose in relation to D-ga lactose on subsequent reduction. Yields of arabinose i n creased i n relation to galactose as the polymer was further reduced.  In addition,  losses which one might expect i n arabinose should have been accompanied by  losses in 4-0-methyl-D-glucuronic acid, since this acid is the major acid lost on mild hydrolysis [1 ], Acid hydrolysis of lemon gum resulted in reduction of 4-0-methyl compared to unsubstituted glucuronic acid. On reducing lemon gum ester v i t h lithium borohydride in DMSO, the percentage of 4-0-methylD-glucose increased as reduction proceeded. Had any oligosaccharides been formed during reduction, they would have been lost during dialysis.  This would  have resulted in lower 4-0-methyl-D-glucose levels as was the case on mild acid hydrolysis. The reduction method as evaluated on lemon gum was successful in reducing 75$ of available uronic acids.  I t seems probable that this method would be  more successful on smaller polymers.  Central portions of a large molecule  l i k e lemon gum may be inaccessible to a reagent such as lithium borohydride. Borohydrides can complex readily with free hydroxyIs, blocking access to the interior of this highly branched polymer. Reduction with lithium borohydride i n DMSO appears to be very useful for convenient detection of the presence of different uronic acids such as glucuronic and 4-0-methyl glucuronic.  I f the structures of the major aldobiouronic  acids are known, then accurate carbohydrate analyses are also conveniently obtained.  The method involves a minimum number of reaction steps and hence would  appear to be a very convenient procedure for i n i t i a l examination of polymer uronic acids.  I f higher degrees of reduction occur in lower molecular weight  or linear polysaccharides, increased use of this reduction sequence could be envisioned.  SIGNAL ENHANCEMENT USING TRIMETHYLSILYL [TMS] DERIVATIVES AND UTILIZATION OF TMS AS A PROBE FOR DETERMINATION OF NUMBER OF HYDROXYLS IN A COMPOUND During gas-liquid chromatographic examination of products from total hydrolysis of periodate oxidized carbohydrates, samples of per silylated polyol were examined by proton magnetic resonance [p.m.r.].  Spectra for car-  bohydrate protons were very complex because of similar electronegativity and environment [see Figure 15].  I t was noted however that s i l y l methyl proton  resonances were very sharp and in most cases, unless s i l y l ethers were in identical positions, resolved from one another. This discovery indicated a possible technique for characterization of polyhydroxy compounds.  Introduction of TMS ethers increases the number of  hydrogens on each hydroxyl by a factor of nine. Consequently detection of the number of hydroxyIs i n a compound i s possible even though the remaining proton signals are lost i n background noise.  In addition, compounds which are  of unknown structure may be given some p a r t i a l assignment of structure. I t may be possible to determine number of hydroxyIs, their positions and degree of substitution at the parent carbon atom. Any compound containing a s i l y l ether would be amenable to this analytical procedure. In examinations of carbohydrate molecules, knowing the number of hydroxyIs can be a useful addition to information about that compound. The analysis of compounds on G.L.C. can result in a confused picture because of wide variation in retention times for partially methylated sugars and polyols.  Although i t  i s possible to bypass many d i f f i c u l t i e s with mass spectrometric analysis, i t may be d i f f i c u l t to distinguish between components by their mass spectra alone.  k6  For example, two methyl sugars may have similar G.L.C. retention times. The mixed mass spectra might be very d i f f i c u l t to interpret.  The s i l y l p.m.r.  spectra however may show two anomers and indicate something about their substitution. FIGURE 15: 100 MH P.M.R. SPECTRUM FOR THE ERYRTHRITOL PROTONS IN TRIMETHYLSILYLATED ERYTHRITOL Z  ERYTHRITOL .' PROTONS (TMS ERYTHRITOL) - J.:  100MHz 1  4.0  X  I l l '  I  —L.  _l  3,0 p. p.m. Li_L_J  I I I I  Figure 16 i s a l i s t of compounds which have been subjected to s i l y l ether analysis indicating the number o f peaks and their degree of separation. Primary trlmethyl s i l y l ether protons resonate at higher f i e l d than secondary trimethyl s i l y l ether protons.  Barring shielding effects, the re-  sonance signal for a trimethyl s i l y l group w i l l reflect electronegativity of the hydroxyl to which i t i s attached.  The wide diversity of signal value can  be seen in almost complete separation of signal for eight different s i l y l ethers present in an unresolved collection of two arabinose TMS ether peaks. This p.m.r. technique provides a non-destructive check on component purity as eluted from the gas chromatograph.  hi  FIGURE 16: -COMPOUNDS SUBJECTED TO P.M.R. [100 MH ] ANALYSIS OF TRIMETHYLSILYL PROTONS Z  TRIMETHYLSILYL • COMPOUND  TRIMETHYLSILYL RM.R. SPECTRUM  k& FIGURE 16: » COMPOUNDS SUBJECTED TO P.M.R. [100 MH ] ANALYSIS OF TRIMETHYLSILYL PROTONS [Continued] Z  TRIMETHYLSILYL COMPOUND  ARABINOSE Opeoks)  GLUCOSE (peok I) 2 mg. a or/S-FURANO  GLUCOSE (peak 2) a-D-PYRANO .  GLUCOSE (peak 3) /9-D- PYRANO  METHYL 2,3-DI-O-METHYLo-D-GLUCOPYRANOSIDE  METHYLfi-DGLUCOPYRANOSIDE  TRIMETHYLSILYL RM.R. SPECTRUM  TMS  k9  Obviously there w i l l be situations where overlap occurs between t r i methyl s i l y l ether proton resonances.  Perfect overlap w i l l occur in cases  such as erythritol where there are two identical primary and two identical secondary hydroxyl positions. tensity.  This results i n only two peaks of equal i n -  Normally some hint of overlap w i l l be v i s i b l e .  For example, two  primary peaks of glycerol overlap but they have, as a result, twice the signal intensity of the secondary TMS ether. In many cases where two compounds produce peaks that overlap, such overlapping w i l l not be absolute making i t obvious that there are two contributing components. In cases where some signals are well resolved, integration of component peak area w i l l indicate percentage composition. This technique for examination of s i l y l methyl proton resonances provides a useful tool i n structural elucidation and detection of component mixtures eluted as a single gas chromatographic peak.  SIGNAL ENHANCEMENT An additional benefit can arise from examination of p.m.r. spectra of TMS ethers. A nine-fold increase in number of protons occurs on s i l y l a t i o n of each hydroxyl position. ulators.  In effect, TMS ethers act as b u i l t - i n signal accum-  The resonance peak for TMS ether protons i s very sharp.  The signal  undergoes only weak proton-proton interactions because of the oxygen bridge between carbon and s i l i c o n .  Normally, proton resonances are interacting,  forming multiplets of lower intensity.  TMS resonances are unaffected by  hydrogen bonding which can broaden hydroxyl proton spectra of alcohols making  50 them even more d i f f i c u l t to observe than normal proton resonances. Jackman [56]  discusses various factors exerting influence on the appearance of  ethanol's hydroxyl proton.  I t can be broadened, s p l i t into a multiplet or  increased i n signal size by the presence of water. TMS protons give a sharp signal on the low side of tetra methyl silane. The signal is nearly ten times as large as the hydroxyl proton signal would have been had i t been present.  This signal enhancing effect allows for ex-  amination of very small samples of TMS ethers.  The number and structure of  hydroxyIs may be determined. A small peak occasionally occurs in glucose TMS ether gas chromatograms. I t represents only about one percent of t o t a l peak area.  Although i t was  thought to be a furanoside anomer i t could not be ruled out that i t was to under s i l y l a t i o n or an anhydro form of glucose.  due  Gas chromatography of a  large amount of TMS glucose resulted in collection of only two milligrams of this compound. Examination of the p.m.r. spectra of TMS protons indicated a compound containing five hydroxyIs.  This ruled out under s i l y l a t i o n or an-  hydro sugars as possible causes of this component peak. The G.L.C. retention time of this component agrees with no other common sugar.  These points strongly  indicate the minor component to be o( or /6 TMS glucofuranoside.  This infor-  mation regarding a minor component was obtained on two milligrams of sample. P.m.r. spectra for the remaining protons of the glucose molecule were completely obscured by spectrometer noise. I t w i l l be possible using TMS proton spectra to obtain number and nature of hydroxyIs on very small samples isolated by gas chromatography. Purity of single peaks may also be evaluated.  51 TRIMETHYLSILYLATION OF GALACTURONIC ACID During studies of lemon gum a method for estimation of aldobiouronic/^ acids was investigated. Work has been done on estimation of f u l l y methylated aldobiouronic acids and on silylated methyl esters of aldobiouronic acids [57]• It was hoped that a procedure could be developed for separation of f u l l y s i l y lated aldobiouronic acids. A reagent was selected which would react with alcohol and acid substituents to form corresponding TMS ethers and esters. The reagent chosen was bis-trimethylsilyl-acetamide [BTMSA] which was known to react with amino acids to form s i l y l esters. Galacturonic acid was chosen as a model compound containing the necessary alcoholic and acidic functions. Silylations were carried out with BTMSA in various solvents; pyridine, dimethyl formamide, dimethyl sulfoxide, hexane and dioxane.  Subsequent gas  chromatography [SF-96 l i q u i d phase] indicated four components. Two components were well resolved, two overlapped to form a central pair of peaks.  Since  under s i l y l a t i o n i s known from our own and other work [58] to produce extra peaks, a check was carried out on the eluted peaks.  Possible lactone form-  ation had to be ruled out as well, since lactones are known to have shorter retention times than parent acids. Other liquid phases were evaluated for gas chromatography of the silylated galacturonic acids. None proved more efficient than standard SF-96 liquid phase used in earlier studies.  Preparative gas chromatography was convenient  with this liquid phase because of a high maximum operating temperature.  Small  samples of the four galacturonic acid peaks were isolated and analysed by p.m.r. spectrometry.  This preliminary examination indicated that each component con-  tained five trimethyl s i l y l groups.  This fact eliminated lactones or under s i l y l -  ation as causes for any of the four components isolated.  For complete p^m.r. spectral analysis large scale sample separations •were carried out.  Analysis of the p.m.r. spectrum  for ring protons should  confirm furano and pyranoside forms for the four peaks.  The well separated  f i r s t and last peak were isolated in a quantity large enough to confirm their structures by p.m.r. spectroscopy. components was extremely d i f f i c u l t .  Large scale separation of central peak In addition degradation was observed due  to prolonged sample collection. An experiment was carried out on an easily isolated peak to determine the exact cause of degradation.  A sample of peak four of TMS galacturonic acid  was treated with a small amount of methanol.  The TMS proton region of the  p.m.r. spectra was monitored prior to and after addition of methanol. spectra revealed a rapid loss of TMS ester.  The  I t i s apparent that although  precautions were being taken to keep the samples dry, loss of TMS ester was taking place. dry chloroform. changes.  Samples of peak one of TMS galacturonic acid were dissolved in Spectra run at intervals over twenty-four hours underwent  The changes in peak height were indicative of changes in substitu-  tion of the galacturonic acid isomer isolated.  These latter changes were  relatively small in comparison to the complete loss of s i l y l ester observed when methanol was added. Losses, however, were large enough to confuse i n terpretation of spectra in an unknown  compound.J  It was possible to completely assign structures to components one and four of TMS galacturonic acid by interpretation of their p.m.r. spectra. Component one was an o<-furanoside and component four used the /3 -pyranoside of galacturonic acid. Component two was isolated in sufficient quantity to indicate that a l l couplings between protons were very small.  This finding is  consistent with a /S- furanoside structure where a l l ring protons can have ~  90  52 For complete p.m.r. spectral analysis large scale sample separations •were carried out.  Analysis of the p.m.r. spectrum  for ring protons should  confirm furano and pyranoside forms for the four peaks. The well separated f i r s t and last peak were isolated in a quantity large enough to confirm their structures by p.m.r. spectroscopy. components was extremely d i f f i c u l t .  Large scale separation of central peak In addition degradation was observed due  to prolonged sample collection. An experiment was carried out on an easily isolated peak to determine the exact cause of degradation.  A sample of peak four of TMS galacturonic acid  was treated with a small amount of methanol. The TMS proton region of the p.m.r. spectra was monitored prior to and after addition of methanol. spectra revealed a rapid loss of TMS ester.  The  I t i s apparent that although  precautions were being taken to keep the samples dry, loss of TMS ester was taking place. dry chloroform. changes.  Samples of peak one of TMS galacturonic acid were dissolved in Spectra run at intervals over twenty-four hours underwent  The changes in peak height were indicative of changes in substitu-  tion of the galacturonic acid isomer isolated.  These latter changes were  relatively small i n comparison to the complete loss of s i l y l ester observed when methanol was added. Losses, however, were large enough to confuse i n terpretation of spectra i n an unknown compound. It was possible to completely assign structures to components one and four of TMS galacturonic acid by interpretation of their p.m.r. spectra. Component one was an c<-furanoside and component four used the /3 -pyranoside of galacturonic acid. - Component two was isolated in sufficient quantity to indicate that a l l couplings between protons were very small.  This finding is  consistent with a ft- furanoside structure where a l l ring protons have small  53 dihedral angles.  Sample purity, however, was insufficient, for assignment  of ring protons.  Low yield and peak overlap made impossible the large scale  isolation of pure component three. had a coupling constant of 4.3 Hz.  The anomeric proton of this third fraction The TMS proton spectra indicated a mixture  of at least two components. The coupling constant 4.3 Hz was consistent with oC-galactopyranosyluronic  acid [59]'  One furanoside and one pyranoside form of galacturonic acid had been isolated and characterized.  Since i t is known that anomeric configuration  has l i t t l e bearing on mass spectral fragmentation,  confirmation of the structure  of the two central peak components was determined by mass spectrometry. A problem known to exist i n interpretation of mass spectra of s i l y l ethers i s the tendency of samples on ionization to undergo ring contraction and expansion. As a result, only small intensity differences exist i n the m/e 191 and 204 mass peaks. These are sufficient, however, to confirm that the i n i t i a l two compounds isolated were TMS galactofuranosyluronic acid esters and the latter two peaks pyranoside forms. A mixture of aldobiouronic acids obtained from lemon gum was silylated with BTMSA. Although p.m.r. spectral evidence indicated formation of TMS esters i n the aldobiouronic  acid mixture, no product could be detected on  subsequent gas chromatography.  I t appears that s i l y l esters are not sufficiently  stable at high temperatures to allow separation of disaccharides.  I t may be  possible, however, that part of the problem i s associated with the gas chromatograph used i n these experiments. Although BTMSA s i l y l a t i o n results i n formation of TMS ethers and esters, i t does not appear to be a useful reagent for analysis of uronic acids. Comparison of obtained results with other methods of s i l y l a t i o n of uronic acids [6o],  indicates a marked change i n equilibrium concentration for the various isomers. Other methods [60] indicate an isomer distribution more like that of the parent sugar galactose.  Galactose has only a small amount of furanose present  at equilibrium.  FIGURE 17 PROTON MAGNETIC RESONANCE SPECTRA FOR THE TRIMETHYLSILYL PROTONS OF SILYLATED GALACTURONIC ACID TMS  GALACTURONATE  100 MHz  TMS  ETHERS  TMS ESTERS TMS  GLC  V  PEAK. I a- FURANO  • • • • • • •• GLC  PEAK 2  P - FURANO  PEAK 3  at - PYRANO  u.  GLC PEAK 4  •  J  I  -i  I  I  20  16  12  ESTERS  IN  LESS  •  •  ^-PYRANO  -I  40 * METHANOL 5%  IN  36  CHCI  3  I  32 REMOVES  I  28  L.  24 TMS  i  8 THAN  i  I  4  0 30  MIN.  ^ r 300  0  100  200  TMS «-D-GALACT0FURANUR0NATE - GLC PEAK I -  rV  5.01  4.51  "PPM  4101  ;  ;  3.51 I  FIGURE 18: P.M.R. SPECTRUM OF TRBETHYLSILYLATED GALACTURONIC ACID [G.L.C. PEAK 1]  CrS  V.1 ON  FIGURE 19:  P.M.R. SPECTRUM OF TRE-IETHYXSILYLATED GALACTURONIC ACID [G.L.C. PEAK 2]  40  60  80  100  120  140  160  180  200  220  240  260  280  300  320  340  360  380  400  420  4 4 0 460 VJi  00  FIGURE 21:  MASS SPECTRUM OF THE TRBLETHYLSILYLATION PRODUCT OF GALACTURONIC ACID [G.L.C. PEAK 1 ]  ~.:z~:  100  7 3 '-  ~ _ r: "  •. E-EE- rz  r: ,zz". zz zzir-zzT zzz z. zzzzz. •  . zZztzzzz  zz z^zzZEizzTE  -"^2 L  •T  —  -  zzzzz  "  _4  —  i  -  —  ,  zzzzz: 1 T  i. ;j  :  •:  E ^  :| r\- • \ zzi  •  :••  -i^^  — ~-  —}._  "T :  j  . !  '_7_~ ™ " i EEEE  ... '  .r':\: •  ;  [  „l! •1 i d ' J 40 60 80  '  1  il  100  ~-V—E:'.E ^  zz zz  .•^ - Jr-.-V  .  • Ef^EEE.  Hiili  120  140  ~E"-'IQ|"'  •: \zzzz}-  160  .  . _ ,-„  204  11 180 200  • i zzzzzzzzz-  i  ;  ;  zz-zELzizzzz  zzzz.  ZZZZZZZZZV—ZZ ~z  n  r  •  ::  ..zzzzzz  f  _..  : zz-zztz-zzzzzzir  \zz::zzzz E l E r l E :  .--:r- .--•fr— »=t ~  5 0 0 520  JO' .5  X3  560 ,  •  Z ZZZ ,  : 1  —  -  :  480  •  = -  .—1  tZzl  i .  —-  19 •• :  ' ;  220  :  ~ Z -  —  <~ zz_~~  —  —2«>2"  ,— ^ 1_ 'ZZ"ZLZZ  I " '  0 BO  i —  f z\zzzzzzzzzzzztzz  EE •. —zz  —{_—  —  Ef=r£i~ zz-zzj-z — ~  —1  z\A  :  ;  .  L -  i  rr  ^ \.. -Z  -: ~ E .E =  -r ~ -'—  r  '  \  Z—ZZZZZZiZZZ  ^— .— -  —i-—i— _  — r  ;  ~:~Z.iZZT.~ZLZZ  T  --:-25-  !.  !  -_— .zizzzzzrzzz  —-—.—  ^ —  zzzzzizz ;-.-K-:r=  .  _  , ,  —  — 217 •: : .. 1  —  t-- -  zzzz  Zzztzz: Z'zZiFl  E i > 'i~'[zlzz 1— .  : r —  t  -  zrz:i :zz  ::. 4  i  .  : E  zzzzzzzzzzzzzzz  — !: ]  25  —  r  :  SZJzZZZZZ ^TTTTi "•" 7,"'—TTT^T  ZZZZZZZZ —•  '  *  zf  _ !  --  1  •  1  --{—4--  ZZZZZZZ  ~£~-z-  ..  rzzzzzrzzxzzr-  zt.  j_  -ZLi—\-.zzr  ;  . _..'J\J  .){-_:: ~y - 1 — — 1 _  •^r— . —  i  —  ~z\ r_z:.\i:~.™ "1 "7T_TE'." r~~ T , ,  ElEEEf:  '  — Ei —-  EEEE  ... —  ''• E.*E--  r  -Z 1Z - . . _  C  PtAK  'S  ~-FzTz'.zzzrl  75  t>L<J  •.•Ev E...  z--'z:.  '~fi-f  TMS " " G A L A C T U R O N A T E  zzizz:  50  zz  :  n 240 260  L-z zz -~---t— 1  " —if  : •1 300  : —- — j — zzzzzz: Z- ::z. ::".::^zr —jzzzzzz_  i  it i1, • Jk . 280  -i  "  A  1 In  320 340  1  III  \ Ml!!  360  380  I 400  II 420 440 460  VJ, VO  FIGURE 22: MASS SPECTRUM OF THE TRD-ffiTHYXSILYLATION PRODUCT OF GALACTURONIC ACID [G.L.C. PEAK 2]  FIGURE 23: MASS SPECTRUM OF THE TRE-LETHYXSILYIiATION PRODUCT OF GALACTURONIC ACID [G.L.C. PEAK 3]  FIGURE 2k: MASS SPECTRUM OF THE TRIMETHYLSILYLATION PRODUCT OF GALACTURONIC ACID [G.L.C. PEAK k]  62 METHYLATION OF LEMON GUM Methylation has been extensively employed i n structural analysis of polysaccharides.  Free hydroxyIs i n a polymer w i l l be converted to methyl ethers  during methylation. Hydrolysis of a methylated polymer w i l l yield carbohydrate fragments characteristic of the original polymer's linkage. The main problems associated with methylation of polysaccharides are yield and degree of substitution.  Methylations using the classical Haworth  [ 6 l ] and Purdie [62] procedures are often long, involving many repeated reactions.  Haworth methylations do not normally achieve a high degree of sub-  stitution on a single reaction.  In addition polysaccharides are i n strongly  basic solutions [20-30$] for prolonged periods o f time.  Purdie methylations  are normally carried out on partially methylated polysaccharides. Silver oxide used i n this methylation adsorbs polymer resulting in poor yields.  This ad-  sorption appears to be more pronounced with poorly methylated starting materials. Table XIV gives literature yields and information on polysaccharides methylated by these procedures.  Losses incurred on complete methylation cast some doubt  on the quality of the methylation analysis.  When large losses occur i t i s  impossible to know i f some structurally significant portion of the polymer has been missed. Many other methylation reactions have been proposed i n recent years. Table XV  l i s t s some of these with references to their use.  Complete methylation of a polymer is necessary to avoid erroneous i n terpretation of the original structure [63]*  Each unblocked hydroxyl detected  in the methylated polysaccharide hydrolysate appears as a linkage point i n the  63 TABLE XIV YIELDS AND DEGREES OF METHYLATION OF VARIOUS POLYSACCHARIDES  $ Yield . Methoxyl Methylated Found Polymer $  Polysaccharide  Ref.  Acacia arabica Gum  56  38.2  [64]  Armillaria mellea f r u i t body heterofcalactan  6l  41.6  [65]  Amylose polyalcohol  65  44.3  [66]  Arthrobacter viscosus extracellular polysaccharide  60  44.7  [67]  Tamarind kernal polysaccharide  54  39.5  [68]  Aeodes orbitosa polysaccharide [desulfated]  26  35.0  [69]  Aldotriouronic acids from Sapote Gum 1  45  N.R.  [70]  Aldotriouronic acids from Sapote Gum 2  13  N.R.  11  Aldotriouronic acids from Sapote Gum 3  25  N.R.  H  Synthetic araban  78  39.8  [71]  A  •  -  Based on starting material weight over methylated polymer weight a no loss : result would be 135-145$. TABLE XV RECENTLY DEVELOPED METHYLATION PROCEDURES  I  Methyl iodide with silver oxide  a] in dimethylformamide [DMF] b]. in dimethylsulfoxide [DMSO]  [72] [75]  II  Methyl iodide with barium oxide or barium hydroxide  a] in DMF b] in DMSO  [74]  Dimethylsulfate with barium oxide or barium hydroxide .  a] in DMF b] in DMF and DMSO  III IV V  Dimethylsulfate with powdered sodium hydroxide Methyl Iodide with sodium hydride  in DMSO a] ether type solvents b] DMF  [73] [75] [75] [76] [77] [77]  64 original polymer. Since molecules such as galactose are known to be d i f f i c u l t to methylate at position four, errors result i n proposed structures.  An ex-  ample of undermethylation being interpreted as a more complicated structure is outlined by Sandford and Conrad [78]. The method chosen to provide optimum y i e l d and degree of methylation was the sodium hydride, DMSO and methyl iodide procedure of Hakomori [79]. Some workers have shown degradation of polysaccharides on use of these reagents. I t was f e l t , however, that a polymer more closely resembling original material would be obtained by this method. Degradations which have been reported have occurred in uronic acid portions of carbohydrate polymers.  Hakomori methyl-  ations were shown to cause O-glucuronides to undergo elimination reactions yielding 4,5-dehydrouronic acids [54 ].  Conversely, Anderson and Cree [80] ex-  amined methylation of some Acacia polysaccharides using this procedure.  They  looked specifically for condensation of ester groups with methylsulphinyl carbanion to  give  a  sulfoxide or ,6 -elimination to unsaturated acids.  evidence was found for either of these reactions.  No  The slight reaction found  with benzyl glucuronide may have been due to unfavourable reaction conditions or simply a higher concentration of uronic acid.  Identification of degradation  product may be easier in a more concentrated system.  Benefits derived from use  of this methylation procedure outweighed any possible minor degradation of uronic acid. One point must be emphasized in the use of Hakomori methylations. In a methylation  of an acidic polysaccharide, uronic acids exist as sodium salts which  limit any degradation to unsaturated uronate [8l]« undergo elimination more readily.  Methyl esters are known to  Uronic acid sodium salts are converted to  65 methyl esters during the course of methylation providing an opportunity for elimination.  I t appears from methylation results that some uronic acid salts  remain after methylation and excess sodium hydride has been destroyed.  Infra-  red spectra for the methylated polysaccharides show the presence of carboxylate. Should a polymer undergo depolymerization due to formation of 4,5 unsaturation then reduction employing lithium borohydride in DMSO would eliminate the cause of the problem. Hakomori methylation produces virtually complete methylation i n a single reaction.  Several possible methods of analysis are available for the methylated  sugars present on hydrolysis. liquid chromatography. possible.  The preferred techniques involve use of gas  Several methods of sample preparation for G.L.C. are  Depolymerization of methylated polysaccharide may be achieved by  methanolysis.  Dependent on the sugars present, the samples may be analysed  directly or further reacted to acetates or s i l y l ethers. Acid hydrolysis give free sugars which may be reduced by sodium borohydride to a l d i t o l s . may be made volatile by acetylation or s i l y l a t i o n .  The samples  A common procedure i s simple  methanolysis, since volatile derivatives are obtained for most partially methylated sugars. Although G.L.C. retention time offers a good indication of the nature of a product i t i s by no means a positive identification. carried out to provide positive identification.  Further tests must be  The classical approach has  been to isolate the components and prepare crystalline derivatives. method of analysis can be extremely d i f f i c u l t .  This  Contaminating products can be  completely missed because of purification during derivative preparation. The need for a more rapid  and smaller scale identification has prompted  many workers to employ mass spectrometry.  Good mass spectra may generally be  obtained on samples which pass through a gas chromatograph. Two procedures  66 have been employed before mass spectral analysis. been silylated for greater thermal s t a b i l i t y .  Methanolysis products have  This method [82]  provides very  precise identification of components. A drawback., however, i s the formation of different ring and anommeric forms.  For example, 2,3,6  tri-O-methyl-D-  galactose w i l l form at least three different derivatives, f a i r l y large amounts of both pyranosides and smaller amounts of a furanoside. These extra peaks can aid and/or hinder analysis of complex mixtures. Lindberg [83]  has developed an alternate procedure employing partially  methylated polyol acetates. Methylated polysaccharides are hydrolysed with 72$ sulfuric acid, reduced with sodium borohydride, acetylated and analysed by gas chromatography. Mass spectral fragmentation i s dependent on methyl ether substitution of the original polyol and provides for very easy interpretation of structure.  Resolution of methylated polyol acetates i s less than for cor-  responding methyl glycosides. This lack of resolution can introduce complications during analysis of complex mixtures.  67 MASS SPECTROMETRY OF METHYLATED POLYOL ACETATES The following i s a brief outline of controlling factors i n mass spectrometry of partially methylated a l d i t o l acetates as outlined by Lindberg [83]. The base peak in almost a l l determined spectra was m/e 43 [CR^CO*]. I t was stated that only peaks of intensity, greater than 10$ of base peak were i n cluded.  I t should be noted that spectra of isomeric alditols having the same  substitution pattern are not sufficiently different to allow for distinction between them. Thus information w i l l sometimes be lost when converting into alditols.  For example, mass spectra from 2,3 and 3,k di-0-methyl pentoses  w i l l be identical. d i f f i c u l t y [83].  Reduction with deuterioborohydride would eliminate this The proposal i s that intense peaks [> 10$ base peak] of the  mass spectrum and retention time of the component on G.L.C. i n combination with sugar composition of the original polymer w i l l , i n most cases, give sufficient evidence for an unambiguous characterization of the methylated sugar. Chizhov et a l . [84] have determined that primary fragments from a l d i t o l acetates arise through fission between two carbon atoms of the carbon chain. Either fragment can carry the positive charge.  Secondary fragments are  formed from primary fragments by elimination of acetic acid [62] and/or ketene [42].  Intensity of primary fragments increases with decreasing mass number.  Chizhov et a l . [84] have shown that intensities of primary fragments given by a l d i t o l acetates are much lower than intensities of corresponding peaks from partially methylated a l d i t o l acetates.  Fission takes place between carbon  .atoms i n p a r t i a l structures V and VI i n preference to structure VII. ions are stabilized by methoxyl groups.  Positive  Primary fragments of high intensity  68 containing two v i c i n a l methoxyl groups are not observed, except when they can be formed by fission between two other vicinal methoxyl groups, indicating that structure V i s cleaved i n preference to structure VI. 0 CH3OCH HCOCH3  HC-OCH3  HC-O-C-CH3  HC-OCCH3  HC-O-C-CH3 li.  0 V  VI  VII  The secondary fragments observed in methylated a l d i t o l acetates are derived  from primary fragments by single or consecutive elimination of acetic  acid [60], ketene [42], methanol [32] or formaldehyde [30]. The lowest molecular weight primary fragment, m/e 45 [A], is produced by substances having a methoxyl group at C - l .  No other polyol produces this p r i -  mary peak. A peak [m/e 45] of low intensity [<5$]  nay be seen in mass spectra  of other substances and is probably a secondary fragment. Primary fragment D, m/e, 117, i s obtained when C - l i s acetylated and C-2 methylated. An analogous primary fragment m/e 131 [E] i s obtained from a 6deoxy hexitol methylated at C-4 but not at C-5« - +  CH =0-CH3 2  HC=0CH3 . H2C-OCCH3  II  HC=0-CH  3  HC-OC-CH3  II  0  0  CH D  3  69 Lindberg [83] reports that two primary fragments have m/e l 6 l , F i and Fa-  Of these, Fx is obtained in low intensity from alditols methylated at  positions 2 and 3.  I f k is also methylated then i t becomes a prominent frag-  ment. Fragment Fa, obtained from alditols methylated at positions 1 and 3> The secondary fragments m/e 129 and 101 are ob-  is always of high intensity.  tained from Fi and Fa by loss of methanol and acetic acid respectively.  Further  loss of ketene from m/e 129 gives m/e 87. Loss of formaldehyde from m/e 101 may give m/e 71. Primary fragment m/e 175 [G] i s obtained from 6-deoxyhexitols methylated i n positions 2, 3 and h. +  HC=0CH  3  I  HC-OCH3 H2C-OC-CH3 II 0  +  HC=0CH3 0  HC=0CH  HC-OCH3  HC-OCOCH3  HC-OCOCH3  HaC-0C0CH3  HC-OCOCH3  HC-OCOCH3  Fi  G  Fa  3  I  •I  CH3  CH3  H2C-OCH3  +  HC=0CH3  I  HC-OCCH3  +  HC =0(3*3  H  I  Primary fragment m/e 189 [H] i s given by alditols methylated at position 3 but not at positions 1 or 2.  Secondary fragment m/e 129 may be formed by  elimination of acetic acid from H.  Loss of formaldehyde or ketene from m/e  129 gives respectively m/e 99 or 87. M/e 203 [I] i s an analogous primary fragment obtained from a 6-deoxy hexitol methylated at position 3 but not h or 5* Three primary fragments of m/e 205 way be expected [Ki, Kg and K ] . The 3  f i r s t , derived from alditols methylated at C - 2 , 3 and k, i s not observed i n high intensity.  Fragment Ka i s observed from alditols methylated at positions  1, 2, h and 55 K3 from alditols methylated at positions 1, 3> h and 5« In either case i f position 5 i s °"t methylated, the corresponding peak appears n  with only low intensity. K  2  and K3.  Several secondary fragments may be obtained from K i ,  Loss of acetic acid from K3 to produce m/e 145 appears to be an  especially important secondary fragment.  70 +  +  +  HC=0CH . 3  HC=0CH  HC-OCOCH3  I  I  H0C-OCOCH3  H2C-OCH3  HC-OCH3  HC-OCOCH3  I  HC-OCH3  HC-OCH3  HC=0CH  I  3  HC-och3  HC=0CH3  HC-OCOCH3  H2C-OCH3  Ki  I  3  I  HC-OCOCH3 HaC-0CH3  HC=0CH  3  . I  HC-OCOCH3  I  HC-OCOCH3  I  K C-OC0CH 2  K3  3  M  Three primary fragments of m/e 233 may be expected. One of these [L] i s obtained from alditols methylated at C - l and 4. A prominent secondary peak [m/e 113] is most probably derived from L by loss of two molecules of acetic acid. The primary peak m/e 2 6 l [M] i s the highest mass number of relatively high intensity.  Alditols methylated at 3 and acetylated at k, 5 and 6 produce  this peak. Lindberg et a l [83 ] have used formation of these primary and secondary fragments to analyse partially methylated a l d i t o l acetates.  Tables of fragments  expected under these rules are presented i n Lindberg's i n i t i a l paper on mass spectrometry.  Knowledge of the partially methylated polyols has aided their  analysis of polysaccharide structure. The Lindberg procedure for analysis was chosen because i t offered an inate simplicity not present i n other analytical schemes. Lemon gum has been analysed by methanolysis [ l ]. I t was f e l t that this sort of complex polysaccharide would give a good evaluation of reduction - acetylation as a method for analysis of methylated polysaccharides.  71 Four polysaccharides were methylated by the Hakomori method and analysed by Lindberghs mass spectrometry - gas chromatography procedure.  The four -were:  1.  Lemon gum  2.  Lemon gum degraded with l/lQN H2SO4  3.  Lemon gum periodate oxidized and borohydride reduced [before hydrolysis]  k.  Lemon gum periodate oxidized and borohydride reduced [after 0.5N H2S0 hydrolysis] 4  METHYLATION ANALYSIS OF LEMON GUM Lemon gum was methylated using the Hakomori method.  Extreme care was.  taken to avoid interfering side reactions. After methylation, uronic acids were reduced with lithium aluminum hydride. Reduction avoided problems associated with formation of aldobiouronic acids on hydrolysis.  Since lemon gum  was f u l l y methylated before reduction, no methylation was carried out to block the six positions in the newly formed neutral sugar. outlined produced high yields.  A l l steps i n the reactions  After reactions to form acetylated-methylated  polyols, analysis was carried out on the gas chroma to graph.  Two columns were  evaluated, one of ECNSS-M, the other butanediol succinate. Resolution of the mixture was virtually identical, with  butanediol succinate being slightly  superior. Samples were collected for analysis on the mass spectrometer since access to a coupled GLC-mass spectrometer was not possible.  The collected components  were analysed by thin layer chromatography [T.L.C.]. T.L.C. revealed that single peaks on the gas chromatograms were, i n fact, at least two components. Using thin layer chromatography, samples of the components could be separated. The samples were then reanalysed by GLC and T.L.C. to ensure their purity.  72  32 MIN.  FIGURE 25:  GAS CHROMATOGRAM OF LEMON GUM AFTER METHYLATION, LITHIUM ALUMINUM HYDRIDE REDUCTION, HYDROLYSIS, BOROHYDRIDE REDUCTION AND ACETYLATION n [140-195°C at 3 ° M - 3  *10  O O  O  OOo o  *9  *a -7 *6 "5  ° 8  CO O O  FRONT  FIGURE 26:  THIN LAYER CHROMATOGRAM OF THE TEN FRACTIONS OF LEMON GUM METHYLATED POLYOL ACETATES  M X3 "2 *1  73 During the isolation and purification of these components, a technique was developed for handling microgram quantities of compounds collected from the gas chromatograph. This procedure for micro manipulation i s discussed in the appendix to the thesis. Mass spectra were obtained on a l l significant components isolated by gasliquid chromatography. Analysis of the spectra indicated that although simple rules had been set down by Lindberg, intensity of some component peaks was altered.  This may be a function of the means of sample introduction.  to be analysed were inserted into the spectrometer via the probe.  Samples  It was not  possible to identify components using the simple rules proposed by Lindberg. It was, however, possible to identify the majority by careful consideration of fragmentations. Care was taken with the spectra to ensure no background contribution which would have lead to misleading peak intensities. spectra.  Two checks were made on the  A background spectrum was obtained and subtracted from the component,  spectra. Analysis of the spectra in their order of production confirmed that major peaks were not contributing to subsequent spectra. The spectra are i n cluded in the appendix and numbered to indicate both order of elution and order of mass spectra determination. Although Lindberg and co-workers u t i l i z e d a coupled gas chromatographmass spectrometer, there are advantages i n using direct sample collection.  The  presence of overlapping peaks is a clear example of need for further separations. Peak broadening also occurs in the gas chromatograph-mass spectrometer separator chamber. The method of micro manipulation outlined i n the appendix has proven extremely valuable in purification and sample analysis.  74 Variation in peak intensity for some mass values has created a problem. Peaks which were expected to be of intensity greater than 10$ were, in fact, less than this.  In addition peaks at lower m/e values had higher intensity  than predicted.  Availability of complete spectra would have aided considerably  interpretation of structure.  '. >  Quantitative estimation of the identified components was not possible due to lack of thermal conductivity molar response factors for partially methylated a l d i t o l acetates.  A relation should exist between subsitution pattern and  molar response factor for a given sugar polyol. A relatively large number of molar response factors are available for flame ionization equipment but none are presently available for thermal conductivity detectors.  METHYLATED POLYOLS FROM LEMON GUM Peak 1: Component one on the gas chromatograph was unidentified. The retention time was less than that reported for the most volatile sugar a l d i t o l polyol.  The increased v o l a t i l i t y of this component was l i k e l y due to elimin-  ation or anhydride formation.  Hydrolytic etherification has been reported for  partially methylated polyols and sorbitol [85].  Strong peaks at m/e 117 and  101 indicated the molecule was terminated by an acetyl group adjacent to two methoxyls. A strong m/e 88 peak was not consistent with any reported peak. Strong even numbered fragments do not often occur i n mass spectra. Peak 2: The spectrum for this component does not correspond to any of the known methylated polyol acetates.  Its structure does, in fact, appear to be  very different from that of a polyol. None of the characteristic primary  75 fragments except m/e 45 can be seen. Although some masses correspond to secondary fragments, i t is d i f f i c u l t to determine their origin. a peak m/e 74 is almost as intense as the base peak m/e Peak 3'»  In addition  43*  T.L.C. indicated that peak three contained two components. I t  was confirmed experimentally that 2, 3, 5-toi-O-methyl-L-arabinitol and 2, 3, 4-tri-O-methyl-L-rhamnitol  acetates overlap as a single peak. Two components  are clearly seen in the mass spectrum. This a b i l i t y to distinguish two components as a result of knowledge about sugar composition and chromatographic behaviour could be used to determine percentage composition.  Only standard  spectra of the two components are required. Peak 4:  This component, present i n only small amounts, i s 2, 3> 4 - t r i -  O-methyl-Lj-arabinitol acetate.  With this component i t became clear that the  level of 10$ of base peak for included peaks was not going to be applicable. In addition to m/e m/e  values of 43, 101, 117 and l 6 l required for identification,  values 45, 5$ and 87 were a l l above 10$ of base peak. Peak 5s  This peak contains two components, which, being very minor con-  stituents, were impossible to separate.  Some information was available from  the mass spectrum of this peak. Additional information may be deduced from G.L.C. retention time.  Since 2, 3, 4, 6-tetra-0-methyl-D-galactitol acetate  comes immediately after these two components their retention times must be less than i t s value.  The only two applicable compounds are 2, 5*and 3>  di-O-methyl-L-arabinitol acetates.  5~  The mass spectrum also indicated the pres-  ence of these two components. Peaks associated with 3> 5-di-Q-methyl-Larabinitol are of greater intensity and correspond i n strength to the second  76 peak of peak 5«  Positive identification of the smaller component as 2,5-di-  O-methyl-L-arabinitol i s clouded by strong signals from 3/5-di-Q-methyl-Larabinitol.  Identification i s based on m/e 233 and 113 peaks. Reduced i n -  tensity of higher mass values increases the d i f f i c u l t y of identification •without pure standard spectra. Peak 6:  This peak contained two components as shown by T.L.C. Peak 6  was collected and separated into i t s components by preparative T.L.C. lation  Reiso-  of the constituent parts by gas chromatography indicated no con-  tamination from peaks 5 or Taration of peak 6 alditols.  Analytical T.L.C. confirmed the clean sep-  The two compounds were designated 6T and 6B on  the basis of position on ascending T.L.C. Peak 6T; 2,3-di-O-methyl-L-arabinitol acetate is the major component of peak 6.  This molecule i s an example of a component that could be derived from  two sugars [2,3-or 3,k-&±-O-methyl-L-arabinose]. which fragment in an identical manner.  Both sugars reduce to polyols  Signal strength for higher m/e peaks  was reduced in intensity. Peak 6B: This constituent proved compounds present as minor constituents in a gas chromatographic peak can be isolated and characterized. Peak 6B is 2,$>h,6-tetra-O-methyl-D-galactitol  acetate.  Except for reduced intensities  for expected peaks the mass spectrum i s very clear. compound coincided i n retention time with peak 6.  A standard sample of this  This retention time con-  firmation aided identification of preceeding chromatographic peaks. Peak 7:  Like peak 6 this fraction contained two components. They were  separated, numbered and confirmed pure i n the same manner as components in peak 6.  77 Peak 7T: Analysis of this compound's mass spectrum showed i t to he a 2,3,4-tri-O-methyl-hexitol.  Since a l l hexitols substituted in a like manner  give similar fragmentation patterns i t was necessary to consider G.L.C. retention times. lemon gum,  Of the two possible 2,3,4-tri-O-methyl-hexitols  glucitol has the shorter retention time.  in reduced  Therefore, peak 7T is  2,3,4-tri-O-methyl-D-glucitol acetate. Peak 7B;  This peak was thought originally to be only one compound.  On  detailed examination of the mass spectrum i t appeared to consist of two components. No one compound could produce the necessary fragments to account for a l l peaks occurring in the mass spectrum. The strong m/e 233 peak must arise from 2,3,6-tri-O-methyl-D-galactitol acetate.  Only two other compounds produce  this peak and they require a strong m/e 189 which was not present. of such intensity that i t had to be considered significant.  M/e l 6 l was  Only 2,4,6-tri-  O-methyl-D-galactitol acetate had a fragmentation pattern compatible with the peaks present.  A l l other compounds which give rise to m/e l 6 l have peaks at  m/e 145 or 189, or are chromatographically unacceptable.  Component 7B consists  of a mixture of 2,3,6-and 2,4,6-tri-O-methyl-D-galactitol. Peak 8:  T.L.C. indicated that this peak was a single component. The mass  spectrum was virtually the same as that for 7T, indicating an identical methyl substitution.  A longer retention time was consistent with the structure pro-  posed, 2,3,4-tri-O-methyl-ri-galactitol acetate. Peak 9:  There were at least two components in this gas chromatograph  peak. The small quantity of these constituents made purification by T.L.C. impossible.  One component appeared to be 2,3-di-O-methyl-D-glucitol  acetate.  The gas chromatogram retention time was identical with an authentic sample. The second component in this peak may be 2,3-di-O-methyl-D-galactitol.  The  78 mass spectrum of this peak would not distinguish between these two compounds. Some peak intensities, however, appear to be too intense, for example m/e 85 and 127.  These fragments should arise from a 3-0-methyl hexitol, however,  the G.L.C. retention times for such compounds are too great. No other polyol accounts for these mass fragments. Peak 10:  This peak typifies the d i f f i c u l t i e s that may be encountered  when trying to interpret mass spectra of methylated polyol acetates. Peaks for mass values which should have been greater than 10$ of the base peak did not reach this value.  Peaks with intensities less than 10$ of base peak can then  not be completely rejected.  Thus the mass peak m/e 233 i  would not normally be considered must be evaluated.  component 10 which  Complete spectra present  a better picture of relative intensities to be expected. table such as given by Lindberg et a l [83]  n  Also, a preliminary  i s a definite aid. I f the table  listed values of peak intensity relative to the base peak, an easier interpretation of spectra might be possible.  Peak 10 i s 2,4-di-O-methyl-D-galactitol  although values of certain fragments do not exceed the required 10$ value. The component compounds from f u l l y methylated lemon gum are l i s t e d in Table XVI, together with peak area for the components.  TABLE XVI METHYLATION ANALYSIS OF LEMON GUM  Compound  Area $  Unknown  . 1  0.51  Unknown  2  0.70 7.02  2,3,5-Me -ARAB 3  2,3,lj.4te -HHM  J  2,3,4-Me -ARAB  3 k  2,5-Me -ARAB  5  1.83  3,5-Me -ARAB  5  3.62  2,3-Me -ARAB  611  2,3,4,6-Me -GAL  6B.  2,3,4-Me -GLUC ..  7T "  2,3,6-Me -GAL  7B  2,4,6-Me -GAL  7B.  2,3,4-Me -GAL  8  3  3  2  2  2  4  3  3  3  3  2,3-Me -GLUC 2  Unknown 2,4-Me -GAL 2  9 " 9 .1 10 -  1.40  21.7 28.5  3.96 3.16 .  ..  27.6  • •  8o METHYLATED DEGRADED LEMON GUM When lemon gum was hydrolysed with l/lON sulfuric acid a polymer was obtained which contained essentially no L-arabinose.  Methylation of this  product gave information about the galactan framework of the plant gum. In addition, i t provided further proof regarding structures of some partially methylated galactitols derived from lemon gum. The acid degraded polymer was methylated using the Hakomori method [79].  Hydrolysis was carried out by  Lindberg's method [ 5 ], uronic acids being removed by ion exchange resin. The acids were not examined further, since they represented only a small amount of acidic material. Neutral methylated sugars were reduced and analysed in a manner similar to that described for lemon gum. There were four principle peaks i n the gas chromatogram. FIGURE 27: GAS CHROMATOGRAM OF DEGRADED LEMON GUM .METHYLATED POLYOL ACETATES [140-205°C at 3°/min.] I/ION H S0 2  4  LEMON GUM  log  4  8  12  16  ~20~  — 1 —  24 24  28  32 MIN.  The peaks were analysed by mass spectrometry with the following results. Peak 1:  The most volatile of the major components exhibited a mass  spectrum characteristic of 2,3,4,6-tetra-O-methylrD-galactitol acetate.  Again,  intensity of some peaks of lower mass was greater than the 10$ cut o f f suggested  8i by Lindberg.  Two such peaks are m/e 55 and 57* The spectrum, however,  was consistent with fragmentation of 2,3,4,6-tetra-O-methyl-D-galactitol acetate in a l l other respects, clearly indicating the value of the method. Peak 2:  This gas chromatographic peak corresponds to 7B from lemon gum.  It contains two inseparable components, 2,3,6-and 2,4,6-tri-O-methyl-D-galactitol. The exact molar balance between these two components w i l l have to await accurate intensity determinations for the two pure compounds. Peak 5; The component contained in this peak was  2,3,4-tri-O-methyl-D-  galactitol, corresponding to peak 8 i n the gas chromatogram of methylated lemon gum. Peak hi  The component of this peak was 2,4-di-O-methyl-D-galactitol  acetate and corresponds to peak 10 of lemon gum.  TABLE XVII METHYLATION ANALYSIS OF DEGRADED LEMON GUM  Compound  G.L.C. Peak  1.09  I n i t i a l k peaks 2,3,4,6-Me -GAL 4  Area i»  1  27.8 16.6  2,3,6-Me -GAL 3  2,k,6-Me -GAL  2j  2,3,4-Me -GAL  3  34.0 '  2,4-Me -GAL  k  20.5  3  3  2  82 METHYLATION ANALYSIS OF LEMON GUM AFTER PERIODATE OXIDATION AND BOROHYDRIDE REDUCTION Methylation of periodate degraded lemon gum should generate information concerning free hydroxyl positions of the remaining intact polysaccharide. Total hydrolysis of the methylated polymer •would produce sugars from lemon gum which contained no v i c i n a l bydroxyls.  Gas chromatographic evidence i n -  dicated that this was achieved to a limited extent.  However, mass spectra  of isolated peaks were very poor. Peak 1;  This component displaying chromatographic characteristics of  2,3,5-tri-O-methyl-L-arabinitol probably resulted from incomplete periodate oxidation  and/or basic hydrolysis during methylation.  In addition, some mild  acid hydrolysis may have occurred during isolation of the borohydride reduced polymer.  I t was not possible to collect this peak because of sample v o l a t i l i t y  and the small quantity present. Peak 2:  Like peak 1 this component was present in very low concentration  in the mixture of methylated polyol acetates. Retention time was similar to that of 2,3,4-tri-O-methyl-L-arabinitol acetate.  The mass spectrum was similar  to peak k of lemon gum but additional mass peaks indicated the small sample collected was impure. Peak 5?  Like the previous two peaks this was a very minor peak in the  gas chromatogram. At most i t represented only 3 percent of total sample. A figure of 1.5 mole percent i s probably more r e a l i s t i c .  The G.L.C. retention  time corresponds to that for 2,5-and 3>5-di-Q-methyl-L-arabinitol acetate.  The  mass spectrum can be assigned to these compounds, however, the intensities make absolute assignment d i f f i c u l t . •  83 Peak hi  The f i r s t of the major peaks, this component had the gas chro-  matographic characteristics and mass spectrum of 2,3-di-0-methyl-L-arabinitol acetate.  Since this product contains vicinal hydroxyIs, blocking groups may  have been removed by elimination reactions during the strongly basic methylation. I t would appear that a small amount of tetra-O-methyl-D-galactitol may be present in this sample. Peak 5: This peak consisted of two components on T.L.C. Attempted separation of the small quantities available was unsuccessful.  Since this peak cor-  responded in R^, to peak 7 of lemon gum, one component should have been 2 , 4 , 6 tri-0-methyl-D-galactitol acetate.  Peaks resulting from this product may be  seen in the mass spectrum, however, there are other minor mass peaks consistent with the other consituents of peak 7 of lemon gum, 2,3,6-tri-0-methyl-D-galactitol and 2,3,4-tri-O-methyl-D-gulcitol. Peak 6:  The major component isolated from this oxidized gum was 2,4-di-  Q-methyl-2-galactitol acetate, accounting for two thirds of the isolated methylated polyols. This information provided further proof that the galactan framework of this polymer is highly branched with many side chains. Analysis of a periodate oxidized borohydride reduced polysaccharide should be an extremely useful analytical tool.  I t appears necessary that more study  be made of parameters involved in possible hydrolysis during methylation. Lemon gum may not undergo complete oxidation. Alginate ' has shown this d i f ficulty.  This underoxidation may account for the presence o f f u l l y methylated  sugars in the hydrolysate after methylation.  FIGURE 28 GAS CHROMATOGRAM OF LEMON GUM SMITH POLYALCOHOL METHYLATED POLYOL ACETATES [l40-205°C at 3 % i n . ]  4  4  1  1  8  1  1  1  12  1  16  1  I  20  1  i  24  I  28  TABLE XVIII METHYLATION ANALYSIS OF LEMON GUM SMITH POLYALCOHOL  Compound  ' * '  Area #  2,3,5-Me -ARAB  . 1  1.03  2,3,4-Me -ARAB  2  1.94  2,5-Me -ARAB  3  0.37  3,5-Me -ARAB  3  3«10  2,3-Me -ARAB  4"  2,3,4,6-Me -GAL  4.  2,3,6-Me -GAL  5"  3  3  2  2  2  4  3  12.9  18.4  2,4,6-Me -GAL 3  2,3,4-Me -GLUC  5.  2,4-Me -GAL  6  3  2  62.3  I  1  r — — T  32 MIN.  85 METHYLATION OF LEMON GUM SI Lemon gum SI was obtained from lemon gum after periodate oxidation, borohydride reduction and mild acid hydrolysis. The reduced and acetylated hydrolysate from methylated lemon gum SI gave a gas chromatogram much like that of lemon gum with some notable Peak 1:  exceptions.  The structure of this component was 2,3,5-tri-O-methyl-L-arabinitol.  The mass spectrum was similar to that from peak 3 of lemon gum with the minor contribution of 2,3,4-tri-0-methyl-L-rhamni t o l removed. Peak 2:  Corresponding to peak 6 of lemon gum i n R^, this peak contained  only one component.  That component was identified as 2,3,4,6-tetra-Q-methyl-  D-galactitol acetate from i t s mass spectrum. Peak 3:  Corresponding to peak 7 of methylated lemon gum this peak has no  tri-O-methyl glucitol constituent.  I t contained, therefore, only 2,4,6-and  2,3,6-tri-O-methyl-D-galactitol. Peak 4: Corresponding to peak 8 of lemon gum this peak was mainly 2,3>4tri-O-methyl-D-galacitol.  The presence of this component indicates the im-  portance of the 1—*"6 linkage i n lemon gum.  The increased signal strength  for m/e 4 5 may be due to a trace of 2,6-di-0-methyl-D-galaeitol  acetate.  This  compound has the correct mobility and might result from undermethylation. Peak 5: Like peak 9 of lemon gum this was a rather odd peak. Its mass spectrum fragmentation pattern indicated the presence of 3-Q-methyl-D-gala c t i t o l acetate.  The spectrum, however, lacks m/e 189 and i t may be that this  component both here and i n lemon gum i s a specific degradation product capable of fragmenting like 3-0-methyl-D-galactitol. 23-di-O-methyl glucitol acetate. during periodate  cleavage.  This peak also appears to contain  This compound may indicate some underoxidation  86 Peak 6;  The f i n a l peak is that of 2,4-di-Q-methyl-D-galactitol. FIGURE 29  GAS CHROMATOGRAM OF LEMON GUM SI METHYLATED POLYOL ACETATES [UO-205°C at 3°/min.] LEMON  GUM ; S \ ' 4  TABLE XIX METHYLATION ANALYSIS OF LEMON GUM SI  Compound 2,3,5-Me -ARAB 3  3,5-Me -ARAB 2  2,3A>6-Me -GAL  G.L.C• Peak  1 Between 1 and 2  Area $  4.69 1.86  1 2.  15.5 15.1  2,3,VMe -GAL  5] 3. k  2,3-Me -GLUC  5 '  29.7 ' 3.4  Unknown  5 . 6  29.0  4  2,3-Me -ARAB 2  2,3,6-Me -GAL 3  2,4,6-Me -GAL 3  3  2  2,4-Me -GAL 2  2  87 The present work on lemon gum is the f i r s t example of analysis of a plant gum by the Lindberg et a l [83] a l d i t o l acetates].  method of mass spectrometry [partially methylated  The complexity of the lemon gum mixture analysed was such  that alternate separations were required on TLC.  Micro manipulation, as out-  lined in the appendex, of less than milligram quantities has facilitated the purification and mass spectrometry of the isolated methylated a l d i t o l acetates. Hakomori methylation [79]  has given high yields and complete methylation im-  proving the r e l i a b i l i t y of methylation analysis.  The combination of Hakomori  methylation and methylated a l d i t o l acetate mass spectrometry has reduced the quantity of polysaccharide needed for methylation analysis to the milligram range.  The a b i l i t y to manipulate these sub-milligram quantities of methylated  hydrolysis products from polysaccharides w i l l be of immense value to workers faced with complex mixtures or mass spectrometers without a coupled gas chromatograph. Methylation analysis of lemon gum and degraded lemon gum are in good agreement with results obtained and the structure proposed by Jones and Stoddart. Analysis of Smith degraded lemon gum i s also in good agreement with one minor exception.  In the previous analysis of lemon gum there was a substantial and  increasing amount of 3>5-di-0-methyl-L-arabinose  in the Smith degraded gum.  The increase has not been confirmed by this work - an unexpected result in view of the generally milder hydrolysis conditions employed in the preparation of the Smith degraded lemon gum.  88 EXPERIMENTAL GENERAL METHODS Paper chromatography was carried out on Whatman No. 1 and 3 paper by the descending method in the following solvent systems: a]  Ethyl acetate-acetic acid-formic acid-water [ 1 8 : 3 : 1 : 4 ] .  b]  Ethyl acetate-pyridine-water [8:2:2] top layer.  c]  Butan-l-ol-pyridine-water [ 6 : 4 : 3 ] .  d]  Butanone-water azeotrope.  Thin layer chromatography was carried out on alumina, and s i l i c a gel [with and without calcium sulfate binder] by the ascending method i n the following solvent systems: e]  Butanone-water azeotrope.  f]  Benzene-methanol [96:4].  g]  Ethyl ether-toluene [ 2 : 1 ] .  Sephadex gel permeation chromatography was performed i n a Sephadex column K25/45 fitted with flow adaptors using Sephadex G-15 fine. the column, G-15 was swollen in water for 16 hours. was maintained by use of a Mariotte flask.  Prior to packing  A constant pressure head  Operation of the column was i n the  ascending mode using water with 0.5$ chloroform as eluting solvent. Samples applied to the column through the flow adaptors using a hypodermic syringe barrel with luer lock adaptor were collected on elution by a; timed fraction collector. Reducing sugars on paper chromatograms were detected with either p-anisidine hydrochloride reagent [86]  or alkaline silver nitrate dip [87].  Non-reducing  carbohydrates and polyhydric alcohols were detected with either the latter dip  89 or alkaline periodate spray reagent [88].  The rate of movement of substances  on paper chromatograras i s quoted relative to the solvent front or an internal standard such as galactose. Visualization of T.L.C. was accomplished by charring [sulfuric acid spray].  When samples were to be isolated, silicone grease was spread in thin  ribbons on a clean glass plate.  When pressed to the T.L.C. plate, a small  amount of adsorbant was transferred, then charred in the normal manner. Melting and boiling points are uncorrected. Unless otherwise stated optical rotations were measured at 21 + 3°C. Infrared absorption spectra were recorded using a Perkin-Elmer model 257 or 21. Samples were examined as KBr pellets, chloroform or carbontetrachloride solutions [5-10$ W/V] in sodium chloride cells, or as thin films on sodium chloride plates. Nuclear magnetic resonance spectra were determined on a Varian HA-100 or A-60 spectrometer. Mass spectra were determined on an Atlas AEI MS9 spectrometer. Gas liquid chromatography was carried out on an F and M model 720 dual column instrument fitted with thermal conductivity detectors. Polyhydric alcohols and sugars were analysed on two columns [8 f t . x 0.25 i n . coiled copper] packed with equal weights [to within 20 mg] of 20$ SF 96 on 60-80 mesh Diatoport S. The columns were held isothermally at 130° for 6 rain, and then programmed at 3° per minute to hold at 220°.  During analyses  for sugars only, the programme was started at 190° and immediately programmed at 2° per minute to hold at 220°.  T h e v i n j e c t i o n port was 270°, the detector  block 295° and the helium flow 88 ml per minute [6.8 sec. for 10 mis].  90 For simple systems of the above compounds other columns may be used. Thus s i l y l derivatives of ethylene glycol, glycerol and methyl  /3 -D-gluco-  pyranoside may be separated on a 2 f t . x 0.25 i n . column of SE30.  This column  was run isothermally at 70° u n t i l the ethylene glycol emerged, then programmed at 10° per minute to hold at 250°C Sugars were dissolved in water containing a small amount of chloroform [0.5$ W/V] and allowed to come to equilibrium.  Aliquots were removed and con-  centrated to dryness at 40° on a rotary evaporator.  Solutions of the poly-  hydric alcohols in anhydrous pyridine were added to give the test solutions reported in Tables II - VI.  These  pyridine solutions [10 parts] were  immediately silylated with hexamethyldisilazane [k parts] and trimethylsilyl chloride [2 parts] and after 5 minutes shaking, portions were injected directly. The values quoted i n the Tables represent the mean of at least five determinations . For the determination of molar response factors separate solutions of the individual compounds with butane-1,4-diol were prepared.  In the case of the  sugars these runs also served to determine relative concentrations of different forms at equilibrium. In preliminary experiments i t was ascertained that none of the compounds involved were lost on passage through Amberlite 1R 120 and Duolite A-4 resins nor on concentration of their aqueous solutions. Methylated polyol acetates obtained from methylated polysaccharides were examined on columns with ECNSS-M and butanediol succinate liquid phases, the latter giving a slightly improved separation. Methylated polyol acetates were analysed on a stainless steel column [6 f t . x 0«25 in.] with butanediol  91 succinate [10$ W/W]  on Diatoport S [6O-8O mesh]. Column temperature was  programmed immediately from 140-205°C at 3° per minute using the standard flow rate and other settings. Peak areas were measured i n i t i a l l y with a Disc integrator with suitable correction for baseline d r i f t .  In later work an Infotronics d i g i t a l integrator  was used. Silylation of galacturonic acid was carried out i n a variety of solvents using bis-trimethylsilyl acetamide as the s i l y l a t i n g reagent  [89].  Acetylated carbohydrates were prepared by two methods. Some samples were dissolved in anhydrous pyridine and an excess of acetic anhydride added. The samples were allowed to stand 16 hours, then heated to 60°C for 1 hour. The solvents were removed by rotary reduced pressure evaporation. Alternatively, samples were dissolved in pyridine and an excess of acetic anhydride added. The samples were sealed i n test tubes and heated at 100°C for 1 hour.  Work up  was similar to the above case. Unless otherwise stated hydrolyses were carried out by either of the following methods. A sample [5-20 mg] was dissolved in sulfuric acid [UN] [5-10 ml] and hydrolysed i n a sealed tube at 100° for 8 - l 6 hours.  Alternatively  a sample [5-20 mg] was solubilized with 72$ sulfuric acid [1 ml] for 1-2 hours at room temperature, then diluted to 7«2$ with d i s t i l l e d water and hydrolysed at 100° in a sealed tube for k hours. After cooling the hydrolysates were neutralized partially with barium hydroxide, then completely with a small amount of barium carbonate. Alternatively complete neutralization was achieved using a slurry of barium carbonate. The precipitated barium sulfate together with  92 the residual barium carbonate -was removed by centrifugation.  The. supernatant  -was then passed through a small bed of Amberlite TJR120 [H + form] ion exchange resin to remove any cations present i n solution.  The column of Amberlite was  often placed in series with a column of Duolite A-4 [OH - form] ion exchange resin to remove sugar acids or other acidic material.  The eluant was con-  centrated to a syrup by evaporation under reduced pressure [ca. 15  mm].  Reduction of free sugars was carried out on the eluant from the ion exchange column [used to remove barium ions s t i l l in the hydrolysate]. A large molar excess [3-5 moles] of sodium borohydride was added to the eluant. After 24 hours excess borohydride was destroyed by addition of acetic acid.  Sodium  ions were removed using a column of Amberlite IR 120 [H + form] ion exchange resin.  The solution was evaporated to dryness under reduced pressure, the  resulting syrup being repeatedly dissolved in methanol and evaporated to dryness.  The repeated evaporation served the dual purpose of removing the boric  acid and traces of acetic acid remaining i n the reduced sugar sample. Reductions with lithium aluminum hydride were carried out on methylated polysaccharides and oligosaccharides i n the following manner. To a 1$  [W/V]  suspension of lithium aluminum hydride i n T.H.F. was added slowly a 2$  [W/V]  solution of methylated carbohydrate in anhydrous T.H.F. The reaction mixture was stirred during the addition and for 16 hours afterwards.  Excess hydride  was destroyed by cautious addition of water to the vigorously stirred suspension.  The tetrahydrofuran solution was filtered and the insoluble residue  extracted continuously with chloroform.  F i l t r a t e and extract were combined  and evaporated to dryness. Moving boundary electrophoresis was carried out by Dr. K. Hunt on a Perkin-Elmer electrophoresis apparatus Model 238  using 2 ml Tiselius c e l l s .  93 Samples were prepared for analysis by dialysis of a solution of sample [1-3$ W/V] in a barbituate buffer [veronal] against the same buffer solution.  This  latter solution was used in the buffer vessels. During the electrophoresis the power output was adjusted to approximately 2 watts. Dialysis of samples was carried out i n viscose tubing against running water when only the polymer was desired or against a static volume when collecting dialysable materials. Dialysis of DMSO solutions resulted i n extensive loses unless water was added to dilute the DMSO before dialysis. The formation of methyl ethers was carried out by one or a combination o f two of the following methods. These were: I] II] III]  Hakamori's method [79] Purdie's method [62] Kuhn's method [72]  A generalized account of these methods as applied to the methylation of poly, oligo and monosaccharides i s given below.  I] HAKOMORI METHOD [79] The methylation procedure was essentially that described by Hakomori [79] wherein the methylsulfinyl anion [90] was used to generate the polysaccharide alkoxide prior to the addition of methyl iodide. Routinely, 1 g. samples of polysaccharide were methylated.  The procedure described i s for this amount.  The methylsulfinyl anion was prepared as follows. Into a dry round bottom flask [250 ml] containing a magnetic s t i r r i n g bar was weighed 1.5 g. of sodium hydride [55$> coated with mineral o i l ] .  The sodium hydride was washed three  times by s t i r r i n g with 50 ml portions of anhydrous petroleum ether [3O-6O ] and 0  94 decanting the wash. After the third wash the flask was fitted with a serum cap. The residual petroleum ether was removed by successive evacuations with a vacuum pump through an l8-gauge hypodermic needle inserted through the serum cap.  After  each evacuation, the flask was f i l l e d with nitrogen [dried by passage through sulfuric acid and sodium hydroxide].  The flask f i l l e d with nitrogen was then  placed in a nitrogen f i l l e d drybox. The serum cap was removed and 15 ml of anhydrous dimethyl sulfoxide was transferred into the flask.  DMSO was dried with  and d i s t i l l e d from calcium hydride under reduced pressure then stored over dried molecular sieve [Linde, type 4A].  The flask was stoppered with a mercury  gas trap and removed from the drybox. The flask was placed in a force draft oven at 60°C and swirled occasionally u n t i l the solution became clear and green and the evolution of hydrogen gas had ceased [ca. 60 minutes]. For generation of the polysaccharide alkoxide, polysaccharide was  first  freeze dried and then dried overnight at 60° under vacuum. Dried material [1  g.]  was added to 50 ml of dried dimethyl sulfoxide in a 250 ml round bottom 2 necked flask containing a magnetic s t i r r e r .  The flask was tightly stoppered with a  polyethylene stopper and removed from the drybox.  The sample was stirred u n t i l  a l l the polysaccharide had dissolved. Polysaccharide and methylsulfinyl anion solutions were placed in the drybox. A 50$ excess over the number of equivalents of base required for hydroxyl plus carbonyl was added to the polysaccharide. The number of equivalents required was calculated on the basis of the polysaccharide structure. Upon the addition of the anion [ ~ 12 ml] to the polysaccharide a gel formed. The flask was stoppered with a serum cap and thermometer, before removal from the drybox. The gel, on s t i r r i n g , l i q u i f i e d and the reaction mixture appeared homogeneous. The reaction was allowed to run for 4 hours, a time indicated as minimum for complete alkoxide formation  [78]'  95 In the methylation reaction the polysaccharide alkoxide solution was cooled to less than 20° i n an ice bath.  Methyl iodide [5 ml] was added to the  stirred solution with a hypodermic syringe at such a rate that the temperature did not rise above 25° [ ~ 10 minutes].  The amount of methyl iodide added was  not c r i t i c a l as long as i t was in molar excess of the base. Shortly after addition of methyl iodide, heat evolution ceased, the solution became clear and the viscosity was markedly reduced.  At this stage reaction was  complete.  The reaction mixture was poured into water, then dialysed overnight against running water. The methylated polysaccharide partially precipitated by water came completely out of solution on dialysis.  The methylated polymer was washed  from the dialysis tubing and dissolved in chloroform.  The chloroform solution  was dried with anhydrous sodium sulfate and evaporated to dryness.  The methyl-  ated polysaccharide was fractionated using chloroform and petroleum ether 60°] extractions.  [30-  The major fractions were dried i n vacuo and analysed by IR  and microanalysis.  II]  PURDIE METHOD [62] The Purdie method was generally applied only to partially, methylated poly-  saccharides.  The partially methylated polysaccharide was dissolved i n methyl  iodide or a methyl iodide/acetone mixture.  The solution was vigorously stirred  and boiled under reflux in the presence of Drierite. portions over a period of 6-l8 hours.  Silver oxide was added i n  The silver oxide was removed by f i l t r a t i o n  through a Celite pad and was continuously extracted in a Soxhlet with boiling . chloroform.  I f acetone was a component of the original mixture the silver  oxide was washed with acetone or methanol instead of chloroform.  The combined  f i l t r a t e and washings were concentrated to dryness and the material was examined by means of an IR.  -  96 III]  KUHN METHOD [72] This method was employed with partially methylated polysaccharides. The  partially methylated polysaccharide was dissolved in a mixture of r e d i s t i l l e d dimethyl formamide and r e d i s t i l l e d methyl iodide. with Drierite.  The solution was stirred  Silver oxide was added in small portions over two hours and  s t i r r i n g was continued for a further 16 hours.  The solid material was removed  by centrifugation and was washed with portions of dimethyl formamide and chloroform. The centrifugate and the washings were poured into a solution of sodium cyanide [1$ W/V] and this solution was extracted with chloroform. Extracts were concentrated to remove chloroform and residual dimethyl formamide was removed by continual co-distillation with water.  In some experiments Drierite  was omitted, in others the methylated polymer was isolated by dialysis. D i s t i l l a t i o n of dimethyl formamide was intentionally carried out without stringent precautions for the exclusion of moisture. Often traces of silver salts remain in the methylated product after Kuhn and Purdie methylations.  These salts were sometimes successfully removed by  redissolving the methylated polysaccharide in chloroform and f i l t e r i n g the solution through a Celite pad.  METHYLATION OF OLIGO AND MONOSACCHARIDES PURDIE AND KUHN METHODS The methods used were similar to those described for the polysaccharides with the following modifications.  During Kuhn methylations a second portion of  silver oxide was added after 4 hours of s t i r r i n g . applicable in these cases.  In addition dialysis was not  97 HAKOMORI METHOD The method described for polysaccharides was followed for mono and oligosaccharides with the following exceptions and changes. Some of the lower molecular weight samples could not be freeze dried. As well the dialysis step was of necessity omitted. water-dimethyl  The samples were isolated by extracting with chloroform the  sulfoxide solution used for dialysis in the polysaccharide case.  After three extractions or continuous extraction the chloroform was washed with d i s t i l l e d water to remove dimethyl sulfoxide. The products were evaporated to a syrup and residual dimethyl sulfoxide was removed by high vacuum d i s t i l l a t i o n of a part or a l l of the sample.  PERIODATE OXIDATION Analytical periodate oxidations were carried out in the following manner. Duplicate samples of the ash-free polysaccharide [•—50 mg accurately weighed] were dissolved in water and the pH was adjusted to 7-  The solutions were  transferred to standard flasks [100 ml] fitted with ground glass stoppers and sodium metaperiodate solution [25 ml] was added. This solution was obtained by dilution of a O.JM sodium metaperiodate solution [10 ml diluted to 100 ml]. The polysaccharide solution was made up to 100 ml and stored in the dark at 0° or room temperature. Aliquots [5 nil] were removed at intervals for the estimation of periodate consumption. Duplicate solutions which contained sodium metaperiodate and water but no polysaccharide were made up i n the same manner to act as reagent blanks.  98 ESTIMATION OF PERIODATE  [91]  An aliquot [5 ml] was added to a solution containing a phosphate buffer at pH 7 [25 ml] and 20$ potassium iodide solution [10 ml].  The liberated  iodine was titrated against standard sodium thio sulfate [COIN] using starch as the indicator. The phosphate buffer contained disodium hydrogen phosphate [5*68 g.] and potassium dihydrogen phosphate [3*62 g.] i n one l i t e r of d i s t i l l e d water.  SIMULTANEOUS ESTIMATION OF POLYHYDRIC ALCOHOLS AND SUGARS The experimental work outlined here was part of a group project involving Reid, Jensen and Gibney [92].  The estimation of erythritol with threitol,  the computer analysis of these results and examinations of the effects of glycolaldehyde were mainly the work of Jensen. Columns employing the following liquid phases were evaluated for their potential use in the separation of galactitol, glucitol and mannitol acetates: Apiezon  J, K, L, M, N  Butane 1,4-diol Succinate Carbowax  2CM  FAPP Neopentyl Glycol Sebacate Silicone DC Silicone GE  QF-1 SE30, 52, SF96, XE-60  Versamide 900 None of these columns would produce the required separation.  The literature  has since reported two systems which w i l l achieve this separation. However, the columns when used on thermal conductivity have resulted in only limited  99 success.  I f large samples are injected the columns overload and the separation  is lost [93].  A certain advantage is apparent when galactose is a minor com-  ponent of the mixture. Columns were examined for the separation of equilibrium mixtures of neutral sugars using the silylation technique of Sweeley et a l [15],  The columns ex-  amined were: Silicone  GE  SF-96  Silicone  GE  SE-30  Silicone  GE  SE-52  Dow  Corning Silicon High Vac Grease  The column having the clearest resolution was that reported in this work [SF-96], It has been shown in the discussion that a scheme can be devised where, knowing the equilibrium values of the components, the total analysis of a sample of the five common neutral sugars may be evaluated.  The column length, the programme  rate and the flow rate were chosen experimentally to give optimum s p l i t t i n g between the two t e t r i t o l s , erythritol and threitol. Samples of the following polyhydric alcohols were purified by vacuum dist i l l a t i o n ; ethylene glycol, glycerol and butane - 1 , 4 - d i o l .  D-Threitol. and  erythritol were recrystallized to literature melting points.  The five neutral  sugars were recrystallized and analysed by paper chromatography and gas liquid chromatography for impurities. was d i s t i l l e d . pellets.  Pyridine refluxed over sodium hydroxide pellets  A narrow.boiling range fraction was stored over sodium hydroxide  Hexamethyldisilazane and trimethylchlorosiline were used as obtained  from Penninsular Chem Research. Analysis of mixtures of internal standard and each of the five neutral sugars was carried out in the following manner. The sugars were dissolved in water containing 0-5$ W/V  chloroform and allowed to equilibrate. A period of  100 2k hours appeared to be ample with no further change being detectable in the equilibrium ratio determined.  The sample evaporation to dryness was  to avoid any change i n equilibrium.  critical  The samples were evaporated under vacuum  on a rotary film evaporator u n t i l the sample just became dry, a further minute of evaporation was allowed and then the sample was removed.  Immediately  a sample of pyridine containing the internal standard was pipetted into the evaporation flask.  The samples were swirled and hexamethydisilazane then t r i -  methylchlorosilane were added. Any variation from this procedure w i l l result in alteration of the equilibrium mixture.  This was especially pronounced  when a component which crystallized easily such as xylose was predominant. The equilibrium ratios found for pyridine are similar to those for water [22] resulting in l i t t l e change i n the equilibrium mixture i f these conditions are r i g i d l y followed. Analysis of single compounds and internal standard butane -1,4-diol yielded data reported in the discussion for molar response factors and equilibrium concentrations for various anomers. Sample size was varied with respect to i n ternal standard over a wide range to confirm that detector response was linear with sample size.  A l l other parameters were held constant to minimize variation  in molar response factors. Sufficient data were evaluated to provide molar response factors and equilibrium anomer ratios [at least three samples were run in duplicate for peaks of the polyols, five samples were run in duplicate for sugars]. Samples were then prepared of the various synthetic mixtures [expected to result from periodate oxidations,of plant polysaccharides]. Samples were analysed with three runs i n each sample determination. discussion.  The data have been presented in the  101 Data were also obtained using erythritol as the internal standard and employing the shorter programme from 190-220° as a method for the analysis of crude hemicellulose hydrolysates as reported in the discussion. Accumulated experimental data indicate that for sugar analysis the following limiting percentages may be determined. .  Xylose  0.5$  Arabinose  0.5$  Mannose  0.5$  Galactose  1.0$  Glucose  0.5$  The value expressed i s normally a practical limit in general hydrolyses.  For  a clearly detected sugar i t may be considerably lower, in the order of 0.01$. Galactose is notably higher because only a single peak containing 30$ of the total peak area is visible in hemicellulose hydrolysates. The f i n a l check on this method was the analysis of three samples prepared independently and analysed by the proposed method. The results were i n good agreement with expected values.  PURIFICATION OF LEMON GUM Lemon gum*[crude] was dissolved in water and filtered through several layers of gauze followed by sintered glass. The solution was poured in a thin stream into a five volume excess of alcohol acidified with acetic acid.  This procedure,  however, l e f t the gum with a high ash so the gum was redissolved and purified by two alternate routes which yielded A  solution  of gum  was  acidified  products with  which  were  hydrochloric  identical. acid  * The sample of lemon gum used i n these experiments was received from the late Dr. E. Anderson.  1G2 and precipitated into 5 volumes of alcohol. of ash.  This product showed only a trace  A second portion of gum solution was deionized by passage through  Amberlite IR 120 [H+].  The solutions were immediately frozen and freeze dried.  Optical rotation of the products gave identical values of [ Oi ]^ +19.2°.  The  polymers were hydrolysed and analysed by G.L.C, for results see discussion. A sample of the freeze dried gum was analysed by Tiselius moving boundary electrophoresis and showed only a single band. The gum was also chromatographed on DEAE cellulose [2.5  x 50 cm] using water [150 mis] followed by  0.05M NaH P0 [150 mis] increasing by equal steps to O.25M NaH P0 . 2  4  2  was eluted during 0.10M  NaH P0 irrigation. 2  4  4  The  gum  The gum tailed somewhat on the  column and as the ionic strength was increased more carbohydrate was eluted from the column. The bulk of the gum was isolated in a volume of 50 ml, tubes 72 to 82.  G.L.C  analysis indicated that the product isolated had the  same neutral sugar analysis as the starting material and an identical optical rotation of [ OC ]  +19.3°•  The neutralization equivalent of the ash free gum was determined by t i t r ation with 0.100N NaOH using a pH meter. The equivalent weight was calculated to be 793 g-  OPTIMUM HYDROLYSIS OF LEMON GUM  - PERIODATE DEGRADED  Analytical periodate oxidation of lemon gum produced results in close agreement with those reported earlier for this gum  [1  ].  Lemon gum [20 g.] dissolved in water [1000 mis], was oxidized with sodium metaperiodate [29 g.] for one week at 5°C.  Dialysis was used to removed low  molecular weight materials after destruction of excess periodate with ethylene  103 glycol.  The polyaldehyde present in the dialysate was reduced with sodium  borohydride [5 &•]  for 48 hours. An excess of acetic acid was added to destroy  unreacted borohydride and aid in break down of borate complexes. Dialysis was again used to remove low molecular weight materials, yield 12.5  g-  Samples of the polyalcohol [20 mg] were dissolved in water [5 ml] and the acid concentration was adjusted so that the f i n a l volume [10 ml] had the f o l lowing sulfuric acid concentrations; was  0.05,  0.1,  0.2,  continued for 24 hours at room temperature.  barium carbonate.  The residual polysaccharide  Hydrolysis  The acid was neutralized with  was  and analysed on the gas chromatograph. The 0.5N  0.5 and 1.0N.  isolated by precipitation  acid solution, as indicated  in the discussion, produced the most satisfactory hydrolysis, as determined by residual glycerol [see page 22  ].  A sample of polyalcohol [200 mg] was dissolved in water and the normality of the acid and volume were adjusted to 0.5N  and 20 mis.  Samples [2 ml] were  removed from the reaction mixture at the following intervals: and 32 hours.  Precipitation of the sample taken was  dilution with alcohol [8 ml].  1, 2, k, 8, 16  carried out by direct  Immediate centrifugation and work up minimized  further hydrolysis. In addition to the samples taken above, a sample of precipitated polysaccharide [32 hours] was dialysed for 2k hours against running water. Neutralization of a sample of supernatant from the 32 hour hydrolysis precipitation provided a fraction containing material lost during hydrolysis. Gas chromatographic analysis was  carried out on hydrolysates of fractions  isolated including a sample of starting material and a sample of unhydrolysed  104 soluble material at 32 hours. in Table X  The data from these hydrolyses are presented  [see page 26].  Lemon gum polyalcohol [9'17 •with 0.5N  H2SO4  [400 ml].  g«] was hydrolysed for the required l 6 hours  The solution -was neutralized •with barium carbonate.  Yield of precipitated polysaccharide designated lemon gum SI on addition of k volumes of alcohol, 3*51 g-  The supernatant fraction was evaporated to a  syrup under reduced pressure.  To this thick syrup was added 30 mis of methanol  and 30 n^ls of acetone [2 times] to extract the very low molecular weight fraction.  The fractions were centrifuged to remove the insoluble higher mole-  cular weight material. product was obtained.  On evaporation of the 120 mis of solution 3*89 g« of The insoluble material was dissolved i n water and freeze  dried, yield 1.06 g. Lemon gum SI [100 mg] was oxidized with an excess of periodate u n t i l the oxidation levelled off [88 hours]. Periodate consumption was 0.40 moles per anhydro  sugar based on galactose and arabinose analysis.  oxidation of 2.30 g. of polysaccharide yielded 1.22  A larger scale  g. of poly alcohol.  Smith degradations carried out as before indicated that an optimum yield of polysaccharide was obtained using the same hydrolysis conditions as outlined for the f i r s t periodate oxidized polysaccharide, namely 0.5N Hydrolysis under these conditions of  2XIO4/BH4 lemon  H2SO4  for l 6 hours.  gum yielded a polysaccharide  containing only a limited amount of arabinose in comparison to galactose as outlined in the discussion.  AW? SMITH FRAGMENTS Direct gas chromatography of the soluble portions from the original periodate oxidized gum programmed to elevated temperature on SF-96 and SE-52 [10$ on 80-100 Diatoport S, 8' x l / V and SE-30 [20$ on 60-80 Chromasorb W, 2' x l/4"] showed only one component in sufficient quantity to isolate and identify.  The column temperature [275°C] was such that maltotriose was eluted  [SE-52 - 80 min., SE-30 - 30 min.] [94]. hydrolysis yielded galactose and glycerol.  Isolation of a small sample and A second sample was isolated, the  s i l y l groups were removed by methanol/water and the sample was methylated by the Hakomori procedure. Hydrolysis, reduction and acetylation produced a partially methylated a l d i t o l acetate with the same mobility as in authentic sample of 2,3,4,6-tetra-0-  methyl-D-galactitol acetate.  Analysis of the mass  spectrum confirmed this assignment.  THE REDUCTION OF ACIDIC POLYSACCHARIDES ESTERIFICATION Lemon gum [1.00  g.] which had been deionized by passage through Amberlite .  IR-120 [H+-form] and freeze dried was dissolved in water [50 mis]. oxide [12.5 bath.  Ethylene  g-] was bubbled into the polysaccharide solution cooled in an ice  The vessel was sealed and stored at room temperature. During the next  fourteen days the sample was cooled daily in an ice bath and the pH of the solution was measured. The solution pH rose from an i n i t i a l value of 3-2 "to slightly over 7 at the end of the fourteenth day.  The gum solution was d i -  alysed against running water for twenty-four hours and freeze dried, yield 0.93 B-  106 Analysis of the degree of esterification was carried out using acid hydrolysis with IN H S0 at 100°C for 20 hours. 2  The quantity of ethylene glycol  4  and what is presumed to be diethylene glycol were in excess of the molar requirements for complete esterification.  REDUCTION OF ETHYLENE GLYCOL ESTER OF LEMON GUM Lemon gum ester [1.00  g.] was dissolved in dry dimethyl sulfoxide [dried  as for Hakomori methylations] [50 ml.]. borohydride [0.5  g«3-  To this solution was added lithium  Such.a large molar quantity was required for interaction  with free hydroxyl functions in the polysaccharide. gel during the f i r s t hours of reduction. persisted throughout the reduction. swirling the mixture.  The solution formed a  The gel was relatively stable and  The gel could be broken down partly by  The reaction was maintained anhydrous and hydrogen gas  was vented through a mercury trap.  The reduction was monitored by daily  analysis of aliquots. A l l samples were worked up in the same manner. Acetic acid was added slowly to the DMSO solution to destroy the excess borohydride and to break down borate complexes. dried, yield O.83 Material [1.00  The samples were dialysed then freeze  g. g.] from a large scale esterification and reduction was  subjected to a second esterification to re-esterify any acids which were saponified.  Salts of uronic acids were converted to free acids by ion exchange  chromatography as in the previous reduction.  Quantities of ethylene oxide and  duration of reaction were as before, yield O.96  g.  This freeze dried ester was reduced by the method indicated previously, yield 0.90  g.  107 The samples were analysed by gas chromatography and the results are reported In the discussion.  SIGNAL ENHANCEMENT Compounds which were examined by s i l y l a t i o n of free hydroxyls [Figure 16] were synthesised by known pathways or purified by preparative gas chromatography as reported in the discussion. Pure starting materials were silylated and purified by both gas chromatography and simple removal of s i l y l a t i n g reagents by evaporation under reduced pressure.  TRIMETHYL SILYLATION OF GALACTURONIC ACID A commercial sample of D-galacturonic acid was silylated using each of the following solvents: pyridine, dimethyl formamide, dimethyl sulfoxide, hexane and dioxane, by warming and triturating the sample in the solvent with added bis-trimethylsilyl acetamide [BTMSA]. in the percentages of sample silylated.  There were significant variations  Those solvents which dissolved the  galacturonic acid showed better yields of silylated product.  In addition,  s i l y l a t i o n was carried out i n pure BTMSA. The percentage of anomers present when these solutions were analysed on the gas chroma,tograph varied widely. The best general yield arid the most reproducible result was obtained with pure BTMSA.  108  TABLE XX ANOMERIC RATIOS FOR BTMSA SILYLATION OF GALACTURONIC ACID IN DIFFERENT SOLVENTS  Solvent  Peak 1  <  Peak 2 and 3  Peak 4  < fi  $  BTMSA  49-5.  24.3  26.1  DMF  52-1  18.1  28.8  Dioxane  43.3  4.2  Pyridine  52.5 18.8  47.8  DMSO  36.2  33.4 33.6  30.2  Hexane  45.O  30-9  24.1  METHYLATION ANALYSIS Lemon gum [1 g.] was methylated by the Hakomori procedure described earlier, yield  0.90 g.  A second methylation of this methylated material [0.70 g.]  was carried out and the DMSO solution -was poured directly into a dialysis sack without dilution by water. The weight of recovered lemon gum was only 0.020 g. A second methylation [5-0 g.] was carried out, yield 6.198 g.  This sample  was fractionated using chloroform in 30-45° petroleum. The extracted fraction insoluble in 20$ CHCI3 but soluble in 30$ CHCI3 contained 6.011 g. of the total sample. Analysis: OMe 39»46$. A small sample [1.0 g.] of methylated lemon gum was acidified with anhydrous hydrogen chloride in chloroform.  A small amount  of solid material [NaCl] was centrifuged o f f and the polysaccharide was precipitated with 30-45° petroleum. over sodium hydroxide.  The polysaccharide was dried under high vacuum  This methylated polysaccharide was given a Purdie methyl-  ation i n order to complete the methylation of the uronic acids.  Analysis: OMe .40.  109 This polymer, although s t i l l showing traces of uronic acid as the salt form-, was reduced with lithium aluminum hydride to give a product showing only a trace of carboxyl, yield 90$. The following polymers [100 mg] were also methylated by the Hakomori procedure: a]  Lemon gum degraded with l/lON H2SO4, yield 105 mg.  b]  Lemon gum periodate .oxidized and borohydride reduced before hydrolysis, yield 82 mg.  c]  Lemon gum periodate oxidized and borohydride reduced after O.5M H2SO4 hydrolysis, yield, 95 ™S«  None of these polymers was reduced with lithium aluminum hydride. A l l four of these methylated polymers were analysed by the method outlined in the introduction to the experimental section. The following monosaccharides were prepared by known routes to f a c i l i t a t e the peak identification on the gas chromatograph: 2,3^4,6 -tetra-0-methyl-D-galactose 2,3>4-tri-0-methyl-D-rhamnose 2, J>, 5 -tri-Q-methyl-L-arabinose 2,3 - d i-0-me t by 1-D - glu co s e 2,3>4-tri-0-methyl-L-arabinose  110 APPENDIX I ESTIMATION OF DEGREE OF POLYMERIZATION During the course of evaluating G.L.C. as a method for the estimation of polyhydric alcohols and sugars, i t became apparent that these procedures could be applied to an estimation of D.P. of oligosaccharides and small polyascharides.  Reduction of the reducing end group -with sodium borohydride produces on  hydrolysis the terminal component which may be estimated with respect to the remainder of the non reduced carbohydrate.  Work by Reid, Rowe, Rowe and Dutton  [95] has shown that a D.P. of 150 may be estimated i n this manner. Previous experience with hydrolysis of solutions containing polyols i n dicated a need to examine closely the hydrolysis of reduced oligosaccharides. Certain factors must be taken into consideration before D.P. may be estimated by the method of Reid et a l .  The compound[s] being reduced must be known. Also  within limits complete reduction must occur.  The complexity of the sugar mix-  ture on hydrolysis must be such that the polyol produced on reduction i s clearly resolved on G.L.C. from the remaining anomeric carbohydrate forms.  Only with  very low D.P. oligosaccharides can analysis be successfully carried out when overlap occurs. action.  I t i s known that sugars decompose during the hydrolysis re-  Previous work i n this laboratory has indicated that dehydration of  polyols to anhydropolyols can also occur [85 ]. A series of maltosaccharides were obtained by enzymatic hydrolysis of starch with  cX-a.mylase.  Isolation of the higher D.P. maltosaccharides [D.P.>  4] i n a pure state was not possible although paper and Sephadex chromatography were employed in the attempted purifications.  Prior to examination of the re-  duced maltosaccharides the effects of hydrolysis on glucitol were investigated.  Ill Prolonged acid hydrolysis confirmed thatdianhydro-D-glucitol was formed as the major dehydration product. findings [85].  This was in agreement with previous hydrolysis  The more volatile component i n the gas  chromatogram was con-  firmed to be 1 , 4 , 3 , 6 dianhydro-D-glucitol by mass spectrometry and  comparison-  with an authentic sample. FIGURE 30 GAS CHROMATOGRAM OF GLUCITOL HYDROLYSED WITH 1.0N H S0 FOR 24 HOURS 2  4  Standard analysis conditions but programmed at 10°/rnin.  20  30  40  min.  During these hydrolysis studies i t was discovered and has now been reported independently by another Laboratory [58],  that undersilylation can occur.  undersilylated product i s eluted before the f u l l y silylated derivative.  The The  silylation of hexitols i s much slower than for lower molecular weight polyols or the parent sugars.  Glucitol i n the presence of excess reagent requires two  hours of reaction before the under silylated component becomes insignificant. Further work is being carried out to confirm the structure of these under silylated hexitols.  112 FIGURE 31 GAS CHROMATOGRAM OF GLYCITOL SHOWING UNDERSILYLATION AFTER 10 MIN. OF REACTION  In order to obtain the maximum D.P. accuracy using reduction, hydrolysis and G.L.C, careful consideration must be taken of carbohydrate degradation and loss of polyol by dehydration.  EXPERIMENTAL A series of glucose oligosaccharides was isolated by enzymatic hydrolysis .of starch [95].  I n i t i a l purification was achieved using a stubby charcoal  column [10 cm in diameter x 2.5 cm deep].  The glucose and maltose were displaced  from the column by gradually increasing the alcohol to water ratio in the elution solvent.  The alcohol percentage was increased to 50$ and the higher  D.P. oligosaccharides -were eluted as a group.  These higher oligosaccharides  were spotted on Whatman No. 2 f i l t e r paper and chromatographed i n solvent C.  113 Components were detected by development of guide strips.  The bands of oligo-  saccharides were eluted with water and the sample was isolated in an easily handled form by freeze drying.  Samples of the major components were rechromato-  graphed on paper and Sephadex. Only i n the case of the t r i - and tetrasaccharide • were relatively pure compounds isolated.  Gas chromatographic analysis of the  reduced oligosaccharides did not produce accurate [+ 5$] D»P» values because of trace quantities of higher oligosaccharides present as contaminants. Samples of glucitol were hydrolysed and analysed by gas chromatography. The presence of l,k;  3,6-dianhydro-D-glucitol was confirmed by comparison of  mass spectra obtained from an authentic sample and one isolated by gas chromatography.  114  APPENDIX II ENZYMATIC HYDROLYSIS OF LEMON GUM  Enzymatic hydrolysis has been employed successfully in the glycolytic cleavage of certain classes of polymers. Notable i s the extensive work which has been carried out on various starches [97]« -A wide variety of different enzymes has been discovered and utilized i n preparing the maltosaccharides for the earlier work on D.P. and amylo 1,6-glucosidase, the enzyme which debranches 1,6 linkages. The enzymatic hydrolysis of cellulose and some of i t s derivatives is well known. The fact that ruminants convert cellulose containing plants into useful food sources, microorganisms destroy cellulosic fabrics, &<3. "the n  host of wood rots decompose wood, has also focussed attention on nature's abundant cellulase activity.  The food industry employs pectinase preparations for  clarifying wines, f r u i t juices and liquefaction of pectin containing gels. Xylans from wood and plant sources have been hydrolysed with pectinase and xylanase preparations to yield a product containing xylose and a series of oligosaccharides [ 9 8 ] • As a result of the success encountered with these enzymatic degradations an examination was carried out on the effects of various general enzyme preparations on lemon gum. Table XXE l i s t s the enzyme preparations employed in this survey. TABLE XXI ENZYME PREPARATIONS EXAMINED FOR ACTIVITY ON LEMON GUM d -Amylase [salivary] Pectinol 41-P concentrate [Rohm and Haas] Pectinase [BDH] Hemicellulase [BDH] HP-150 [Rohm and Haas] Cellulase [BDH] Cellulase 36 [Rohm and Haas] A -Glucosidate [BDH] Xylanase [BDH] BDH - B r i t i s h Drug House  115 Although some of the preparations listed in Table XXI  -were not expected  to show activity they were examined because of possible activity of minor impurities.  Two of the preparations [Hemicellulase and HP-150] were expected  to show the highest activity.  This was especially true of the latter material  since i t is designed to reduce viscosity in plant gum solutions. Assay of the o< -amylases, pectinases, jQ -glucosidase and xylanase with lemon gum showed no activity.  The enzymes present in these preparations were  active as was evidenced by formation of maltosaccharides and a series of xylose oligossacharides from birch xylan [98]. assayed by dialysis [99]  The remaining enzymes could not be  due to high cellulase activity.' The dialysis tubing  was attacked, resulting in release of glucose oligosaccharides and rupture of /the tubing.  Activity^of these preparations on lemon gum was examined after  the substrates had been in contact for two days. by a short heat treatment.  The enzyme was inactivated  There were several minor low molecular weight com-  ponents in the solutions. They were, however, attributable to contamination of the enzyme preparation or autohydrolysis of the lemon gum substrate. Lack of activity with these latter preparations was unexpected. A sample of HP-150 was fractionated with ammonium sulfate into a series of precipitates. Assayed against lemon gum these fractions were s t i l l not active. Fractionation was also carried out on Pentinol fractions.  4 IP.  Ko activity was found in the isolated  The only oligosaccharides present on dialysis of fractionated HP-150  or Pectinol 4lP treated lemon gum were attributable to autohydrolysis. . This fact was verified by running blanks containing only lemon gum and d i s t i l l e d water. During these experiments a parallel set was being conducted on mesquite gum by Rowe [100]. Equally negative results were obtained.  In addition, i t was  116 discovered that HP-150 had transferase activity capable of forming arabinose oligosaccharides.  Since, on hydrolysis of either lemon or me3quite gums  large amounts of arabinose are expected, any oligosaccharide isolated -which contained arabinose would be suspect.  The lack of activity against lemon gum  and the presence of transferase activity would appear to rule out convenient hydrolysis of lemon gum. When the role of the transferase activity is more fully known [100]  i t may  be possible to u t i l i z e HP-150 for the hydrolysis of oligosaccharides obtained by hydrolysis of lemon gum.  Careful examination w i l l be necessary to ensure no  other transferase activity [for example galactose transferase] exists. The closing discussion is speculation regarding low activity against a polymer such as lemon gum.  Lemon gum is a highly branched and extremely com-  plicated molecule containing many different subunits. damage to the tree bark.  The gum is exuded on  To be effective as a barrier the exudate must not be  readily attacked by organisms.  In an evolutionary sense those plants which  exuded a barrier which was d i f f i c u l t to degrade would have had the best chance of survival.  The inability of any of these enzyme preparations to degrade lemon  gum may be a reflection of this evolutionary protection. An interesting experiment would be the examination of micro-organism growth on media containing lemon gum.  When samples of gum have become contaminated in our laboratory, after  a short growth period further growth seems to stop.  This growth stoppage may  reflect a lack of nutrients such as nitrogen or complete utilization of the carbohydrate available as a result of autohydrolysis.  It is possible also, that  a pH change or formation of a metabolite may be factors which are limiting growth.  Experiments designed to test these factors may indicate whether there  is limiting growth rate or a b i l i t y to u t i l i z e lemon gum as a carbon source.  117 EXPERIMENTAL ENZYMATIC HYDROLYSIS OF LEMON CUM The enzyme preparations listed in Table XXI were screened for activity on lemon gum by the following procedure. [^1.0  A small sample of ash free gum  g.] was dissolved in water [50 ml] and a sample [5 ml] was pipetted  into solutions of the various enzyme preparations.  The samples were allowed  to interact for KQ hours, then excess enzyme and polysaccharide were precipitated with alcohol. The reactions were carried out in centrifuge tubes [50 to f a c i l i t a t e work up.  ml]  A second solution of lemon gum was adjusted to pH 7 with  sodium bicarbonate and a similar series of analyses repeated. Paper chromatograms were spotted with samples from the concentrated supernatants of the two experiments, with a blank from a pure gum solution and a blank of each enzyme. The chromatograms showed no spots which could not be attributed to either autohydrolysis of the lemon gum or contaminating  carbo-  hydrate from the enzyme preparations. Samples of the enzymes and lemon gum were also examined by dialysis  [99L  since i t was possible that a certain concentration of oligosaccharide could inhibit further enzymatic hydrolysis. The results as before were negative except with lemon gum acid which showed autohydrolysis products.  Four enzyme  preparations were not amenable to dialysis as a means of oligosaccharide isolation.  Hemicellulase, HP-150 and the two cellulase preparations show such  high cellulase activity that the viscose tubing, even though forming a heterogeneous reaction'site, was weakened-to the point that i t ruptured.  In addition  new compounds formed by enzymatic hydrolysis of viscose tubing were released into the solution.  118 These four preparations were examined by a second dilute reaction solution and the enzyme was inactivated by heating.  Alcohol precipitation and concen-  tration of the whole solution were both used to prepare samples for paper chromatographic examination. The results for the dialysis and the latter experiments were as before negative showing no increase in oligosaccharides which could be attributed to enzymatic hydrolysis.  FRACTIONATION OF PECTINASE, CELLULASE AND HP-150 Pectinol  41-P concentrate, Cellulase and HP-150 were a l l fractionated  using ammonium sulfate by the following general scheme. Enzyme  preparation  [10 g.] was dissolved in water [200 ml] containing sodium bicarbonate [4 g.]. Additions of ammonium sulfate were made to the solution in approximately 10 g. lots.  When a precipitate formed i t was centrifuged off and further additions  of ammonium sulfate were made. No attempt was made to determine quantitative data on various fractions. Rather a general qualitative result was determined.  PECTINOL 41-P CONCENTRATE A significant portion of enzyme preparation was insoluble in water because of f i l l e r added to the preparation.  The prepared enzyme fractions  were reacted with dilute solutions [1-2$] of lemon gum. A l l samples gave results similar to an autohydrolysed gum sample.  119  PECTINOL 41-P FRACTIONATION  Ammonium Sulfate [g.]  Samples Retaining Molish Color  Molish on Enzyme  Amount ofPpt  0  +  ho  ++  50  ++  —  small  60  ++  —  fair  70 80  ++  ++  fair  ++  —  fair  90 100  ++++  ++++  fair  ++++  +++  fair  110  +++++  +++++  fair  Final Solution  +++  large  —  small  ++++  CELLULASE 36 A slightly cruder fractionation was obtained on this enzyme.  Ammonium Sulfate [g.]  0 60 120  120 [over night] Supernatant  Amount of Ppt large [ f i l l e r ]  Molish Test +  small  +++  fair  +10  small  ++++ ++  Examination of activity against lemon gum was similar to that for Pectinol l+l-P and showed no specific increase i n components isolated over autohydrolysis.  120 HP-150 A similar crude fractionation was obtained for this enzyme preparation and no activity increase was noted in any of the five fractions examined. Extensive purifications of this preparation were carried out by Rowe [100] but because of the transferase activity noted, work was not carried further.  121  APPENDIX III CARBON-CARBON BOND CLEAVAGE, DIALYSIS AND DESTRUCTIVE PURIFICATION AN UNDERGRADUATE ORGANIC EXPERIMENT Carbohydrates are among the most abundant natural products.  They are  widely distributed in both the plant and. animal kingdoms. Their variety is vast, ranging from cellulose and starch to glycogen, deoxyribonucleic acids, streptomycin and blood group glycoproteins.  Carbohydrates have unique and  varied biological activity, ranging from stores of potential energy in animals to supporting tissues in plants.  Bacterial polysaccharides  frequently carry  the dominant immunological character of the bacterial c e l l and, in the case of pathogenic species, may form essential ingredients of vaccines. Experiments in undergraduate laboratories with carbohydrate compounds have been very limited-. Recently a procedure for conversion of polymeric glucose into erythritol was developed.. Since this carbohydrate experiment involved a variety of techniques and reactions not often utilized in undergraduate laboritories i t was f e l t to be of possible general interest. This experiment, as described below, can be used to illustrate  hetero-  geneous and hemogeneous reactions, oxidative carbon-carbon bond cleavage, d i alysis, reduction, destructive  purification, decolorization, crystallization,  melting point and chromatography. This experiment allows for a development of varied pathways to the f i n a l product.  Through the use of prepared samples the duration of the experiment  may be shortened from one involving several lab periods to only two lab sessions. The quantity used in the oxidations may be large or sufficient for only chromatographic identification of the f i n a l product.  Table XXII below l i s t s the various  reagents and purification steps involved in the experiment. Not a l l pathways are possible,therefore, i f desired a student may be allowed to determine a feasible pathway.  122 TABLE XXII VARIABLE STEPS IN PREPARATION OF ERYTHRITOL  Polymer  Oxidant  Cellulose  NaI0  Soluble starch  HT0  4  4  Purification Reduction NaBH  Dialysis  4  Filtration  Purification  Isolation  Dialysis  Precipitation  Filtration  Freeze drying Not necessary  Insoluble starch  a] filtered o f f  Glycogen  b] next reaction on solution  Hydrolysis  Neutralization  Time and acid strength may be varied  Ion Exchange Ba[OH] - BaC0  Decolorization  Characterization  Short charcoal column  Melting point mixed melting point  Repeated Crystallizations  Chroma to graphy - gas liquid - paper - thin layer  3  As a result of the -wide variety of pathways different points may be i l l u s trated with the experiment.  The carbon-carbon bond cleavage may be carried out  as a homogeneous or heterogeneous reaction depending on the solubility of the • glucose polymer. Depending on the homogeneity of the reaction, products from the oxidation and reduction may be purified by dialysis or f i l t r a t i o n .  As an  alternate, soluble polymers may be purified by precipitation i f periodic acid is  the oxidant.  the oxidation.  Barium hydroxide w i l l precipitate iodate ions remaining after  123 The following experimental procedure outlines the necessary details to convert one of the polymers into erythritol.  The quantities and time may be  altered by the use of prepared samples and method of characterization. Where prepared samples are used students should carry out the reactions, but pool their sample at each stage i n order to supply subsequent laboratory periods with prepared samples. Alternate reaction pathways differ from the experiment below as noted. Starch [l6.2  g.] was dissolved by heating in d i s t i l l e d water [400 ml].  Sodium metaperiodate [23.4 g.] was added to the cooled solution.  The reaction  was allowed to stand in the dark for 2k hours [Note 1] at room temperature. At the end of this time the reaction was stopped by the addition of ethylene glycol.  The solution was poured into a piece of cellulose tubing double knotted  at one end. knotted. 2].  The a i r was expelled from the tubing and the top end was double  The tubing was completely immersed in running water for 2k hours [Note  Sodium borohydride [4.0 g.] [Note 3] was added to the dialysate and after  2k hours the excess reducing agent was destroyed by the addition of a small amount of acetic acid [5-0 ml].  Dialysis was employed to remove the sodium  borate [Note k] and the solution was concentrated to a small volume [approximately 100 ml]. 3 hours.  The solution was made 2N with sulfuric  acid and re fluxed for  The acid was neutralized with slightly less than an equivalent amount  of barium hydroxide.  The slight excess of acid was neutralized with a small  amount of barium carbonate.  The precipitate was removed by suction f i l t r a t i o n  through a pad of diatomaceous evaporator to dryness.  earth. The f i l t r a t e was concentrated on a rotary  The resulting syrup was dissolved in boiling methanol  and the solution filtered to remove traces of. inorganic salts.  The solution was  124  diluted with an equal volume of water and passed through a stubby charcoal column [101]  [Note 5']. A wash solution of the same solvents removed small  amounts of erythritol from the column. The column eluate was concentrated dryness and the clear syrup was dissolved in a small amount of methanol.  to On  cooling erythritol crystallized from-the solution. Yield 5.Q g' m.p.t. 121.5.  NOTE 1: ' Reaction rates with soluble or insoluble polymers may be reduced to 3 hours [102], NOTE 2:  Insoluble polymers when oxidized may be purified by f i l t r a t i o n rather than dialysis.  Washes with 50 ml. of water and intermediate  filtration  are necessary u n t i l essentially free of iodate, usually involving about 6 washes. NOTE 3:  I t has been recommended that a l 6 fold excess of borohydride. be used when reducing oxycelluloses [103]. For this reaction however, a small amount of unreduced product w i l l not effect the results. It i s recommended as well that insoluble polymers be shaken or stirred during reduction to prevent material being l i f t e d out of the reduction solution.  NOTE 4:  Insoluble polyalcohols are f i l t e r e d out, washed with water, steeped for 30 minutes in 10$ acetic acid, and washed several more times with water.  NOTE  Purification may be achieved by repeated crystallizations but the colored matter produced during hydrolysis i s d i f f i c u l t to remove.  125 CHARACTERIZATION The crystalline erythritol may be characterized by melting point and mixed melting point.  The effectiveness of the oxidation may be investigated by chrom-  atographic means. Examination of the hydrolysis solution w i l l show up to three components; glucose from unoxidized polymer, erythritol,and from terminal glucose residues, glycerol.  The amount of glycerol present  of branching in the polymer be.shown on paper  [10^3  [953*  is an indication of the degree  The presence of these three components may  or thin layer chromatograms  [1053*  Gas liquid chromato-  graphy can be employed to obtain quantitative results regarding the effectiveness of the oxidation and the degree of branching of the starting polymer  [95].  126  APPENDIX IV LEMON GUM:  DEGRADATION WITH O.IN SULFURIC ACID  Further proof of the galactan branch on branch structure of lemon gum may be seen on analysis of the residual polysaccharide isolated after hydrolysis Of lemon gum with O.IN sulfuric acid. tains essentially ho L-arabinose units. percent galactose as neutral sugar.  The residual polysaccharide con-  This polymer contained 98.2 mole  This material was further analysed by  methylation as reported earlier.  EXPERIMENTAL Lemon gum [20 g.] was dissolved in water. to the desired O.UT  The gum solution was adjusted  sulfuric acid strength and a f i n a l volume of 250 mis.  solution was refluxed for 12 hours.  The  The residual galactan was obtained on  precipitation of the neutralized solution with k volumes [1000 ml] of alcohol, • yield 8.20 g.  Analysis of the galactan showed only a trace of L-arabinose  [D-galactose 98.2 mole # ] . ' ' ' si  AUT0HYDR0LYSIS OF LEMON GUM Lemon gum contains a large amount of uronic acid [approximately one residue in five].  Free acids were obtained when the gum was acidified or deionized with  ionexchange resin prior to precipitation.  The pH of the gum acid is nearly  three and when a solution is heated extensive hydrolysis occurs. were isolated from autohydrolysis and from 0.1N  Oligosaccharides  sulfuric acid hydrolysis. The  components from both hydrolysis procedures were identical in their chromatographic behaviour and composition.  Close comparison of the oligosaccharides isolated  127 by these tvio procedures indicated they were identical chromatographically in several solvents. Table XXIII i s a l i s t of the oligosaccharides Isolated. Included in this.table are two neutral D-galactose containing oligosaccharides obtained when the 0.1N sulfuric acid residual polysaccharide was subjected to stronger acid hydrolysis. and  ref. therein].  A l l oligosaccharides had been isolated previously [ l  Identification  was achieved by comparison with known  mobility on paper chromatograms. The pure oligosaccharides were isolated from paper chromatograms and hydrolysed to indicate the component sugars. Oligosaccharides containing uronic acids were reduced in DMSO with lithium borohydride prior to hydrolysis.  TABLE XXIII OLIGOSACCHARIDES ISOLATED ON HYDROLYSIS OF LEMON GUM. 1] 3 -0-yS -D-galactopyranosyl-D-galactose  2]  6-0-/3 -D-galactopyranosyl-D-galactose  3]  4- Q-[4-O-methyl- o< -D-glucopyranosyluronic acid]-L-arabinose  4]  0-[4-0-methyl- oc-D-glucopyranosyluronic acid]-[l —> 4 ] - 0 - & ? L-arabinopyranosyl-[1—>5]-L-arabino s e k_o-[4-0-methyl- c<-D-glucopyranosyluronic acid]-D-galactose  6]  6-0-[ fS> -D-glucopyranosyluronic acid]-D-galatose  128 EXPERIMENTAL The pH of a lemon gum solution [10 g. i n 80 ml of water] was measured as 3«17  for a sample of Amberlite IR-120 deionized and freeze dried gum.  ized lemon gum  [l6 g.] was dissolved in water [200 ml].  Deion-  The solution was  placed in a dialysis tube previously inserted in a column [ 9 9 ]  fitted with  a side arm to allow intake of liquid and a bottom outlet to remove the d i The unit was wound with 8 feet of  alysate silution as i t passed by the bag.  heating tape and the temperature was maintained at 70-8o°C [measured on a thermometer inserted between the heating tape and the column]. After one week of dialysis the solution in the tube had expanded sufficiently to prevent liquid from passing around the dialysis sack. A small amount of liquid was removed from the dialysis sack and dialysis was continued for a further ten days.  The polysaccharide removed earlier and this f i n a l solution were pre-  cipitated into alcohol and a total of 11.90  g. of polysaccharide was recovered.  The collection of solutions from the autohydrolysis had been made in four separate fractions. Paper chromatography indicated that a l l four fractions had a similar composition.  Fractions 2,  3 and 4 were combined and fraction 1 was  further divided into an acid and a neutral fraction by absorption on Duolite A-4  [OH] and subsequent elution with 10$ acetic acid. 1  OLIGOSACCHARIDES Oligosaccharides isolated from the 0.1N  sulfuric acid and the autohydrolysis  were chromatographed on paper and on inspection proved to be v i r t u a l l y identical.  Only minor variation could be detected in the concentrations of some  of the components isolated.  129 Samples of the oligosaccharides present were isolated by elution from paper chromatograms. Since a l l compounds isolated were known,data are present in Table XXIV.  TABLE XXIV NEUTRAL AND ACIDIC COMPONENTS OBTAINED ON HYDROLYSIS OF LEMON GUM OLIGOSACCHARIDES Neutral Components  GAL Solvent [A] R  Components on Hydrolysis  Rhamnose  2.15  Rhamnose  Arabinose  1.30 1.00  Arabinose  0.53 0.37  Galactose  Galactose  1 2  Components. On Hydrolysis  Galactose Galactose  Acid Components  GAL" Solvent [B]  Reduced Components on Hydrolysis  3  1.13  arabinose  arabinitol 4-0-Me-Glucose  4  O.69  arabinose  arabinose, arabinitol 4-0-Me-Glucose  5  0.75  galactose  galactitol, 4-0-Me-Glucose  6  0.23  galactose  galactitol, glucose  130  APPENDIX V  A TECHNIQUE FOR HANDLING MICRO SAMPLES COLLECTED FROM THE GAS CHROMATOGRAPH A problem exists when samples are collected in capillary tubes on their elution from the column of a gas chromatograph.  I f these samples are to be  used for TLC or Mass Spectra they may be dissolved in a solvent which can then be evaporated away on the TLC plate or on the probe for the Mass Spectrometer. However, for micro analysis, IR and often with the Mass Spectra i t is imperative to avoid contamination of the sample with any solvent.  To avoid  this problem and s t i l l allow 25 jug samples to be handled requires a technique which avoids losses due to adhesion to loops, wires or other transferring systerns. A system has been developed which can handle small l i t t l e or no loss.  quantities with  The technique involves manipulation of the sample in a  glass collecting tube i n the same way i t was collected, heat and, flowing gas. I f a sample can be handled on the gas chromatograph without degradation i t may be handled by this technique.  I f the sample i s reasonably stable i t may  be manipulated using only heat since, as the sample i s heated i t s viscosity is reduced and the sample w i l l flow as the tube i s drawn through the furnace. A gentle flow of dry nitrogen or gravity may be used to increase the movement' of the'sample for less stable compounds, minimizing any degradation. In reality a portion of the gas chromatograph can be taken to the analyst or mass spectroscopist.  In addition, purity of collected  samples  can be  determined by analysis on the gas chromatograph, TLC or IR spectrometer. In order to handle 25 ju. g samples i t i s necessary to reduce the bore of the melting point capillary to the size of a vacuum leak capillary. be  done  on  a l l samples  since  the  capillary  action  This can helps  draw the  131 compound to the tip of the melting point tube.  Positioning i n the fine capillary-  facilitates removal of the sample to the t i p of the mass spectrometer probe, for spotting on a TLC or IR plate.  In Mass Spectroscopy, since only o. JAZ  sample i s required ample sample for examination by other techniques i s s t i l l available. This technique of moving the condensed sample i n a melting point capillary by the use of a thermal push overcomes the problems faced by workers using one of the simplest and yet s t i l l efficient collection techniques. tube acts as an a i r condenser and sample container.  The capillary  Samples may be stored by  simply sealing both ends in an oxygen -natural gas flame.  Should the sample  spread in the tube on long standing, slow insertion of the tube i n the heater w i l l allow the sample to flow and collect i n a small band.  Even i f the sample  should crystallize, i t s melting i n the oven w i l l cause i t to flow i n the tube. The melting point capillary should be f i r e polished at both ends for clean insertion through the holed septum i n the exit port of the gas chromatograph. By inserting the tube i n this manner a l l the carrier gas and compound w i l l pass through the tube and maximum collection w i l l be achieved.  The fine capillary  must be drawn after the collection because the extremely fine bore desired on the end of the tube would r e s t r i c t gas flow and increase the pressure drop across the column. The minimum sample that can be handled this way was weighed at 25yug and represented an approximate one inch high peak on an F and M 720 gas chromatograph at maximum sensitivity.  The upper limit of size on the col-  lection of a peak, even when the tube has been blown slightly would appear to be about 7-10 mg.  A sample larger than this i s simply blown through the tube.  132 FIGURE 32 COLLECTION OF GAS CHROMATOGRAPHY SAMPLES IN GLASS CAPILLARIES AND OVEN DESIGN FOR THEIR MICROMANIPULATION SWAGELOCK  NUT  SEPTUM SAMPLE •EXJT  CAPILLARY  TUBE  PORT  VARJAC  POWER  UNIT  SPIRAL.  WOUND  CORE  I f gas i s required to help move the sample, a lecture bottle of pure nitrogen may be mounted on the board with the small oven. Extreme care must be used when using gas as i t i s very easy to blow the sample out of the micro capillary t i p .  A large opening in the gas line, such that when open there i s  no gas flow out the exit tube and into the capillary i s an excellent safety valve and avoids loss of sample due to blow through.  This opening was closed  with an oversized rubber stopper, since i t was easily removed when the sample neared the t i p of the drawn tube. A small section of rubber tubing can be used as a bulb to force out sample. The tubing can be placed on the capillary without forcing sample out. A finger over the tubing end w i l l then move as much of the sample out of the tube as desired. U t i l i z i n g this collection  133 FIGURE 33 OVEN AND GAS SUPPLY FOR MICROMANIPULATION  technique and method of removal,it has been possible to analyse a single run on the gas chromatograph of a complex mixture of partially methylated a l d i t o l acetates.  Individual components varied in size as much as  100:1.  This method of micromanipulation w i l l have wide application to the collection of gas chromatography samples for the entire range of organic compounds This technique w i l l permit analysis of gas charomatograph runs much in the way one would with a GLC - Mass Spectrometer combination.  134 APPENDIX VI COMPUTER SEARCHING THE CURRENT CARBOHYDRATE LITERATURE The information explosion has affected a l l chemists.  As journals mul-  tiply and papers increase i t has become increasingly d i f f i c u l t to cover a l l the journals which contain important references.  Services such as "Chemical  T i t l e s , " published by the American Chemical Society, have eased the need to search for journals among different library branches, finding on occasion that they are missing, borrowed or have not arrived. I t i s , however, a prodigious task to review a l l the probable journals and scan the key words to ensure that one has not missed interesting or pertinent references.  I t is now possible,  however, to u t i l i z e one of the new computerized Chemical T i t l e search systems to retrieve a comprehensive l i s t of t i t l e s of papers of interest to carbohydrate chemists.  Not only the laborious work of searching but also of writing  is immeasurably reduced since the computer print out provides the subscriber with a printed reference sheet for each t i t l e retrieved. The programme developed here is designed to cover carbohydrate polysaccharides.  chemistry from synthetic to  Two restrictions were eventually imposed on the developed  programme because of interests and economy of terms.  The nucleotides were re-  moved from the search profile as well as certain specific names. The names were included i n i t i a l l y but later removed when i t was clear that there were few retrievals that could not have been picked up by checking the key words of Chemical T i t l e s .  The extra possible profile words were more valuable in  limiting retrievals in more d i f f i c u l t areas.  The profile development w i l l be  outlined in order that individuals with specific or broader interests who  may  wish to develop their own search profiles w i l l be able to improve and shorten their profile development period.  135 The search profile was designed to retrieve references to carbohydrate chemistry using the CAN/SDI^" Project for.computerized searching of Chemical Titles.  Since nomenclature for the f i e l d of carbohydrates i s systematic, i t  is possible to use these systematic endings and structural words to retrieve a l l references to carbohydrates. Profile words used in the search are given a letter code. of terms were prepared. of common letter groups.  Initial lists  These were shortened by truncation and u t i l i z a t i o n Truncation allows retrieval of words using letter  groups preceeded or followed by other letter groups. Truncation • 1.  Retrieval  OX*  2.  *0X  3*  OX  4.  *0X*  ox, oxen, oxide ox, box, fox, Xerox ox a l l the above, plus , hydroxy, foxes, boxes, dioxide, etc.  The l i s t of words of desired retrievals should be scanned for common letter groupings, for example: Cerebr Gangli Furan Pyran Glyc  o o o o o  side side side side side  s s s s  '  ' ' •  Incorrect truncation w i l l result i n excess retrieval. •KSIDE-* besides the terms above picks up side, consider, residence, prusside, sidewise, inside and sidechain, however, *OSIDE* picks up none of these.  National Science Library, National Research Council of Canada  136  TABLE XXV COMPUTER PRINT OUT OF PROFILE ONE jus * •G9  P 526 T  A * ALD*  PROFILE  OUITON. G G T T  I  T  T I ~J  T  T T  "IT T  r r I T T T T  T I T  r j  T T T T  nr.-' T  r  T  C *CELL* "D""*ER YTHR*~" E-_*FUC*_ F *HEX* G *KE T* H *P ENT* 1 *RIB* J * SORB* K *TAL* 'L~"*TETR*' M * THRE* N *XYL* 0 *ALTR* _  T T T T  -  T "T" T "T" T  1  _Q_*FRUCT_*__ R * GAL AC T* S *GUL* T *LYX* U *MANN* V *RHAMN* W_*GLUC* X *GLYC* Y CARBOHYDR* Z EXUUAlh* AA GUM* _ ' _ A8 MUC* AC_*MYCIN* AO. *NUCLEOT I D * " AE POLY HYDROXY  T T  Q  _  :  i yy  TAUTA1/-BM j  :  A  X ~ * T X J R X N U ' * "  AM * I T O L * "AN ~*L A C T u N E * AC) * 0 N 1 C * ~AP~*OS AMINE"*" AO * O S E *  : ATTVpyRA-jTO*  T -y T T T  AS * S I O E * Ar~*~U'R ON * AU AGAR* AV A L G T N * AW C A R R A G E E N A N *  ~T T  AX AY  -j T  AZ DE'X TR* ""' BA_1NULIN*_ _ _ _  "T T  BB BC  T T T I  BD N E U R A M i N* BE P E C T * BF S I A L I C * " ' BG TEICHO'IC*  "" BH A C E T O L Y S I S BI E P G X I D * ~BTJ RTTTiYirAI i U N * i BR M U I A R U T * . T ~BL~ K L E B S I C L L A : "T "~ BM- P E R IOOAT.t T "EOF - qFA" | " B — N y ^ X A T f A K — A T T E02 99 (GjP-V) E03 I w is E 0 4 T 99 X|X "E0'5T" 99" "(YU-AT) E 0 6 T 99 (AJ]ALI AMjAOjAPJARjAT) E 0 7 ~ T " "99" "A"Q'i~A"Q : E08 T 99 AS | AS  h09  AT P U L Y U L * AG_*SACCHAR* "AH"*ST A R C H * ~ A1_*SUGAR*_ "AJ *AR I C * AK * A S E *  :  CHTTTN* CHONDRUIT I N *  KERATAN* LEVAN*  137 Another example: Muc ilage Muc i c Muc o polysaccharide MUC * however, picks up as well mucosa, mucosal, mucous, mucoid, muconic, mucor; however, MUCI* and MUCO are completely selective. The f i r s t search profile was over-truncated and the pairing of most terms was not sufficient to eliminate many unwanted references. Profile Word *ALD**AMYL *•  Thus:-  Unwanted Retrieval aldrin, diels-alder carbamylation  XCELL*  cells, sub-cellular  KHEX-*  hexane, hexanol  *PENT*.  pentoxide  *RIB*  distribution, ribosomyl, ribosomes  *SORB*  adsorbed  •*TAL*  metal, skeletal, congential, d i g i t a l i s , orbital, crystalline, etc.  Removal of the prefix truncation on a large number of the root words was attempted with some degree of concern since i f the root were in a compound name, such as trimethyl-D-ribose, the computer might view this as an unbroken word and no retrieval would take place.  Fortunately this was not the case and a l l  roots preceeded by a hyphen were printed out. The following additional changes were made i n the preparation of the second profile. AO *0NIC* from E 06 because of terms ending in "onic" electronic, anionic, embryonic, paraconic, carbonic, etc. AC  * MYCIN*- because of too many references to drug use.  1J8 TABLE XXVI COMPUTER PRINT OUT OP PROFILE TWO  U 5 26" A T T' B C T. 0 T: T E F T" T G T "" H T I T J K T T L' T M N T ' 0 T P T ' " T Q R T T S T " T T U T V w T X T Y T ... _ T AA T AB AC T T "" AD T AE  DUTTON» "G G S " " P R O F I L E ALD* AMYL * AF PCLYOL* T CELLULOSE I T AG *SACCHARI* ERYTHR* ~ "j T " " * AH *STARCH* FUC* A l •SUGAR* T HEX* ~ ~ "7 "~ , AJ • A R I C * .T •• KET* T AK * A S E * PENT* . , "AL " * FUR ANO* T RIB* T AM * I T O L * S O R B * " T~~ AN •LACTONE* T AL* ) AO *ONIC* T TETR* " • • • • •T AP *OS A M I N *. THRE* AQ *OSE* T XYL* T "AR *PYRANO* *ALTR* T AS *S IDE* - AT •URONIC ARAR* ~ :_ _ •FRUCT* : . T • AU AGAR* *GALACT* "AV A L G I N * " 'T. GUL* T AW CARRAGEENAN* *LYX* AX C H IT I N * T MANN* AY CKONDKO IT IN* T T ~ * RH AMN* "" AZ DEXTR* BA INUL IN* T *GLUC* T •GLYCO* ' BB KERATAN* CARBCHYDR* T BC LEVAN* EXUDATE* ' T BO NEURAKIN* BE PECT* GUI"* ' T MUC* T B F S I AL I C * GLYCAN* T BG T E I C H O I C * *NUCLEOTID* POLY HYDROXY .  T BH T BI BJ" T T BK BL T BM T E O l T 99 E 0 2 T 99 E0 3" "T 99" EC4 T 99 EC5 T 99 E06 T 99 E07 T 99 EO£ T 99 EC 9" T" 99" E 10 T 99 E l l T 99  ACETOLYSIS 1 EPCXID* METHYLAT I O N * MUTAROT* . KLE6S IELLA ' ' ' PER IODATE "(AlBlD-NJCCAJIAk-AT)"™ (GlP-V) / wI w X1 X (Y | Z-AC) . (AOIAE-AI) ~" ( A J ] AL i AM 1 A P I AR | A t ) AQ | AO AS1AS , (AUIAV-BM)  (  :  T  1  !  ,  C)C  139 X *GLYC* and replacement with * GLYCOL and GLYCAN* because of references to glycine, hypoglycemic, etc. AT  HURON* to *URONIC  because of neuron, pleuroncipes.  AG *SACCHAR-* to AG *SACCHARI* avoids saccharomyces. AP C  -tfOSAMINE* to AP *0S  AMIN*  CT word splitting.  *CELL* to cellulose to many words with  CELL  EQUATIONS EOl  Remove C *CELL* to new equation E l l  E05  Made into two equations E05 and E06 to many retrievals  E06 - » E07  Remove term AO because of terms inding in onicj electronic, anionic, embryonic, paraconic, carbonic, etc.  PROFILE NUMBER 2 EVALUATION EOl  S t i l l too many t i t l e s which contain, by chance, two of the required terms, i.e.  THRE, ASE Triplet State effects in dye lasers at threshold. HEX, OSE Binding energy and compressibility of body centered cubic and close packed hexagonal sodium.  Therefore at the risk of losing a few references [2] EOl was s p l i t into two search equations.  The equations were selected so that the [3] EOl  contained a l l those root words which were found to commonly occur in other  TABLE XXVII COMPUTER PRINT OUT OF PROFILE THREE  U 526 T  T ~  T T"' T "T T T T "T T T'~ T  " ~ "  T  T ~- T T T~ • T T T  ! : ; ;  ...  j...  ;  T  i  t T T T  -  -  -  -j-  T T T  i i  _  - - - A ALD* 6 ERYIHK*. C HEX* " " D KET* E PENT* " F RI R * " ~ " G TETR* H'THRb* • I AMYL* J FUC* K SORB* L " T A L * "' M XYL* N *AL 1 K* ; 0 ARAB* P *FRUCT* Q *GALACT* R GUL* S LYX* T MANN* U RHAMN* V * G L U C * ~ " W *GLYCO* " ~ X T. ARBOHYDR* Y EXUDATE* L GLYLAN* AA GUM* ~A8 MUC * . ' " " ~ AC * N U C L E O T I D * ~AD" P O L Y H Y D R O X Y AE P O L Y O L  E O l ' T " "99 E02 T 99 E"03~T" 99" E 0 4 T 99 "" "E05 T""99 E 0 6 T 99 F 0 7 "T -go. E 0 3 T 99 - - q - - 99 E l O T 99 E11'"'T "99 E12 T 99  -  E  0  T  PROFILE  DOTT'O'NT, T. G S 1 T T " T r . T  AF * S A C C H A R 1 * AG * S T A R C H * "AH * SUGAR* Al *ARIC* AJ' * A S E * " "~ ~ AK * F U R A N O * AL * I T l . ) L * i T AM * L AC TONE* i T "AN~*ONTC* AO *OS AM I N * ! AP *OSE • i T : T AQ * n S F S AR" *PYR A N O * " ' 1 : T AS * S I O E * , . . . . . . . . . AT *URONIC ! T .AU A D I P O S E * AV C L O S E * T T AW D O S E * 1 AX MANN I L H AY S I D E * T "AZ" METABOL I SM T~"'" T ~ T BA * E N E * BB GLUCONEOGENESTS """"", 1 T BC, G L Y C O L Y S I S BD MUCOSA '' i BE C E L L U L O S E * ! T Bh CKBKA ~" ' " i.e. j. T BG P H O S P H * !  1  ] T  :  j  (A J B'-H )'£ ( A I | A K J AL I "AO | A P |~A"Q fA'R\ A T}-»("AU ] AV j "A W | A I) ( I |J-M)&(AI | AJ-ATJMAU|AVlAWlAY|AZ )  (N|U-U)MAX|AZ|Bti)  BE | BE ""' V-.IAZI BB 1 BG) WMAZlBAIBC) (X|Y-AB)-.(AZ|0n) <AC| A D - A H ) - - ( A Z ) AK|AL1 AO|AR|AT ( AP | AQ ) -»( AU | AV | AW ) AS-AY 1  BF|BF  ~~ "  Ikl  fields of chemistry. ase ,  The following very common endings were removed: lactone ,  onic ,  side  In addition certain not terms were added to remove some commonly occurring words. adipose, close, dose, metabolism The remainder of [2] EOl was the same as before with the addition of the not words above and the word  AY  SIDE* .  Equation [2] E02 formed [3] E03 with the not terms MANNICH, METABOLISM and PHOSPHX added. A number of not terms were added to the rest of the equations in order to eliminate commonly occurring non-carbohydrate retrievals, i.e. *ENEK was added as a not term for the equation [3] E06 to remove terms such as polyethylene glycol. In order to reduce the profile to 60 terms and to incorporate the required not terms i t was necessary to delete the terms AU to BG of profile No. 2. As a check on this completeness of the retrieval of references the coden for the Journal Carbohydrate Research has been given as the last equation in the profile in order to pick up any t i t l e s not previously printed in order to see why they have not been included.  PROFILE NUMBER 3 The following changes were made in profile No. 3 to produce the f i n a l profile.  142  Profile No. 3 Term AB AC  MUC*  Profile No. 4 Term AB  MUCO  * NUCLEOTIDE  *SIDE*  AY  SIDE *  AZ  METABOLISM  BB  GLUCONEOGENESIS  BC  GLYCOLYSIS  BD  MUCOSA  Too many non-carbohydrate words Removal of nucleotides ref. from profile  AC AS  Reason  MUCI  AS *OSIDE*  MUCI and MUCO replace MUC Improved truncation Replaced because of improved AS term  AY  GLUCAGON  New not term  AZ  METABOLI*  Better truncation now w i l l pick out metabolic as well Term AB * ENE K covers this term  BB  INSULIN  New not term  BC  GLYCOLY-K-  Better truncation [glycolytic] No longer needed [AB]  BD  CORTICO*  New not term  BH  TRANSPORT  New not term  The equations for the search expressions were modified to include the new not terms.  Amylase references have been eliminated as well as most references to  carbohydrate phosphates.  The equations are, however, open to general refer-  ences such as sugar phosphates.  143 TABLE XXVIII COMPUTER PRINT OUT OF PROFILE FOUR  U "52"6 " •. 0 9 0 6 6 9 P R O F I L E DUTTON, G G S T A ALP* . T 8 ERYTHR* t AF * S A C C H A R T * _._ T C_HEX* T AG_*STARCH*_ "T , " ~D' K E T * " | f " AH '* SUGAR* T E__PENT* _JT A l *AR.IC* T "F R I B * " f* AJ~*ASE* T G TETR* ' T AK .* FUR ANO* _ T H THRE* T AL * I T O L * T I_ A M Y L * • T AM * L A C T O N E * f" j "F(JC*" ' f AN'"*'ONIC* T _ _* I. AO_*OS A M I N * "J" ' L TAL* ' " ~ f "AP"' *OS'E T M_XYJL* T AO *OSF.S T N *ALTR* T AS * O S i n " E ' * T 0_ARAB* J _ AR_*PYRANO* _ ~ P ~ *"FRUC'f* " " ~J "AT •URONIC*" T Q__*G AL A C T * AU_AD I P0S F _ _ f " " R GUL* : ~T AV C L O S E * T S LYX*_ . ' T AW DOSE* T T MANN* T AX MANNICH _ T U Rl-lAMN* T_ AY_GLIJCAGON_ _ T " ' "V * G L L T C * " ." ; f ~AZ~ META B O L I * T W_*GLYCO* | T __BA_*ENF*_ f X C ARBOHYDR* "\ "f BB INS1JL I N T Y EXUDATE* • j T BC G L Y C . O L Y * T 7. G L Y C A N * T BD C O R T I C O * T " AA GUM* ; | T . BE C E L L U L O S * ' "f A B "Mucn " . "' I C B F " CRBRA T AC_MUC_I* ' T 8G P H G S P H * y- AD POLY HYDROXY • •" i T AE P O L Y O L | ' K  S n R R  T  :  T  T  T BH T R A N S P O R T * ', ' E O l T 99 ( A 1 B - I ) G ( A K | A L I AO 1 A P 1 A Q | ARI A S ] A T ) ^ ( A U I A V ) A W 1 A Z 1 B G ) EO? T 99 ( J 1 K-M ) & ( A I I A J - A T )-. { AU I A V I AW I AZ I BG ) E 0 3 T 99 ( N I O - U J M A_X I AZ I B G ) _ .... " E 0 4 " t ~ ' 9 9 B E IBE E 0 5_ T 9 9 y> ( AZ I 80 I BG I AY 1 8A l_BD I B_H) "EQ6 T 99" AZ | B A I DC I BB I R G) E 0 7 T 99 (X|Y-AC)~»(AZ|BB| BH) E08 T 99 I AD | A E — A H )-»(AZ|BB|BH) E09 T 99 I AK I AL J AO | AR | AT ) M A Z J BG |_BHJ ¥.10~f~99 ( A P | AQ ) -»( AU 1 AV 1 AW I AZ 1 BB | B G) E l l T__9_9 AS->(AZ 1 BB 1 BG I BH ) E l ? " ! 99 BF'I BF t  TABLE XXIX.  EVALUATION SEARCH PROFILES  PROFILE NO. 1  Search Expression  1 2 3 4 5 6 7 8 9  Total $ of Total  C.T. No. 12, 1969  C.T. No. 11, 1969 Yes  12 8 15 16 50 5 12 4 7 109 18.5  No  87 46 34 65 69  94  62 13 11 481 81.5  Total 54 49  99j 99* 74  17 18 590  Yes  18 10 6 10 29 6 13 3 5 100 20.5  No  81 52 24  35 53 78 39 10 15 387 79.5  Total  99* 62 30 45  82 84 52 15 20  Maximum number of retrievals 99•  PROFILE NO. 2  Search Expression  1 2 3 4 5 6 7 8 9 10  Total $ of Total  C.T. No. 13, 1969 Yes  13 9 8 15 12 22 2 11 7 6 105 32.3  No  61 10 22 19 7 20 7  42  15  17  220  67.7  C.T. No. 14, 1969  Total 74  19 30 34 19  42  9 53 22 23 325  Yes  No  23 18  72 11 33 25 16 26 7 35 15 19 259 65.9  19  11 12 18 5 15 6 7  134 34.1  Total 95  29 52 36 28 44 12 50 21 26 393  TABLE XXIX  [Cont'd]  PROFILE NO. 2  Search Expression  1 2 3  4  5 6 7 8 9 10  Total $ of Total  C.T. No. 15, 1969 Yes  20 13 6 12 9 23 6 11 5  No  Total  79 10 26 29 16  '  .  41  44  21 60 18 9 312 74.1  109 25-9  99* 23 32 25 67 27 71 23 13  421  C.T. No. 16, 1969 Yes  16 7 15 10 6 16 1  4  2 7  84  27.9  No  Total  81  65 7 29 15 7 21 8 34 17  14 44  25 15 37 9. 38 19 21 301  14  217 72.1  PROFILE NO. 3  Search Expression  1 2 3  4  5 6 7 8 9 10 11  Total $ of Total  C.T. No. 17, 1969  C.T. No. 18, 1969  Yes  No  Total  Yes  16 6 7 12 8 11  4 4  20 10 12 23 28 27 19 43 6 20 21 229  10  13 2  4  5 94  41.0  5 11 20 16 9. 30 4  16 16 135  59.0  No  7 13 1 2 9. 5 6 11 - 21 7 14 12 10 2 18 9 1 6 2 11 2 13 114 68 37.4 62.6  Total  C.T. No. 19, 1969 Yes  20 3  8 1  17 28 26 12 27 7 13 15 182  15 3 7 5 17 2 3  14  4  l  66 35.5  No.  7 9 5 • 11 18 8 5 34 1 13 9 120 64.5  Total  15 10 9 26 21 15 10 51 3 16 • 10 186  TABLE XXIX  [Cont'd]  PROFILE NO. 4  C.T. No. 20, 1969 Search Expression  1 2 3  4  5 6 7 8 9 10 11 12  Total $ of Total  Search Expression  1 2 5 4 5 6 7 8 9 10 11 12  Total $ of Total  No But Valid  Yes  No  14 4  1 2 1 0 0 2 0 3 3 3 0 0  0 0 7 10 9 2 9 1 2 2 1  9.2  31.6  13 10 8 8 10 10 2  4  2 5 90 59.2  14  4  48  C.T. No. 21, 1969 Total  19 6  14  • 17 18 19 12 22 6 9 4  6 152  Yes  No •  9 1 9 7 6 8 7 28 1 5 2 0 83  0 5 1 0 0 5 0 0 0 7 0 0  41.7  C.T. No. 22, 1969 Yes  11 3 10 10 9 10 3 13 2 2 1 3 77 43.3  No  0 0 2 0 0 2 2 2 1 6 0 0 15 8.4  No But Valid  6 •1 7 12 17 8 8 12 2 9 4 0 86 48.3  14  7-0  No But Valid  6 ' 2 11 8 17 15 7 23 1 2 10 0 102 51-5  Total  15 6 21 15 23 26  14  51 2  14  12 0 199  C.T. No. 23, 1969 Total  Yes  No  17 4 19 22 26 20 13 27 5 17 5 3 178  8 2 13 9  0 2 0 0 0 5 0 3 2 3 0 0 13 7.8  14  6 3 15 1 6 7 0  84  50.6  No But Valid 4  0 2 9  14  9 0 12 2 8 9 0 62  41.6  Total  12 4  15 18 28 18 530 5 17 16 0 166  Ikl The f i n a l search profile shows extremely good reference retrieval. The references which were not useful retrievals have been broken down into two subgroups, references retrieved because of carbohydrate words and references retrieved because of other factors. Many of the non-useful references are legitimate retrievals but outside our fields of interest.  The retrieval has been based on the presence  carbohydrate word in the t i t l e of the a r t i c l e .  of a  One example for each search  expression i s l i s t e d below. E01  HEX, OSE  ... A Genetic Reappraisal of Hexose Transport by Kidney and Intestine  E02  SORB, ITOL  ... Effects of Ethanol, Sorbitol and Thyroid Hormones  E03  ARAB  L-Arabinose Binding Protein ...  E04  CELLULOS  Chromatography ... on Columns of Benzoylated d i ethylamino ethyl cellulose  E05  GLUC  Effect of Serotonin on Glucose ... i n Rabbit Brain  E06  GLYCO  Catabolism of Plasma Glyco Proteins ...  E07  CARBOHYDR  ... Chickens Fed Carbohydrate Free Diets  E08  SUGAR  ... Effects on Sucrose Yield of Sugar Beets ...  E09  ITOL  Effect of Myo Inositol on the Prevention ...  E10  OSE  Nature of Lactose Fermenting Salmonella, ...  Ell  OS IDE  Radio Denaturation of DNA Deoxy Nucleosides.  The other terms which are non-useful are normally less than 10$ of the retrievals and f a l l into one of the following groups of retrievals. 1]  Accidental combination of two words XYL, ASE  Xylene, bases  AID, FURANO  Diels-Alder, furanophane  148 2]  3]  Truncation retrievals GALACT  Galactic  SACCHARI  Saccharin  OSE  Rose, purpose, those, etc.  Specific words found in other fields of chemistry OS, AMIN  Di ethyl nitros amine  GLYCO  Glycolate  POLY, HYDROXY  Poly hydroxy phenylenes  It is clear that the f i n a l profile w i l l retrieve a highly significant portion of the carbohydrate literature present i n any issue of Chemical T i t l e s . The profile may be modified as well to be more specific and to retrieve references missed by altering the profile words and search equations. I f at f i r s t glance the number of non-useful retrievals seems large i t should be borne in mind that i t i s a matter of moments to peruse the print out and discard those not wanted.  In practice a high percentage of marginal  material can be tolerated before the mechanical retrieval system ceases to be competitive with a manual search of the literature.  The computer print out  has in effect carried out a preliminary screening and "short l i s t e d " possible references for closer attention.  Furthermore, each entry of the print out i s  on a separate [perforated] sheet of paper which may serve as the basis of a personal f i l i n g system.  The time saved i n avoiding the necessity of trans-  cribing references of interest should also be considered when assessing the merits of the method. The use of the journal coden for Carbohydrate Research allows a check on the  percentage of papers which would not be retrieved by the profile.  Of over  two hundred references in Carbohydrate Research only 8.6$ would not have been  149 retrieved by the profile.  A portion of these were intentional exclusions by  not terms and a few references would have been obtained by examination of Chemical Titles keyword l i s t s for the twenty or so profile words eliminated to include the not terms and shorten the profile [compare profiles No. 1 and No. 4]. trieved.  I f these references were included only 5'4$ would not have been reExamination of several t i t l e pages of Carbohydrate Research w i l l i n -  dicate the type of references which w i l l not be retrieved by the search profile. In general, i t w i l l be seen that the t i t l e s often give no clue to their carbohydrate nature.  In terms of this evaluation the profile proposed i s 95$  effective in retrieving desired references from journal t i t l e pages. The same profile has also been used to search Chemical Abstract Condensates with good results although the percentage of undesirable retrievals is higher. This situation could now easily be improved by the addition of a profile word PATENT as a not term. 4 was developed.  This possibility was unavailable at the time profile  150 APPENDIX VII LEMON GUM in : 0.1 Li  in .  .  .  lio-r "F 1 CO CI 0) •? 1 1 •> O -4LT\ •\ o w OJ OJ  ill  liiL  II  i'r'  ! i;  i!H .,,.  o  JT  •': !o sit: 0) li w :iJ! r I Li mi  X :•  1 HI'  3  i o l  •o'  "T  jllifpii IT'IS~: ;.i:opi,  !1TI  g lO 1  ro  . I.  *  o o  o in  —  ili'tl' . •!  !  P4  o  X  50  100  150  FIGURE 36: LEMON GUM PEAK 5 greater amount  x  2,5-Me -ARAB  o  3,5-Me -ARAB  2  2  200 m/e  153  50  100  150  . 200 m/e  FIGURE 38: LEMON GUM PEAK 6T 2,3-Me-ARAB 2  §  156  IM  LliUl  -III I !M  !Mi  •Mil I: i! i :  CM  o 3  O  Ml Ml!  o  cvj •  O O  O). i.o>l  M  ! .031 M II::  tii- hi  I i M-  ,41-1  Ml MM  o o  i?  f -=r  s 3 K\ w w •» 3 CL. CVJ  ...!..  llii.  to  8  a  jfil,, .1.1  §  O w  J_MJ  o m  158  .1  50  100  150  200  m/e  FIGURE 43 LEMON GUM l/lON H2S0 LEMON GUM . SMITH POLYALCOHOL LEMON GUM LEMON GUM SI 4  PEAK 10 PEAK 4 PEAK 6 PEAK 6  [DIFFERENT SAMPLES SHOTTED ONLY MINOR INTENSITY CHANGES] 2,4-Me-GAL 2  H  162  t;T|  1  il  Hi!  i.L  lll:.,  rrtt  r  I! 'j •  ill  8 CM  4j. iliii .a  ;ir !i:i i Hi L;3  iiniiip  -H !i  o  Am.  O  I Hi .  I. !  8 w ^ rl < IA  H! N!! 1  [ill  .lijil  ^>  ill  i|'0>.,  o o  f  i  Ah ; i  Till .iI  LU  fl  'III liiitii I:: • I :..I-:i. I,,' I : 'l.l: O  w  rH (X, CM  I .i! i i  si--  i  co QJ  3  to.  ~i • ill!, ii'i  g  Mil o m  FIGURE Vf:  SMITH POLYALCOHOL LEMON GUM PEAK 2 2,3,4-Me -ARAB 3  100  43 ox  i-  - -- j  1.'. ' X-. -\":-S :  t'l-Tti'.f^-il-r  i  :  T^?;T:4-r{->";zr- ;!=r.t'.-. |l:'-":~4 r  1  R.I.  50  200 m/e  FIGURE 48: SMITH POLYALCOHOL LEMON GUM PEAK 3 2,5-M2-ARAI5 [greater amount] e  3,5-Me -ARAB 2  ;  100  200  m/e  FIGURE 49: SMITH POLYALCOHOL LEMON GUMPEAK 4 o  2,3,4,6-Me -GAL  x  2,3,-Me -ARAB  4  2  $  43ox  -  1  zizz  Z.Z  ^T7l  .  "  ^  -  ——t  777  —  :  — -. — — <—'.*.-. 1 - r — ZZTZZZ, LZZJlZZZ — 7_f. t „  .  zzz'.. -. „  — J —  ZZZZ  I'  7777777X77;  -  z l~  "T  50  ——  L_ — . "--T~ - tj!T ;; , —  .  - -  77  :  —  •.....^  .  7 177  L  „ ——  I  1  • -  _ • ; ';;  . I  i  1  _L b~7m „— i  ~ •LvX r—;•  :  ;  1  -t—^' -f—— " 1  1  ri  .  1 :  i  j.  7777J  .zzzz ' — T™ ii  -!-r- . ,  7  „I_-L4. —  777X77777  •;—**•  i —_—i.—1_„„  ' i •  . , •i j, •' • •,'  r  -_: . — L-^™ -—^ E—-r-( -—  fTTT™  •  1771777  .+ - F . z h -  45 ox zz _: -  z  7.-77zi •  '  zzz^. i  1  r ~~  h-  - I  —  —  ; •IZ:  _;_  .zzzz L  ,  1  , ——  .. J.  ~7717 —' -| — _;~ _ .  50  1 ll 1  _ _:  u. ::  , . .7 — — ' ~? - T:  I  ||  87ox  „  .1  77 .77—  Z.ZZZ  ~r.  1  lOlox  ^ & A  i  - .  • i  •  —  i  ,.„-.  r . . ..  —  .  r  r  1  :^  — |  l l II  j.  :  —  -  1  J  j  150  ,  _4_.  ,  -  1  „  f,. •  "i  i  i _ .—:—i—  .  —.  vzz  _j  '  : f. _ : •  •  1  i  -i~' :  i  -  ^^^^^i  1  7-~77-77X7189 ~'  i-  i  .' • • !  '.  777  r-f— "  ^ „.—— 1  i .  :  .,  _f  1  v  1  —.  R  f .  -i ' . 1  . .„i ,  ,  ^„L .„  „ •  •  .77777  (  -r" ~~ " ' •7T7":?',7" .','  . . ' f  • • •!r  '  -  i— —r~: ,• • •  f  zzzzzzz777777 r r r t~*~ : .'- — ; —T7..7-—U;—  1  ~  f  1  r  i  —"7 ...  F  .7 .777 .77 1777:^777777^7 ... « 'I i' • • • ' 1 , f. .  I  — y—  ..  ..  „ i  . _  .: _.7 ' .7_ , ' —: _r— _r — ! 777777 _ .1 777^7X71727^ 7~77 zz. 7•~ 7i 7r_17.~ "7—77 ,  ;  "7 "  >  ; _- . ;:4. .— ' ' 1610  " 1  .  . _..  j„  |  zz: ^zzzzzzzzz. z'z-zzzzrrr "zi : r Z"JZZZ.ZZZZ- zzzzZ.zz.zJ^z  - „*l*^ : \7 :. _—... —  •.  i  i  [  (  •  1  • 77 77 77 777 7—. 77-;:-7™77 77. ~7_ l l 77 1 1 l 100 _-  i  1  .-TTTT  77777777 :.-7771 U tz~:~~ ~r~~ 7 7777777"— j ^ •'•r±rxr.:•  y~—'•  1  j  . Z—Zi:~ "7777 .7. ..777 L - . — - i — . ~7_.777_T77_• . -~_777777|jJ"  •  : .  ;. i 1  . . . i • ^TTTTT^—rlr_7.„>  1 . 1j T r i " !•  ^. 71 J777 _  ;. . . .!  _  \ .  :  f- *  . ^ f  j  H7rtv n  —  -.-  —_ i  :  r—~"'X " l ?.9_ r t "  •— •_ i — -_ 7.7; -L  , \r  ~^-rr— - \  1  :—i  ;z~rt7:7:r|""_H i  .. |... „;  t- — • • —f  -  H  trrr:--_r  i — i —1____„_..  - —  l- - - { . _  --j —p ;—. 1 T —  I •  i  —  • ._  r  ;  1  1  — i  r  -j *.  ^. ,  -  .:  1  J — .  ;  ?  —.— .— —i—.—^_  -  : . ** i  ,—j  ,-Z...  _- ;-~"zErLL ''•zTzi-lz-i.r  r~ "1— —j - - r r . "  u,- :- - j- -,- - T i-  —  • — 1  .. : : *. 1  'z  r  r if " • ". ,  —  77ZT —r— 7777^ ~ ......  i .  ~t  ,'77  . • ,f"7..  r  { - -i  ~rr~-J-7--  : -  4inzTT-f:7r777 ..  rE-.f:r—irr-rr  1  "  -—•  f ~ • - -,. -  ,  zz  _l "1 ll II If j! II  ' .  _.j ....  —  -  zz ' z~ zz z  i  -f — -~-—  .J-  ,  7777.4:4^~l:T""v--f ZZ jr-T-7j' f] : : \ M z l z z s z z z ^ — , — I — . —  r  -  '  -77r-r  ....  • . ". . - , .7. 77  7777^! - -77:  -— r-7i: 7~ I7777_ 1 _7 -77JU7 777777.7; 7.1777717771 ~|7~ "\.^~. j--; - ; --• • —'- -P" ~! 7 7 7 1 i 4 1-.-' ~f -r*-1— -777.77±~7.777 —— • r ~ 7 7 _}"_ 7 7  -ZZZTJ.ZZZZ  R.I.  -.-77^  - ._ •  • -•  -  §7  17-I-I  7I7-~7~_™7~  -  r77_  •. •;• •! •i ; . , • .I" -  I  -J.  ——j —  r r_r - t  t77"7 t77__  : —™ i_ .  , —  .  ——I  ^  233x  •' .--7 7.-7777-7-77—7 -777777-7r7.7-_777.-7 200 m/e  •-  FIGURE 50: SMITH POLYALCOHOL LEMON GUM PEAK 5 2,3,4-Me -GLU 3  x 2,3,6-Me -GAL 3  0  2,4,6-Me -GAL 3  ^  o £3  50  100  150  200 m/e  FIGURE 51: LEMON GUM SI PEAK 1 2,3,5-Me -ARAB 3  H ON  50  100  150  FIGURE 52:  200 m/e  LEMON GUM SI PEAK 2 2,3,4,6-Me -GAL 4  H  8  50  .150  100  200  FIGURE 53: LEMON GUM SI PEAK 3 o  2,4,6-Me -GAL  x  2,3,6-Me -GAL  3  3  m/e  50  100  150  FIGURE 54:  200 m/e  LEMON GUM SI PEAK 4 2,3,4-Me -GAL 3  H O  172 BIBLIOGRAPHY 1.  J.F. Stoddart and J.K.N. Jones, Carbohydrate Res., 8, 29 [1968].  2.  J.M. 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