Analytical Methods for Determining Pectin Composition - ACS

2 Analytical Methods for Determining Pectin Composition Landis W. Doner

Downloaded by UNIV LAVAL on June 20, 2014 | http://pubs.acs.org Publication Date: June 5, 1986 | doi: 10.1021/bk-1986-0310.ch002

Agricultural Research Service, North Atlantic Area, Eastern Regional Research Center, U.S. Department of Agriculture, Wyndmoor, PA 19118

Chemical and modern chromatographic methods have been applied to the determination of pectin composition and structure. The dominant structural feature of pectin is a linear chain of galacturonic acid residues, some of which are esterified with methyl groups. Chemical methods (including reactions with carbazole and sub stituted phenols) and chromatographic methods (GLC and HPLC) are available for galacturonic acid determination. Methyl ester levels are determined either chemically (after oxidation to formaldehyde) or by GLC after pectin ester saponification. A variety of neutral sugars are present in pectin, mainly rhamnose, galactose, arabinose, and xylose. Effective GLC procedures are available for their determination, and applicable liquid chromatographic methods have been developed. Measurements of less the less-common substituents, such as O-acetyl and O-feruloyl esters have been achieved by colorimetric and titrimetric 13

procedures. Developments in infrared and C-NMR spectro scopy have resulted in these being applied to structural analysis in pectin. Pectin polysaccharides and the hemicelluloses are matrix components i n the c e l l walls of higher plants. T r a d i t i o n a l l y , these classes of carbohydrates have been defined operationally by t h e i r presence i n fractions obtained by sequential extraction of c e l l walls. The p e c t i c substances are extracted with water, d i l u t e a c i d , or with calcium chelating agents, such as EDTA, ammonium oxalate, or sodium hexametaphosphate. But c l a s s i f i c a t i o n of polysaccharides i s best based on s t r u c t u r a l components rather than on the method used f o r i t s i s o l a t i o n . According to structure, the pectic substances would include galacturonans, rhamnogalacturonans, arabinans, galactans, and arabinogalactans which possess a l i n e a r β-1,4-Ι) -galactan backbone. A recent c l a s s i f i c a t i o n (1) describes the p e c t i c polysaccha rides as those polymers found i n covalent a s s o c i a t i o n with galacturonosyl-containing polysaccharides. The hemicelluloses are those carbohydrate polymers which are noncovalently associated with c e l l u l o s e . Diverse categories of pectic polysaccharides occur not only among plant sources, but among tissues i n a given source. This chapter not subject to U.S. copyright. Published 1986, American Chemical Society

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.


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CHEMISTRY AND FUNCTION OF PECTINS

A comprehensive (2) review on the structure of p e c t i n has recently been published. Pectin was described as consisting of a branched block, i n which the main galacturonan chain i s interrupted by rhamnose u n i t s . Many of these rhamnoses carry arabinan or galactan chains; the galactan chains are sometimes further substituted with arabinan segments. These heavily branched galacturonan chains alternate with unbranched blocks i n which rhamnoses are r a r e l y present. Methyl esters of galacturonic acid are also present as blocks, a l t e r n a t i n g with sequences of n o n - e s t e r i f i e d galacturonic acid. This review w i l l describe the a n a l y t i c a l methods a v a i l a b l e to determine the s t r u c t u r a l components of p e c t i n . These features determine the important p h y s i c a l , chemical, and b i o l o g i c a l propert i e s of p e c t i n . Included w i l l be discussion of galacturonic acid determinations, degree of e s t e r i f i c a t i o n with methyl groups, the neutral-sugar composition, and analysis of some less-common e n t i t i e s , such as 0-acetyl and O-feruloyl linkages. Quantitative Analysis of Galacturonic Acid i n Pectin The dominant and u n i f y i n g s t r u c t u r a l feature i n pectins i s a l i n e a r 1^4-a-linked D-galactopyranosyl-uronic acid chain. a-L-Rhamnosyl residues are inserted at i n t e r v a l s i n the chain, and v a r i a b l e proportions of the uronic acid residues are e s t e r i f i e d with methanol. Neutral sugars other than rhamnose are present, and neutral sugar l e v e l s often t o t a l about 20%. The other neutral sugars are mainly D-galactose, L-arabinose, and D-xylose, and these are l i k e l y to be attached i n branches to the rhamnose residues i n the main chain. In a l a t e r section, various chromatographic approaches f o r determining the l e v e l s of i n d i v i d u a l neutral sugars w i l l be described. Chemical Methods. Determinations of galacturonic acid of p e c t i n usually includes both the free and e s t e r i f i e d forms, since strongly a c i d i c media are employed i n the c o l o r i m e t r i c methods. Procedures which continue to be used widely are modifications of those described e a r l y by Dische. One i s based upon reaction with cysteine (3) and the other with carbazole (4). One modification of the carbazole method, which gave a doubling i n s e n s i t i v i t y , along with an increased s t a b i l i t y i n color and greater reproducib i l i t y , was reported i n 1962 (5). Inclusion of borate into the assay medium was responsible f o r the enhancement of the method. In a l l the c o l o r i m e t r i c methods, galacturonic acid i s l i b e r a t e d during the assay by hydrolysis of polymeric p e c t i n . An applicat i o n of the carbazole method, a f t e r e x t r a c t i o n of p e c t i n from various f r u i t s and vegetables, has been described (6), as has been an automated carbazole method f o r monitoring uronic acid l e v e l s i n p e c t i n f r a c t i o n s (7). A more rapid and somewhat simpler procedure f o r uronic acid determination was described i n 1973 (8). This method i s advantageous f o r determining galacturonic acid i n p e c t i n , as i n t e r f e r ence by neutral sugars i s reduced. This method i s based on the color formation which accompanies the addition of m-hydroxybiphenyl to heated solutions of uronic acids i n s u l f u r i c acid/boric



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acid. This assay has been applied to the determination of galacturonic acid i n food pectins (9,10), and interference by neutral sugars was minimized (9). Another phenol, 3,5-dimethylphenol, has been found (11) to be more s e l e c t i v e than m-hydroxydiphenyl when large amounts of neutral sugars are present i n the sample. This method has recently been employed i n studies of the degree of methylation of pectin i n plant c e l l walls (12). A procedure employing c o l l o i d a l t i t r a t i o n has been used f o r the determination of galacturonic acid i n p e c t i n , and i n d i r e c t l y , also for determining the degree of e s t e r i f i c a t i o n (13). Samples are t i t r a t e d with poly-N,N-dimethylallylammonium chloride, and a d i s t i n c t f l o c c u l a t i o n occurs, the endpoint of which i s determined by use of t o l u i d i n e blue i n d i c a t o r . In a duplicate sample, ester methyl groups can be saponified, and t o t a l galacturonic acid determined; by difference, the degree of methyl e s t e r i f i c a t i o n i s calculated. The quantitation of t h i s c o l l o i d a l t i t r a t i o n method i s more precise with pectins of high degrees of polymerization. In another t i t r i m e t r i c method, t o t a l galacturonic acid and the degree of e s t e r i f i c a t i o n i s determined by copper-binding before and a f t e r s a p o n i f i c a t i o n (14). The bound copper i s determined by atomic absorption spectrometry. A p p l i c a t i o n of t h i s copper-binding approach to the analysis of c e l l - w a l l polysaccharides i n many f r u i t s and vegetables has been reported (15). Decarboxylation with hydroiodic acid [16) was the basis f o r a procedure used i n determining uronic acid l e v e l s i n dietary f i b e r fractions (17). The carbon dioxide from decarboxylation was p u r i f i e d , trapped i n a c e l l containing standard sodium hydroxide, and conductivity changes were measured using an Ingold electrode. Studies comparing the d i s t r i b u t i o n of free carboxyl groups i n enzymatically and chemically d e - e s t e r i f i e d pectins are important because the g e l l i n g behavior of r e s u l t i n g products i s a function of the method used. Enzymatically d e - e s t e r i f i e d pectins have a blockwise d i s t r i b u t i o n of non-esterified galacturonic acid residues, and gel with calcium at higher degrees of e s t e r i f i c a t i o n than do acid d e - e s t e r i f i e d pectins, which possess a more random d i s t r i b u t i o n of free carboxyl groups. Free carboxyl d i s t r i b u t i o n has been studied (18) by f i r s t e s t e r i f y i n g by reaction with ethylene oxide ( g l y c o l a t i o n ) , and then t r e a t i n g the sample with a mixture of pectin enzymes. The glycolated fragments are unreactive toward these enzymes. F i n a l l y , the hydrolysis products are separated from the glycolated fragments by ion exchange chromatography, and a f t e r deglycolation, chain s i z e i s determined by g e l f i l t r a t i o n . An a p p l i c a t i o n of t h i s approach i n studies of orangepeel pectin has been reported (19). -

Physical Methods. Infrared (IR), Raman, and nuclear magnetic resonance (NMR) spectroscopic methods have been applied to struct u r a l analysis of polysaccharides such as pectin. These applications have been reviewed (20), and reference IR spectra of p e c t i c substances have been published (21). Quantitative IR has been used to estimate acid d i s s o c i a t i o n constants of polyuronides from the r a t i o of -CCLH to -CO^- as a function of pH (22). Also, by


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CHEMISTRY AND

use of s o l u t i o n IR i n D 0, 2

FUNCTION OF PECTINS

the r a t i o of -C0 H to -C0 CH (free to 2

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e s t e r i f i e d ) groups i n pectins can be determined (23).

The ester-

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carbonyl stretching band i s observed at 1740 cm and a carboxylate -1 stretching band at 1650 cm . C-NMR spectroscopy has also been useful i n determining r e l a t i v e proportions of free and e s t e r i f i e d carboxyl groups i n pectin (24). In t h i s approach, the r a t i o of peak areas at 172.8 ppm (-C0 H) i s determined r e l a t i v e to the areas e i t h e r at 171.3 ppm (-C0 CH ) or 53.7 ppm (-0CH ). In 13 addition, pectin from sugar-beet has been examined by C-NMR, and 0-acetyl, carbons-one of the minor constituents galactose and arabinose, and carbon-six of rhamnose can be discerned (25). Chromatographic Methods. The high l e v e l s of galacturonic acid (free and e s t e r i f i e d ) i n p e c t i n has resulted i n the development of chemical methods which are rather non-specific. With corrections applied for neutral sugars however, reasonable estimates of i t s l e v e l s can be made. The c o l o r i m e t r i c procedures are conducted i n strongly a c i d i c media, which r e s u l t s i n some loss of galacturonic acid by decarboxylation to L-arabinose. Also, a l l uronic acids respond to the various c o l o r i m e t r i c t e s t s , so for analyzing mixtures of uronic acids, gentler h y d r o l y t i c steps and f a r more s e l e c t i v e assays are needed. To t h i s end, several g a s - l i q u i d (GLC) and high-performance l i q u i d chromatographic (HPLC) methods have been developed for determining galacturonic acid. Some have employed s p e c i f i c enzymes f o r depolymerization as an a l t e r n a t i v e to acid hydrolysis. Combinations of p e c t i c enzymes and acid c a t a l y s i s are required to q u a n t i t a t i v e l y hydrolyze p e c t i n . This section w i l l describe applications of modern chromatographic methods for determining galacturonic acid i n pectin. For excellent and comprehensive descriptions of sugar chromatography i n general, review a r t i c l e s on GLC (26) and HPLC (27,28) have been published. A s e n s i t i v e GLC procedure for determining galacturonic acid i n p e c t i n has been developed (29) from an e a r l i e r described method for analyzing uronic acids (30,31). Pectins, a f t e r extract i o n from plant tissues with ammonium oxalate, are depolymerized with pectinase. The l i b e r a t e d galacturonic acid i s then reduced by sodium borohydride to galactonic a c i d , which i s converted to L-galactono-1,4-lactone. The t r i m e t h y l s i l y l d e r i v a t i v e of the lactone gives a sharp peak on SE-30 stationary phase, and p e r - t r i m e t h y l s i l y l x y l i t o l i s used as an i n t e r n a l standard. Often, the decomposition of monomeric sugars which r e s u l t from the resistance to acid hydrolysis of polysaccharides containing a c i d i c groups such as p e c t i n i s overcome by f i r s t reducing the uronic acids to neutral sugars. This reduction method (32), which should be repeated at l e a s t twice for quantitative conversion, i s widely used i n polysaccharide s t r u c t u r a l a n a l y s i s , and has been applied i n a GLC procedure for galacturonic acid (33). A f t e r a c t i v a t i o n of p e c t i n carboxyl functions with a water-soluble diimide, reduct i o n with sodium borodeuteride converts the galacturonic acid residues to 6,6-dideutero-galactose d e r i v a t i v e s . Then, a f t e r acid h y d r o l y s i s , standard gas chromatography-mass spectrometry 1 3

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methods to resolve neutral sugars are applied. The galactose (dideutero) which had been generated from galacturonic acid i n pectin i s distinguished from galactose ( d i p r o t i o ) n a t u r a l l y present i n p e c t i n by mass spectrometry. These GLC methods are c h a r a c t e r i s t i c a l l y very s e n s i t i v e and e f f i c i e n t . Liquid chromatographic procedures have been developed more recently, and some are quite e f f e c t i v e f o r determining galactu ronic acid i n p e c t i n . An automated anion-exchange chromatographic system (34) allows the separation of i n d i v i d u a l uronic acids, including galacturonic acid. Column e f f l u e n t s were s e n s i t i v e l y analyzed for uronic acids by post-column reaction with o r c i n o l and monitoring at 420 nm. More rapid HPLC approaches have been described. By using strong anion-exchange columns, 0.7M acetic acid mobile phase, and r e f r a c t i v e index detection, galacturonic acid was separated from mannuronic and glucuronic acids i n less than 15 minutes (35). S i m i l a r conditions were employed to separate oligogalacturonic acids of up to DP-8, and Bio-Gel P-2 has been used f o r the same purpose (36). In another report (37), strong anion-exchange HPLC also was used, with a mobile-phase consisting of b o r i c acid/potassium hydroxide b u f f e r , and post-column f l u o r o metric detection (2-cyanoacetamide), allowing the r e s o l u t i o n and s e n s i t i v e detection of four uronic acids, including galacturonic acid. Polygalacturonic acid has been subjected to methanolysis (reaction with methanolic hydrogen c h l o r i d e ) ; multiple peaks r e s u l t i n HPLC chromatograms, due to the presence of α,β-mixtures of the methyl glycosides (38). Although the procedure i s not i d e a l f o r q u a n t i t a t i o n , i t i s useful f o r q u a l i t a t i v e analysis of uronic acid composition i n polysaccharides. In a recent report (39) polygalacturonic acid was subjected to both acid and polygalac turonase catalyzed hydrolysis. The hydrolyzates were analyzed by +

HPLC on a cation-exchange column of HPX-87-H , and galacturonic acid was eluted i n 8.5 minutes. The advantages of enzyme over acid-catalyzed hydrolysis were apparent. The y i e l d of monomer was greater, no monomer degradation products were present, and a far lesser quantity of oligogalacturonic acid chains were produced. Determination of Methyl, A c e t y l , and F e r u l o y l S u b s t i t u t i o n i n Pectin Pectin consists mainly of polygalacturonate chains, and the carboxyl groups are s i g n i f i c a n t determinants of i t s chemical and b i o l o g i c a l properties. In plant c e l l w a l l s , more than 50% of the carboxyl groups are often e s t e r i f i e d with methanol. The degree of e s t e r i f i c a t i o n l a r g e l y determines the ion-exchange, water-binding, c r o s s - l i n k i n g , and hydrogen-bonding capacities of pectin. S i m i l a r l y , properties of p e c t i n i n c e l l walls are some times modified by low l e v e l s of hydroxyl e s t e r i f i c a t i o n with acetyl groups. The d i s t r i b u t i o n of a c e t y l groups i n p e c t i n i s unknown, but i n sugar beet, pear, and apricot p e c t i n , a c e t y l levels approach 4%. In addition, a l k a l i - l a b i l e f e r u l i c acid groups are found i n ester linkage to p e c t i n ; they are believed to be c a r r i e d by arabinose and/or galactose residues on neutral side chains. This section w i l l describe recent methods to determine p e c t i n s u b s t i t u t i o n with methyl, a c e t y l , and f e r u l o y l groups.




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Methyl Esters In Pectin. A t i t r a t i o n method has been reported (40), i n which methyl ester l e v e l s are calculated from the number of equivalents of standard sodium hydroxide required to neuturalize the pectin sample before and a f t e r saponification. The copper t i t r a t i o n procedure described e a r l i e r for determination of galacturonic acid residues i n pectin (15), i s also used to determine methyl ester l e v e l s from the increase i n copper-binding a f t e r hydrolysis of the esters. An accurate and sensitive colorimetric method (41) i s rather time-consuming, but several samples can be run i n p a r a l l e l . Samples are saponified, the released methanol oxidized to formaldehyde, and the formaldehyde determined by spectrophometric assay (4l2nm) of i t s condensation product with pentane-2,4-dione. GLC procedures are widely used for methyl ester determinat i o n s ; a f t e r saponification with 0.5N base, methanol i s measured by GLC on columns of Poropak Q at 120°C (42), or on Carbowax 1500 at 125°C (12). In the l a t t e r study, analyses were conducted on small samples of i s o l a t e d plant c e l l w a l l preparations. In studies on the enzymatic incorporation of methyl groups from S -adenosyl-L-methionine into pectin (43), ^C-methyl-labelled 14 substrate was used. The C-methanol, a f t e r release from p e c t i n , was determined by GLC on Carbowax 300 using a r a d i o a c t i v i t y counting detector. The coupling of a n a l y t i c a l p y r o l y s i s to GLC has resulted i n the detection of c h a r a c t e r i s t i c fragments from macromolecules, such as pectin. This topic has been reviewed (44), and correlations between degree of methyl e s t e r i f i c a t i o n and i n t e n s i t y of some of the peaks have been made (45). Acetyl and F e r u l o y l Esters i n Pectin. A colorimetric method f o r determining degrees of acetylation i n pectins from various sources (46), has been shown to be rapid and quite s e n s i t i v e . Hydroxylamine i s reactive toward both the methyl and acetyl esters i n p e c t i n , and f e r r i c ion complexes with the r e s u l t i n g hydroxamic acids are red. The pectin complex i s insoluble and removed by f i l t r a t i o n ; the i n t e n s i t y at 520nm i n the soluble f r a c t i o n , consisting of the f e r r i c complex with acetohydroxamic a c i d , i s a measure of acetyl content. The accuracy of the method was demonstrated i n determinations of 0-acetyl l e v e l s i n standard per-acet y l a t e d polysaccharides. Another method (47) involves a l k a l i n e hydrolysis of the acetyl groups from p e c t i n , followed by d i s t i l l a t i o n of acetic acid and i t s t i t r a t i o n with standard base. In a study of the structure and functions of feruloylated pectins i n primary c e l l walls i n spinach, about one f e r u l o y l group was found per s i x t y sugar residues (48). F e r u l i c acid was determined a f t e r a l k a l i n e hydrolysis by the Folin-Ciocalteu phenol reagent. Neutral Sugar Composition of Pectin The neutral sugars, with the exception of L-rhamnose, are attached e x c l u s i v e l y i n sidechains, and include D-galactose, L-arabinose, D-xylose, and less frequently, D-glucose, D-mannose, L-fucose, 2-0-methyl-D-xylose, 2-0-methyl-D-fucose, and D-apoise. Whether



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the sugars are determined by GLC or HPLC methods, i t i s e s s e n t i a l that the polysaccharide f i r s t be hydrolyzed to i t s monomeric sugars. Acid-catalyzed h y d r o l y t i c methods are most often used, but the various linkages have d i f f e r e n t s u s c e p t i b i l i t i e s , as do the various sugars when released upon hydrolysis. These problems have been discussed, along with a review of the sugar GLC l i t e r a ture p r i o r to 1973 (49). I t was stated that "no one method of hydrolysis w i l l necessarily cleave every linkage and give each component i n quantitative y i e l d . " The Saeman hydrolysis (50), which employs 72% s u l f u r i c a c i d , or 2N t r i f l u o r a c e t i c acid"T51) are used most often. When p o s s i b l e , enzymatic approaches i n combination with acid hydrolysis are preferred f o r polysaccharide hydrolysis. A f t e r h y d r o l y s i s , the most widely used methods for sugar determination are based on GLC of s u i t a b l y v o l a t i l e derivat i v e s . The derivatives i n which the anomeric center i s eliminated so that s i n g l e peaks r e s u l t are most e f f e c t i v e . Single-peak sugar derivatives which allow the r e s o l u t i o n of sugar constituents i n p e c t i n include the t r i m e t h y l s i l y l a t e d methyloximes (52), acetylated a l d o n o n i t r i l e s (53), t r i m e t h y l s i l y lated a l d i t o l s " T 5 4 ) , and acetylated a l d i t o l s (51). A comprehensive review a r t i c l e on GLC of sugars has been published (26). In studies of polysaccharides structure, the a l d i t o l acetate procedure remains the most widely used GLC procedure. The advent of high-resolution glass c a p i l l a r y columns has allowed very e f f i c i e n t separations. Recent applications of these columns to a l d i t o l acetate separations have been described (55-57). The a l d i t o l acetate procedure requires reduction of the sugars with sodium borohydride. A f t e r removal of boric a c i d , the sample i s acetylated by conventional means. Various polar stationary phases have been used i n GLC separation of a l d i t o l acetates, i n both packed and c a p i l l a r y columns. A low-polarity phase was used i n a report (54) which demonstrated the separation of t r i m e t h y l s i l y l a t e d a l d i t o l s , and the neutral sugars i n a hemicellulose sample were resolved. A l i q u i d chromatographic system has been applied i n a study of monomer composition i n c e l l - w a l l polysaccharide hydrolyzates (58). A strong-base anion-exchange column was eluted with a borate buffer step-gradient. Post-column reaction with o r c i n o l allowed the s e n s i t i v e determination of the sugars rhamnose, xylose, arabinose, glucose, galactose, and mannose. An improved two-step HPLC procedure f o r t o t a l r e s o l u t i o n of the above neutral sugars has been published (59). On aminopropyl s i l i c a with acetonitrile-water as mobile phase, rhamnose i s separated, but the pentoses xylose and arabinose are not w e l l resolved, nor are the hexoses glucose, galactose, and mannose. These two m u l t i component peaks are c o l l e c t e d and re-chromatographed on Aminex HPX-87P, a heavy metal cation-exchanger i n the lead form. Total resolution of the f i v e monosaccharides r e s u l t s . Acknowledgment R e f e r e n c e to a brand or f i r m name does not c o n s t i t u t e endorsement by the U.S. Department of A g r i c u l t u r e over o t h e r s of a s i m i l a r n a t u r e not mentioned.


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Pectin Composition Determination 21

33. Lau, J. M.; McNeil, M.; Darvill, A. G.; Albersheim, P. Carbohyd. Res. 1985, 137, 111-125. 34. Spiro, M. J. Anal. Biochem. 1977, 82, 348-352. 35. Voragen, A. G. J.; Schols, Η. Α.; Devries, J. Α.; Pilnik, W. J. Chromatogr. 1982, 244, 327-336. 36. Thibault, J. F. J. Chromatogr. 1980, 194, 315-322. 37. Honda, S.; Suzuki, S.; Takahashi, M.; Kakehi, R.; Ganno, S. Anal. Biochem. 1983, 134, 34-39. 38. Cheetham, N. W. H.; Sirimanne, P. Carbohyd. Res. 1983, 112, 1-10. 39. Hicks, K. B.; Lim, P. C. Haas, M. J. J. Chromatogr. 1985, 319, 159-171. 40. Schultz, T. H. in "Methods Carbohyd. Chem."; Whistler, R. L.; BeMiller, J. Ν., Eds.; Academic: New York; 1962, Vol. 5; pp. 189-194. 41. Wood, P. J . ; Siddiqui, I. R. Anal. Biochem. 1971, 39 418-428. 42. Knee, M. Phytochemistry 1978, 17, 1257-1260. 43. Kauss, H.; Hassid, W. Z. J. Biol. Chem. 1967, 242, 3449-3453. 44. Bayer, F. L.; Morgan, S. L. in "Pyrolysis and GC in Polymer Analysis", Liebman, S. Α.; Levy, E. J., Eds.; Chromatographic Science Series; Marcel Dekker: New York, 1984, Vol. 29, pp. 277-337. 45. Zamorani, Α.; Roda, G.; Lanzarini, G. Industrie Agrarie 1971, 9, 35-41. 46. McComb, Ε. Α.; McCready, R. M. Anal. Chem. 1957, 29, 819-821. 47. Schultz, T. H. in "Methods Carbohyd. Chem."; Whistler, R. L.; BeMiller, J. Ν., Eds.; Academic: New York, 1962, 5; pp.187-189. 48. Fry, S. C. Planta 1983, 157, 111-123. 49. Dutton, G. G. S. in "Advances in Carbohydrate Chemistry and Biochemistry"; Tipson, R. S.; Horton, D., Eds.; Academic: New York, 1973, Vol. 28, pp. 11-160. 50. Saeman, J. F.; Moore, W. E.; Mitchell, R. L.; Millett, M. A. Tappi 1954, 37, 336-340. 51. Albersheim, P.; Nevins, D. J.; English, P. D.; Karr, A. Carbohyd. Res. 1967, 5, 340-345. 52. Laine, R. Α.; Sweeley, C. C. Anal. Biochem. 1971, 43, 533-538. 53. Varma, R.; Varma, R. S.; Wardi, A. H. J. Chromatogr. 1973, 77, 222-227. 54. Bradbury, A. G. W.; Halliday, D. J.; Medcalf, D. G. J. Chromatogr. 1981, 213, 146-150. 55. Oshima, R.; Yoshikawa, Α.; Kumanotani, J. J. Chromatogr. 1981, 213, 142-145. 56. Hudson, J. R.; Morgan, S. L.; Fox, A. J. HRC & CC 1982, 5, 285-290. 57. Henry, R. J . ; Blakeney, A. B.; Harris P. J . ; Stone B. A. J. Chromatogr. 1983. 256, 419-427. 58. Kato, K.; Noguchi, M. Agr. Biol. Chem. 1976, 40, 1923-1928. 59. Blaschek, W. J. Chromatogr. 1983, 256, 157-163. RECEIVED December 20,

1985


Analytical Methods for Determining Pectin Composition - ACS

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