Carbohydrate Polymers 17 (1992) 205-207 Determination of gulose and/or guluronic acid by ion chromatography and pulsed amperometric detection L. H. Krull & G. L. Cote* Biopolymer Research. National Center for Agricultural Utilization Research. US Department ofAgriculture. T Agricultural Research Service, 1815 North University St. Peoria. Illinois 61604. USA (Received 8 June 1990: revised version received 28 December 1990: accepted IS January 1991) A method for the determination of guluronic acid in polysaccharides which may also contain either glucuronic or mannuronic acid has been developed. The method makes use of an ion chromatographic instrument equipped with a pulsed amperometric detector. The method will detect concentrations in the range of0·5 to 50 ppm. This technique has been applied to the detemlination of mannuronate/guluronate ratios in commercial samples of alginic acid. as \vell as to the stmctural analysis of an extracellular bacterial alginate. INTRODUCTION arising from the reduction of D-glucose. Although alginic acid itself contains neither glucose nor Complete and quantitative acid hydrolysis of poly­ glucuronic acid. this may not always be the case when saccharides containing uronic acid residues is one is working with neVi. uncharacterized poly­ notoriously difficult to attain (Adams. 1965). For this saccharides. reason. the uronic acid residues in such polymers are There are ways to circumvent this ambiguity. usually first reduced to their corresponding neutral including the use of per-O-acetylated aldononitrile sugar analogues (Taylor et at.. 1976). Once reduced. the (PAAN) derivatives rather than per-O-acetylated neutral polysaccharide can then be analyzed by more alditols (Lance & Jones. 1967). and the direct HPLC conventional means. i.e. acid hydrolysis. reduction to determination of the free sugars released from the the corresponding alditols. and acetylation. The per-O­ polysaccharides upon acid hydrolysis. However. both acetylated alditols can then be quantitated by gas­ of these procedures have their drawbacks. While the liquid chromatography (Sloneker. 1972). and further use ofPAAN derivatives allows one to work with much characterized by GC-MS. Ambiguities can enter the smaller samples due to the high sensitivity of gas­ analysis. however. when two different uronic acids can chromatographic methods. it introduces another give rise to the same alditol acetate. due to the loss of derivatization step into the procedure. Although the asymmetry during the reduction step. One example of direct HPLC detection ofthe free sugars released from this is seen when L-guluronic acid. a component of the acid-hydrolyzed polysaccharide is a simpler method. alginic acid. is reduced to L-gulose. and then to the the instruments and detectors in common use (i.e. corresponding alditoL which is identical to the alditol refractive index) are often not sensitive enough for work with small samples. *To whom correspondence should be addressed. carbohydrate makes use of an ion chromatograph TThe mention offirm names or trade products does not imply that they are endorsed or recommended by the US Depart­ coupled with a pulsed amperometric detector (Dionex ment of Agriculture over other firms or similar products not Corp.. Sunnyvale. CAl. We have taken advantage of mentioned. this instrument's capabilities to develop a simplified 205 206 L. H Krull. G. L. Cote method for the determination of monomer compo­ Inos sitions of acidic polysaccharides. particularly those of Gulose Inos the alginate type. 20 ppm Gulose Man 0.05 ppm Gulose Glc Man + 10 ppm Inositol Gal Glc + 10 ppm Inositol Xyl Gal Xyl EXPERIMENTAL Materials All carbohydrates and reagents were purchased from Sigma Chern. Co.. St Louis. The production. purifi­ .r:'" cation. and properties of the extracellular poly­ a: .r:a:'" saccharides from Azotobacter chroococcum have been recently described (Cote & Krull. 1988). The reduction of the uronic acid functions was carried out by a I....._--I.I__.L.I _--,I IL-_...L'__.L.I_---l' modification ofthe procedure ofTayloret at.. (1972). in o 5 10 15 0 5 10 15 which a solution of the polysaccharide in water was Time, min treated with an approximate IO-fold excess of solid l-methyl-3-(3-dimethylamino-propyl )-carbodiimide Fig. I. Chromatography of standard sugar mixtures including L-gulose at two concentrations (20 and 0·05 ppm). hydrochloride. The pH was held at 4·75 by automatic on an HPIC AS6 carbohydrate column with 100 mM NaOH titration with 0·1 N HCI over about 2 h and the reaction as eluting solvent at flow rate of0,5 mllmin. 50 pi sample size was allowed to proceed for 2 h until no further and room temperature. Inositol (10 ppm) was present as an hydrogen ion uptake was observed. The intermediate internal standard. was then reduced by the dropwise addition of an aqueous solution ofsodium borohydride (NaBH ) with 4 sugars. even at very low relative amounts. The separation the pH held at 7·0 with 0·1 N HCI over about 2 h. The is rapid. being completed in less than 15 min. reduced polysaccharides were acidified to pH 3. freed Figure 2 shows the separation which occurs when of salts and lyophylized. Hydrolysis was carried out the eluant is 10 mM NaOH. Gulose is separated from with 2 N tritluoroacetic acid (TFA) at 100°C and the galactose. glucose. and xylose. but not from mannose acid removed under a stream of nitrogen. The samples under these conditions. With either eluant. using were diluted with water and run directly on the ion inositol as an internal standard. the response ofgulose chromatograph. in the range of 0·05 to 50 ppm was linear. The lower limit ofdetection. for practical purposes. was 0·05 ppm. Chromatographic analysis Solvent gradients and other NaOH concentrations A Dionex Advanced Chromatography Module with a HPIC AS6 carbohydrate column. coupled to a Pulsed Amperometric Detector (PAD) with a gold electrode (Olechno et at.. 1987) was used in this study. Two sets of chromatographic conditions were employed. The first Inos conditions consisted of a 100 mM concentration of NaOH at a tlow rate of 0·5 ml/min. The HPIC AS6 Gul + Man columns were run at room temperature. The PAD settings were: E I: 0·1 volts. E2: 0·6 volts. E3: -0,8 volts. tl: 2. t2: 2. and t3: 5. The second conditions were identical to the first. except that the NaOH concen­ tration was reduced to 10 mM. RESULTS AND DISCUSSION 30 Time. min Figure I illustrates the separation of a mixture of monosaccharides under the first set of conditions. Fig. 2. Chromatography ofstandard sugar mixture. including L-gulose. on an HPIC AS6 carbohydrate column with 10 mM using 100 mM NaOH. Although xylose. galactose. NaOH as eluting solvent. at a flow rate of 0·5 mllmin. 50 pi glucose and mannose all chromatograph as a single sample size and room temperature. Inositol (10 ppm) was peak. gulose is clearly separated from the rest of these present as an internal standard. Detemtination ofgulose and/or guluronic acid 207 Table 1. Composition of alginates Alginate Reference Percent carbohydrate o-Mannuronic L-Guluronic acid acid Macrocystis pyr[fera (Sigma) High viscosity 45·3 54·7 Low viscosity 78·6 2104 EPS-II Cote & Krull (1988) 87·9-92·0 12·1-8·0 Laminaria digilala 8-61 53-6 46·3 Knutson & Jeanes (1968) 57·8 40·8 Haug & Larsen (1962) 61·6 3804 Laminaria digilala I-52 50·1 49·9 Knutson & Jeanes (1968) 49·0 48·6 Haug & Larsen (1962) 55·6 4404 Laminaria hyperhorea 12.61 39·0 61·0 Knutson & Jeanes (1968) 38·3 61·7 Haug & Larsen (1962) 37·5 62·5 were unsuccessful in the separation of all mono­ mation. and simpler sample preparation. The tri­ saccharides and gulose. The gulose was either fluoroacetic acid used for hydrolysis is simply unseparated or the peaks hroadened and resolution evaporated. the sample diluted and injected. Any TFA was lost. It was apparent from this information. that no that did not evaporate would suhsequently he neutral­ single set ofchromatographic conditions was sufficient ized hy the NaOH eluant. and its signal is suppressed for the separation of all monosaccharides from one electronically. another and in order to determine concentrations of mannose in the presence of other monosaccharides would require two runs. For a sample whose com­ ACKNOWLEDGMENTS ponents are known. it would he possihle to use the 100 mM system for quantitative analysis ofthe mannose We express our gratitude to Mr Clarence Knutson for and gulose. Tahle I illustrates the results from several samples ofalginates and Mr EricJohnson for technical alginate samples. The results are compared to results assistance. from calculations from spectophotometric data (Knutson & Jeanes. 1968) and chromatographic data (Haug & Larsen. 1962). A high-viscosity alginate from REFERENCES the alga Macrocystis pyr(fera (Sigma) was found hy this method to have a guluronic acid composition of 54· 7% Adams. G. A. (1965). In Methods in Carhohvdrate Chemistrv. while a lower viscosity sample was found to have a Vol. V. ed. R. L. Whistler. Academic Press. New York. guluronic acid content of 21-4%. This is the range of p.269-76. Cote. G. L. & Krull. L. H. (1988). Carhohvd. Res.. 181, 143-52. results found hy other methods (Penman & Sanderson. Haug. A. & Larsen. B. (1962).Acta Chem: Scand.. 16, 1908-18. 1972: Morris et al.. 1980). A numher of extracellular Knutson. C. A. &Jeanes. A. (1968).Anal. Biochem.. 24,482-90. alginate preparations from Azotobacter chroococcum Lance. D. G. &Jones. J. K. N. (1967). Can. J Chern.. 45,1995-8. (see Tahle I. EPS-II) were also analyzed. and were Morris. E. R.. Rees. D. A. & Thorn. D. (1980). Carhohyd. Res.. found to vary from hatch to hatch. with guluronic acid 81,305-14.