ANALYTICAL BIOCHEMISTRY 115, 410-418 (1981) 4889 Differential Gas-Liquid Chromatography Method for Determination of Uronic Acids in Carbohydrate Mixtures JACOB LEHRFELD Northern Regional Research Center. Agricultural Research. Science and Education Administration. U. S. Department of Agriculture.' Peoria. Jllinois 61604 Received March 17, 1981 An integrated gas-liquid chromatography method is described for the quantitation of mix­ tures containing simple monosaccharides in addition to mannuronic, glucuronic, and/or ga­ lacturonic acids. A hydrolyzed sample is divided into two portions. One portion is analyzed by the standard aldononitrile method. Glucuronic, galacturonic, and mannuronic acids are converted into compounds that do not chromatograph in the region of the standard aldononitrile acetates. Thus, this analysis gives an accurate estimation of the neutral monosaccharide con­ tent. The second portion is analyzed by a modified alditol acetate procedure. The reduction step is repeated three times to convert mannuronic, galacturonic, and glucuronic acids to their corresponding alditols via their intermediate lactones. The results of this gas-liquid chro­ matography analysis reflect the sum of the monosaccharides present plus their corresponding uronic acids. The difference between the values obtained by the aldononitrile acetate method and the modified alditol acetate method, therefore, is a measure of the uronic acid(s) present. Heteropolysaccharides containing only heating time must be carefully controlled, neutral sugars are readily analyzed by the as well as the temperature of the reaction alditol acetate (1-4) or the aldononitrile ac­ when the sulfuric acid is added. The presence etate (5-9) procedures. However, the pres­ of other neutral sugars requires the addition ence of uronic acids complicates the analysis. of a correction factor that will vary with the Out of necessity, a supplemental spectro­ amount of sugar present. Additionally, the photometric (4) reaction generally is run on ability to differentiate between the various the mixture to determine how much uronic uronic acids in mixtures is limited (13). acid is present. For example, uronic acids, Other more specific methods have been when treated with sulfuric acid and either reported. For example, Spiro (14) reported carbazole (10,11) or harmine (12), develop that an Aminex A-25 column can separate a color quantitatively. However, if the so­ a number of uronic acids. Quantitation is lution is colored to start with, the analysis accomplished by coupling the column to a is difficult or impossible to perform. Technicon sugar analyzer using the orcinol Furthermore, the carbazole analysis is reagent. Enzymic methods are also avail­ very sensitive to slight procedural changes able. For example, D-galacturonic acid (IS) (13). For example, small amounts of im­ can readily be analyzed. These supplemen­ purities in the sulfuric acid, carbazole, or tary methods require additional equipment absolute ethanol can markedly change the and/or specialty reagents. results. The temperature of the bath and In contrast, the differential method re­ ported here uses the same basic methodol­ , The mention of firm names or trade products does ogy, reagents, and equipment used for not imply that they are endorsed by the U. S. Depart­ standard gas-liquid chromatography (glc)2 ment of Agriculture over other firms or similar products not mentioned. 2 Abbreviation used: glc, gas-liquid chromatography. 0003-2697/81/120410-09502.00/0 410 Purchased by U.S. Dept. of Agriculture for Official Use. Copyright 1981 by Academic Press. Inc. All rights of reproduction in any form reserved. URONIC ACID DETERMINATION IN CARBOHYDRATE MIXTURES 411 analysis of neutral sugars by alditol acetate periods of time.) The solution was heated at and aldononitrile acetate procedures. In ad­ 100°C for I h and then cooled. One-half dition, the uronic acids can readily be dif­ milliliter of acetic anhydride (0.5 ml) was ferentiated be they glucuronic, galacturonic, added and the solution was reheated at or mannuronic. 100°C for I h. The resultant solution was satisfactory for glc analysis. A word of cau­ MATERIALS AND METHODS tion is needed: Hydroxylamine has been shown to be a mutagen and, as such, should Materials. Extracellular polysaccharides, be handled with caution (16). NRRL B-1973 and NRRL B-1459, were a Preparation ofalditol acetate. A standard gift from C. A. Knutson of the Northern containing up to 30 mg of carbohydrate (in Regional Research Center. Carbohydrate a 16 X 125-mm Teflon-lined screw-capped standards D-galactose, D-glucose, D-man­ tube) was dissolved in a solution of 25 mg nose, D-ribose, D-arabinose, D-xylose, ga­ sodium borohydride in 1 ml water. The so­ lactitol, D-glucitol, D-mannitol, ribitol, D-ar­ lution was kept at room temperature for 1.5 abinitol, xylitol, D-glucono-I,5-lactone, D­ h. Sufficient 10% acetic acid in water was glucurono-6,3-lactone, D-galactopyranuronic added dropwise (10-20 drops) until bub­ acid monohydrate, and sodium D-gluconate bling stopped. The solution was percolated were obtained from Pfanstiehl Laboratories through a column made from a Pasteur pipet Inc. Potassium D-gluconate, D-mannuronic and I to 1.5 ml of a cation-exchange resin. acid-6,3-lactone, L-mannonic acid-I A-lac­ The resin was washed with 4-6 ml of deion­ tone, and D-galactonic acid-I A-lactone were ized water and the combined eluate and obtained from Sigma Chemical Company. washings were evaporated to dryness. Meth­ Sodium borohydrate was obtained from anol (4 ml) containing HCI (0.1%) was Ventron Company. Barium carbonate, silver added to the residue and the solution was carbonate, hydroxylamine hydrochloride, shaken while being heated at 50-55°C for pyridine, and acetic anhydride were obtained about 5 min and then was flash evaporated. from Fisher Scientific. The cation-exchange This procedure was repeated twice. A Buch­ resin was AG 50W-X8, 200-400 mesh, H+ ler Vortex evaporator can rapidly and ex­ from Bio-Rad Laboratories. The X8 is a peditiously handle up to 72 samples. The better choice than the X4 because it has a reduction and deionization were repeated higher capacity, i.e., 1.7 mEq/ml vs 1.2 twice. mEq/ml. The final residue was dissolved in 0.5 ml HI-EFF 3BP (neopentyl glycol succinate) pyridine and 0.5 ml acetic anhydride and on Gas-Chrom Q, 100-120 mesh, and JXR heated at 100°C for I h. This reaction mix­ (methyl silicone) on Gas Chrom Q, 100-120 ture is suitable for glc analysis without fur­ mesh, were obtained from Applied Science ther treatment. Inc., and SP 2340 (Cyano-silicon) on Su­ glc analysis. A Packard Instrument Model pelcoport, 100-120 mesh, was obtained from 428 gas chromatographic unit equipped with Supelco Inc. dual F.LD. and dual electrometers was out­ Preparation of aldononilrile acetates. A fitted with two glass columns 2 mm X 2 m. standard containing up to 30 mg of carbo­ Column A was used to separate the aldon­ hydrate (in a 16 X 125-mm Teflon-lined onitrile acetate and contained 3% HI-EFF screw-capped tube) was dissolved in 0.5 ml 3BP on Gas-Chrom Q, 100-120 mesh. Col­ pyridine containing 30 mg hydroxylamine umn B was used to separate the alditol ac­ hydrochloride. (Solutions containing 60 mg etates and contained a I: I mixture of 3% hydroxylamine hydrochloride/ml pyridine HI-EFF 3BP on Gas-Chrom Q, 100-120 can be prepared readily and stored for long mesh, and 3% SP 2340 on Supelcoport, 100- 412 JACOB LEHRFELD 120 mesh. A helium flow rate of 25 milmin been used for over a year under the condi­ was maintained. Temperature programming tions described under Materials and Meth­ was employed for maximum resolution in ods with little loss in resolution. minimum analysis time. For simple mixtures The aldononitrile acetate method for neu­ of hexoses, an initial temperature of 210°C tral sugars is simple. The sugars are dis­ was maintained for 3 min and then the tem­ solved in pyridine containing hydroxylamine perature was programmed upward at 2°C hydrochloride. Solutions more than a year per minute to 234°C. A complete analysis old have been used with no problem. The took 10-12 min. Complex mixtures were in­ solution is heated (IOO°C) for a short time jected into the column kept at an initial tem­ (1 h) to convert the sugars to their corre­ perature of 184°C for 3 min and pro­ sponding oximes. Then, acetic anhydride is grammed upward at 2°C/min to 234°C. added and the oxime is dehydrated to the Trimethylsilylated lactones were analyzed corresponding nitrile. Actually, an initial on a column of 3% JXR on 100- to 120-mesh heating period of 1-2 min is all that is re­ Gas-Chrom Q 2 mm X 2.5 M. This column quired. For example, glucose is completely was kept at 184°C for 5 min and then pro­ converted into its oxime within 1 min. Pyr­ grammed up to 204°C at 2° Imin. idine solution containing xylitol, glucose, and hydroxylamine hydrochloride was heated RESULTS AND DISCUSSION at 100°C (Fig. 1). Samples removed after lOs and after 1 min were treated with an Accurate and meaningful component excess of acetic anhydride. Any D-glucose analysis of a polysaccharide presupposes ho­ that was not converted into its oxime ap­ mogeneity of the sample and a hydrolysis peared as glucose pentaacetate; any oxime technique (4,17,18) that results in complete present at the time acetic anhydride was breakdown of the polymer and, at the same added was converted into the corresponding time, minimal degradation of the compo­ D-glucononitrile. After lOs, 40% of the glu- nents. Once this is accomplished, the sample then can be treated as described to obtain useful component composition data.