18
Electrophoresis 1990, 1 I , 18-22
H. Scherz
5 References [ 11 Cheung, W. Y., Science 1980,207, 19-27.
[21 Wang,J.H. and Waisman.D. M.,Curr. Top. Cell. Regulat. 1979.15, 47-107. 131 Klee, C . B. and Vanaman, T. C., A&. Prolein Chem. 1982. 35. 213-321. 141 Means, A. R., Tash, J . S. and Chafouleas, Jl. G., Physiol. Rev. 1982. 62, 1-39. (51 Kretsinger, R. H., CRC Crit. Rev. Biochem. 1980,8, 119-174. 161 Satyshur, K. A., Rao, S. T., Pyzalska, D., Ilrendel, W., Greaser. M. and Sundaralingam, M., J . B i d . Chem. 1988,263, 1628-1647. 171 Szebenyi, D. M. E. and Moffat, K., J. B i d . Chem. 1986. 261, 8761-8777. 181 Herzberg, 0. and James, M. N. G., Nature 1986,313,653-659. 191 Babu, S. Y., Bugg, C. E. and Cook, W. J., J . Mol. B i d . 1988.204, 191-204.
Heimo Scherz Deutsche Forschungsanstaltfur Lebensmittelchemie, Garching
1101 Plancke, Y. D. and Lazarides, E., Mol. Cell. Biol. 1983. 3. 14 12- 1420. I I 11 Runte, L., Jurgensmeier, H.-L., Unger, C. and Soling, H. D.. FEBS Lett. 1982,147, 125-130. (121 Jorgenson, J . W. and Lukacs, K. D., Anal. Chem. 1981. 53, 1298-1302. I131 Jorgenson, J. W. and Lukacs, K. D., Science 1983.222,266-272. I141 Terabe, S., Otsuka, K., Ichikawa, K., Tsuchiya, A. and Ando. T.. Anal. Chem. 1984,56, 113-1 16. I151 Jorgenson, J. W., Anal. Chem. 1986,58,743A-760A. I161 Lauer,H. H. andMcManigill,D.,Anal. Chem. 1986,58,166-170. I171 Hjerttn, S., Elenbring. K., Kilar, F., Liao, J., Chen, A. J. C. and Zhu, M., J . Chromatogr. 1987, 403,47-61. I181 Cohen,A.S.andKarger,B.L.,J. Chromatogr. 1987,397,409-417. I191 Grossman, P., Colburn, J. and Lauer, H.,Ana/. Biochem. 1988.173, 265-270. l201 Compton, S. W. and Brownlee, R. G., BioTechniques 1988. 6, 432-440.
Thin-layer electrophoretic separation of monosaccharides, oligosaccharides and related compounds on reverse phase silica gel The electrophoretic mobilities of monosaccharides, oligosaccharides, sugar alcohols and sugar acids were determined in 0.3 M borate buffer, pH 10, using thin-layer electrophoresis on silanized silica gel, pretreated with octanol- 1. A rapid separation of a number of sugars, occurring in foods, could be achieved. Using a 0.05-0.1 M neutral solution of barium acetate as electrolyte, thin-layer electrophoresis allowed excellent and rapid separation as well as identification of all common uronic acids which are constituents of many acidic polysaccharides.
1 Introduction Zone electrophoresis is applied in the field of carbohydrate chemistry as an alternative separation method to paper and thin-layer chromatography. Whereas charged molecules such as sugar acids and sugar phosphates migrate in the electric field without any modification, neutral carbohydrates must be converted into ionic forms. Usually, neutral carbohydrates are transformed into borate complexes which in aqueous solution can easily be generated by addition of borate ions at alkaline pH. Under these conditions borate ions react with vicinal hydroxyl groups with resultant formation of complexes, migrating in the electric field. Since the specific chemical structure of carbohydrates strongly influences the formation of such borate complexes, great variations of their mobilities in the electric field are observed, providing a basis for the electrophoretic separation of carbohydrate mixtures. Similar to many other electrophoretic separations, the electrophoresis of carbohydrates also requires an inert solid support for stabilization. In the past, chromatography paper was widely used as a stabilizing matrix and, especially under conditions of ~
~~
Correspondence: Dr. Heimo Scherz, Deutsche Forschungsanstalt fur Lebensmittelchemie, LichtenbergstraRe 4, D-8046 Garching. Federal Republic of Germany
0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
high voltage electrophoresis, good separations were obtained I1,21. Other supports were cellulose acetate 131and glass fiber paper 141, which had been applied preferably to the separation of polysaccharides. However, the above approaches had the following disadvantages, limiting the applicability of electrophoresis to the separation of carbohydrates: (i) need of expensive equipment with an efficient cooling system in the case of high voltage electrophoresis, (ii) limited choice for visualization, especially revelant to chromatography paper and cellulose acetate, excluding all reactions requiring aggressive reagents, and (iii) adsorption effects and strong electroendoosmosis, especially in the case of glass fiber paper, resulting in strongly distorted patterns. Although silanization of glass fiber paper eliminates these undesirable attributes, the currently available procedures result in mechanically unstable sheets [ 5 , 61. Recently, for the separation of polysaccharide -containing thickening agents, a thin-layer electrophoretic procedure was developed, using as support a silanized silica gel, pretreated with octanol-1 [71. The silanized and pretreated silica gel affords a stable inert system on which adsorption and electroendoosmosis effects are strongly diminished and aggressive carbohydrate reagents can be applied for visualization. Good 01 73-0835/90/010 1-0018 $02.50/0
Electrophoresis 1990. I I , 18-22
separation of polysaccharides was obtained 171 and also the successful separation of some low molecular degradation products of the hydrothermolysis of cellulose biomass, e.g. dihydroxyacetone, glyceraldehyde, glucose and aromatic carboxylic acids, has been described [81. In this report electrophoresis on silanized and octanol-1 pretreated silica gel is applied to determine the electrophoretic mobilities of monosaccharides, oligosaccharides, sugar alcohols, sugar acids and sugar phosphates.
2 Materials and methods 2.1 Materials
Thin-layer electrophoresis of monosaccharides and oligosaccharides
19
neutral detergent (Tween 20, Serva). The detergent was added to prevent the formation of a thin film of octanol- 1 on the water surface which would reduce water evaporation and accelerate the desiccation of the plates. A few crystals ofthymol were added to prevent microbial contamination. The side walls of the desiccator were taped with wet filter paper, thus establishing a saturated moist atmosphere with resultant reduced water evaporation from the surface of the plates. After incubation for 3 h the plates could be used for electrophoresis. In most experiments freshly prepared plates were used. Storage under the described conditions should not exceed 48 h.
2.3 Electrophoresis
The following materials were obtained from the indicated sources: silanized silica gel, 60 H, for thin-layer chromatography (Merck, Darmstadt, FRG), octanol- 1 (analytical grade, Merck), glass fiber paper (Whatman GC/F), polyvinylpyrrolidone K-90 ( M , 350 000, Fluka, Buchs, Switzerland), 1,3-dihydroxynaphthalene(naphthoresorcinol; Serva, Heidelberg, FRG), omega-hydroxymethylfurfural (Merck). The following compounds were used as reference substances. Monosaccharides: L-arabinose, D-ribose, D-xylose, D-galactose, D-mannose, D-fructose, L-sorbose were obtained from Merck, L-rhamnose, D-fucose from Serva, and D-lyxose from Sigma (Munchen, FRG). Oligosaccharides: sucrose, lactose, maltose, trehalose, raffinose, melibiose were obtained from Merck, sophorose, gentiobiose, isomaltose, cellobiose from Serva, lactulose, melezitose, palatinose turanose, stachyose, maltotriose from Sigma. Sugar alcohols: xylitol, ribitol, mannitol, sorbitol, erythrol, meso-inositol were from Merck, arabitol from Serva, dulcitol, threitol from Sigma. Sugar acids: galacturonic acid was obtained from Merck, gluconic acid, arabonic acid K salt, ribonic acid, L-y-lactone from Serva, galactonic acid- 1,4-lactone,mannuronic acid-3,6-lactone, glucuronic acid Na-salt from Sigma. Polysaccharides: Naalginate and gum arabic were from Roeper (Hamburg, FRG). All samples were dissolved in the respective buffer. In the case of barium acetate the sugar acid lactones were hydrolyzed prior to separation with a few drops of 0.1 M Ba(OH),. Polysaccharides were hydrolyzed with sulfuric acid, followed by neutralization and removal of sulfate with 0.1 M Ba(OH), 191.
The 0.3 M borate buffer, pH 10,wasprepared according to 171. Crystallized boric acid 18.54 g and Titriplex I11 7.44 g were dissolved in approximately 400-600 m L distilled water, followed by addition of an aqueous solution of 2 M NaOH until a p H of 10 was reached. The solution was then made up to 1000 m L with distilled water. Barium acetate: Equivalent amounts of barium acetate were dissolved in distilled water and made up to 1000 mL (the respective amounts were 12.77 g for 0.05 M, 17.88 g for 0.07 M and 25.54 g for 0.1 M solutions). Electrophoresis was carried out in the Desaga Double Chamber according to [7]. Both electrode vessels were filled with buffer and the silica gel plates on the cooling block were connected with the buffer vessel with strips of glass fiber paper pressed onto both edges of the silica gel plates with 200 x 20 x 1 mm glass plates. On top of these glass plates, second glass plates (200 x 20 x 4 mm) and on these a standard glass plate (200 x 200 x 4 mm) were placed to reduce the evaporation of water during electrophoresis, which might result in irregularities of the electric field. After equilibration for 30 rnin at 350-400 V, 10-15 mmlonggrooves weremadeinthelayerwiththetipofa needle, at a distance of 30 mm from the cathode, and 2-5 pLof the sample, dissolved in the running buffer, were deposited into the grooves with a Hamilton syringe. After replacing the cover plate, electrophoresis was carried out for 60-120 rnin at 350-400 V, with the temperature of the cooling block set at 16- I 8 “C. Electroendoosmosis was determined in each run using omega-hydroxymethylfurfural as uncharged marker.
2.2 Preparation of thin-layer plates
2.4 Visualization
Thin-layer plates were prepared as previously reported [7,81, with some modifications. The improved procedure is briefly described. A slurry of 15 g silanized silica gel in 50 mL dichloromethane or methyl acetate, containing 6 g* octanol- 1 was stirred for 40 rnin using a magnetic stirrer. The solvent was evaporated at 50 “C, using a Rotavapor (Buechi, Flavil, Switzerland), until a lump-free powder was obtained. T o the dry powder in a mortar (diameter: 160 mm) 10 mL of a 2 % solution of polyvinylpyrrolidone in buffer, followed by 35 mL buffer, were added stepwise with intense stirring with a pestle. After stirring for about 10 min, the slurry was spread on 200 x 200 mm glass plates using the Desaga equipment for thinlayer chromatography (Desaga, Heidelberg, FRG). Immediately after coating, the plates were transferred to a desiccator containing, on the bottom, distilled water with a few drops of a
Following electrophoresis the plates were dried at 90 “C for 30 min. Visualization was carried out with the following reagents: Naphthoresorcinol-sulfuric acid prepared according to I 7 I : 0.4 g naphthoresorcinol were dissolved in 50 mLethano1, containing 2.5 mL concentrated sulfuric acid and 1.5 mL octanol1. The reagent must be freshly prepared. The dry, octanol-free plates were sprayed with this reagent and heated at 1 10 “C for 10- 15 min. Monoaldoses, their phosphates and uronic acids appear as deep blue spots, ketoses and their phosphates as red ones. The spots of oligosaccharides are different according to their compositions. The periodic acid-benzidine reagent for sugars and sugar alcohols was prepared according to i101.Solution “a” contained 0.25 g sodium periodate, dissolved in 50 m L distilled water, and solution “b” contained 1.8 g benzidine in 50 mL ethanol, supplemented with 20 mL acetone and 10 mL 0.2 M HCl. The dry plates were sprayed with solution “a” and after 5 rnin with solution “b”. The visualized components appear as light spots on a deep brown background.
*
Valid for new batches of silanized silica gel due to their higher grade of silanization; old batches need only 4 g 171.
20
Electrophoresis 199O,Il, 18-22
H. Scherz
3 Results An electropherogram of a mixture ofselected mono- and oligosaccharides is shown in Fig. 1. Most of the carbohydrates, namely glucose, fructose, maltose, lactose, sucrose and raffinose are typical elementsin food, whereas mannose and rhamnose are widespread components of some natural polysaccharides. All these compounds can be well separated in a single run of 120 min. The relative electrophoretic mobilities of monosaccharides, oligosaccharides, sugar alcohols, sugar acids and sugar phosphates were determined in 0.3 M borate buffer, p H 10, using D-ghCOSe as reference substance (Tables 1,2 and 3). The electrophoretic mobilities; of mono- and oligosaccharides are similar to those obtained in high voltage electrophoresis on chromatography paper or glass fiber paper [ 1, 21 (Table 4). The mobilities of sugar acids and sugar phosphates in the same buffer are higher than those of the neutral compounds (Table 3). Hexosephosphates can easily be separated from the neutral compounds as well as from each other. IJsing a neutral medium, this electrophoresis system enables rapid separation ofthe acid compounds without interference from the neutral sugars
Table 1. Relative mobilities (MG)of monosaccharides and sugar alcohols in 0.3 M borate buffer, pH loo) Monosaccharides
MG
Sugar alcohols
D-Glucose D-Mannose D-Galactose D-Fructose L-Sorbose L- Arabinose
1 .oo
0.69 0.92 0.86 0.95
Manriitol Sorbitol Dulcitol Xylitisl Adonitol Arabitol Threitol Erythrol meso-Inositol
D-XylOSe
D-Ribose L-Rhamnose D-Fucose D-LyXOSe
1.oo
1.01 0.70 0.53 0.95 0.67
a) Reference substance: D-ghCOSe.
and sugar alcohols which do not migrate under these conditions. In pure aqueous solutions of barium acetate, an excellent separation of all uronic acids, occurring as monomers in acidic polysaccharides, can be achieved. Separation of sugar phosphates is also possible in the medium (Table 5 ) . The relative electrophoretic mobilities of uronic acids, with D-glucuronic acid as reference substance, were determined at three different concentrations of the electrolyte (Table 6). The mobilities of galacturonic acid and guluronic acid strongly depended on the electrolyte concentration.
Table 2. Relative mobilities (MGofoligosaccharidesin0.3 M borate buffer, pH 10a) Oligosaccharides
*G
1 2 4-Disaccharides Maltose Lactose Cellobiose Lactulose
0.39 0.42 0.32 0.6 1
I 2 6-Disaccharides Isomaltose Gentiobiose Melibiose Palatinose
0.62 0.73 0.79 0.48
1 2 3-Disaccharides Turanose
0.64
1 2 2-Disaccharides
0.90 0.80 0.94 0.79 0.87 0.75 0.87 0.68 0.48
Sophorose Sucrose
0.35 0.20
1 2 I-Disaccharides Trehalose
0.22
Trisaccharides R affinose Melezitose Maltotriose
0.29 0.28 0.26
Tetrasaccharose Stachyose
0.37
a) Reference substance: D-glucose.
origin -
Figure I . Thin-layer electrophoresis of mono- and oligosaccharides on silanized silica gel pretreated with octanol-1, in 0.3 M borate buffer, pH 10. Electrophoresis:400V, 120min, 18 OC.Lane(1) sucrose, (2) rafinose, (3) maltose, (4)lactose, (5) rhamnose, ( 6 ) mannose, (7)fructose, (8) glucose, (M) mixture of all eight compounds. Sample loading: 10 bg of each compound. Visualization with naphthoresorcinol-sulfuric acid. Origin indicated.
21
Thin-layer electrophoresis of monosaccharides and oligosaccharides
ElfctrophorePis 1990, 11, 18-22
Table 3. Relative mobilities (MG) of sugar acids and sugar phosphates in 0.3 M borate buffer, pH 10”)
Table 5. Relative mobilities of sugar phosphates ( M t i ~in) aqueous barium acetate solution, 0.05 M ~ )
Sugar acids
Mti
Sugar phosphate
MtiA
Glucuronic acid Galacturonic acid Mannuronic acid Gluconic acid Galactonic acid Ribonic acid Arabonic acid
1.39 1.28 1.21 1.21 1.23 1.17 1.30
Glucose- 1 -phosphate Glucose-6-phosphate Fructose-6-phosphate Fructose-l,6-diphosphate
0.77 0.86 0.8 1 1.38
Sugar phosphates
Mti
Glucose-6-phosphate Glucose- 1 -phosphate Fructose- 1-phosphate Fructose- l,6-diphosphate
1.30 1.06 1.21 1.44
a) Reference substance: D-glucuronic acid.
Table 6. Relative mobilities (MGA)of uronic acids in pure aqueous barium acetate solutions of different concentrationsa) MGA
a) Reference substance: D-glucose.
Table 4. Comparison of MG-values of some important sugars and sugar alcohols on silanized silica gel-octanol-1 plates with those on chromatography paper and glass fiber paper, in 0.3 M borate buffer, pH loa) Compound
L-Arabinose D-Xylose D-Fucose L-Rhamnose D-Galactose D-Glucose D-Mannose D-Fructose Cellobiose Maltose Gentobiose Saccharose Rafinose Sorbitol Mannitol
Silanized silica gel pretreated with octanol-1
Chromatography Paper
Glass fiber Paper
1.oo 1.01
0.96 1.oo 0.89 0.53 0.93 1.oo 0.72 0.90 0.29 0.34 0.72 0.18 0.28 0.89 0.90
0.94 1 .oo 0.88 0.49 0.9 1 1.00 0.67 0.88 0.26 0.30 0.69 0.15 0.23 0.82 0.95
0.95 0.53 0.92
1 .oo
0.69 0.86 0.32 0.39 0.73 0.20 0.29 0.80 0.90
a) Reference substance: D-glucose.
Glucuronic acid Mannuronic acid Galacturonic acid Guluronic acid
0.05 M
0.07 M
0.1 M
1.oo 0.80 0.42 0.22
1.oo 0.74 0.26 0.06
I .oo 0.68 0.14 -
a) Reference substance: D-glucuronic acid.
The usefulness of this electrophoresis system is demonstrated for a mixture of galacturonic acid, glucuronic acid, mannuronic acid and guluronic acid, along with their individual compounds, and for a mixture of the hydrolysate of sodium alginate and gum arabic as a practical example for the identification of uronic acids in polysaccharides (Fig. 2). In the case ofgum arabic the acid and neutral compounds can be well resolved. The latter are shifted toward the cathode, presumably as a result of a weak interaction between the carbohydrates and the barium ions.
4 Discussion Thin-layer electrophoresis, on a silanized and octanol- I pretreated silica gel, of low molecular weight carbohydrates and some related compounds, affords the following advantages over the preceding procedures: (i) The separation can be car-
Figure 2. Thin-layer electrophoresis of uronic acids on silanized silica gel, pretreated with octanol-1, in 0.07 M barium acetate solution. Electrophoresis: 400 V, 150 min, 16 “C. Lane ( I ) galacturonic acid, (2) mannuronic acid, (3) glucuronic acid, (4) hydrolysate of gum arabic (glucuronic acid), (5) hydrolysate of sodium alginate (mannuronic and guluronic acid), (M) mixture of (1-3) and guluronic acid, (H) omega-hydroxymethylfurfural. Sample loading: 10 kg of each individual compound. Visualization with naphthoresorcinol-sufuric acid. Origin indicated.
22
Electrophoresis 1990,l I , 18-22
H. Scherz
ried out at 350-400 V, instead of the 2000-5000 V necessary in high voltage electrophoresis on chromatography paper, thus obviating the need for expensive equipment and a cooling system. (ii) Silica gel as inert support material permits the application of sulfuric acid containing reagentsfor carbohydrate visualization. These visualization reactions are more sensitive and specific than those used for cellulose supports. (iii) The zones of separated carbohydrates display a better defined geometry than those on other supports such as chromato graphy paper or glass fiber paper. Thin-layer electrophoresis of carbohydrates may be an alternative to thin-layer and paper chromatography, both still the prevailing methods in the field of qualitative analysis of low molecular weight carbohydrate compounds, despite the recent developments of gas chromatography and high pressure liquid chromatography, which are more useful in the field of quantitative analysis. In some important applications, paper and thin-layer chromatography give unsatisfactory results. The identification of mannose in a mixture with glucose and fructose is difficult due to the small differences in Rfvalues of all common development systems [ 11, 121. By contrast the relative electrophoretic mobility of mannose MG 0.69 differs distinctly from that of glucose and fructose (MG 1.00 and 0.86), enabling a clear identification of this compound even in an excess of both latter substances. A similar case is the identification of lactulose ( M ~ 0 . 1) 6 in the prersence of an excess of lactose (MG0.42), which is important for the assay of heated milk 1131. Another important application is the separation of mixtures of common uronic acids, which may prove difficult and time-consuming by paper or thin-layer chromatography due to the small differences in Rfvalues [ 14, 151. Also, in this area thin-layer electrophoresis enables an excellent separation and even partial identification of these substances in a short time. The individual electrophoretic mobilities of the monosaccharide borate complexes strongly depend om the position of the vicinal hydroxyl groups in the molecules and correspond to those on chromatography and glass fiber paper [ 1,2]. For oligosaccharides the mobilities depend pritriarly on the linkages between the sugar units. The relative electrophoretic mobilities of sugars with 1 + 3 and 1 + 6 linkages are generally higher than those with 1 + 1, 1 -+ 2 and 1 + 4 1ink.ages. This is also in good agreement with results obtained on electrophoresis on chromatography paper and glass fiber palper I 1,21. The great differences in mobilities of uronic acids in presence of barium ions can be attributed to the strength ofthe barium-uronic acid complexes. The mobilities of guluronic acid and galacturonic acid strongly depend on the barium-ion concentration (Table 6), with 0.07 M found to be the optimum for the separation of these compounds. As previously shown for Ca-complexes 161, the position of the OH at the C4 of the uronic acid molecule determines the binding strengths - which are higher for guluronic and galacturonic acid, where the OH-groups are in the cis-position to the carboxylic groups at C6. The low
relative electrophoretic mobilities of both compounds can be explained by a partial neutralization of the negative charge of the carboxylic groups by the complex formation with barium ions. Their strong dependence on the barium ion concentration in the electrolyte supports this assumption. It is interesting to compare the application of thin-layer electrophoresis on a pretreated silanized silicagel to the separation of a low molecular weight carbohydrate with capillary zone electrophoresis I 171, recently applied to the separation of reducing monosaccharides as N-pyridylglycamines in 0.1 M borate buffer at pH 10.5 [ 181. Most of the common monosaccharides and uronic acids can be separated and quantitated in about 30 min, which clearly presents an advantage over the prevailing quantitative methods of sugar analysis, such as gas chromatography and high pressure liquid chromatography. In contrast, thin-layer electrophoresis on a pretreated silanized silica gel completes the field of qualitative analysis, especially for the screening of multiple samples, by allowing fast and reliable qualitative and often also semiquantitative determinations. Spot tests and thin-layer chromatography are the methods of choice here. Both methods have their merits and symbolize progress in the area of sugar analysis. Received July 20, 1989
5 Literatur [11 Weigel, H., Adv. Carbohydr. Chem. 1963,18,61-97. 121 Forster, A. B., Adv. Carbohydr. Chem. 1957,12, 81-1 15. 131 Padmoyo, M., and Miserez, A,, Mitt. Geb. Lebensmittelunters. Hyg. 1967,58,3 1-49. [41 Briggs, D. R., Garner, E. F. and Smith, F., Nature 1956. 154-155. 151 Jarvis, M. C.,Threlfall, D. R.and Friend,J.,Phytochemistry 1977.16, 849-852. 161 Schafer, H. and Scherz, H., 2. Lebensm. Unters. Forsch. 1983. 177, 193- 195. [71 Scherz, H., Z. Lebensm. Unters. Forsch. 1985. 181.40-44. 181 Bonn, G., Grunwald, M., Scherz, H. and Bobleter, O..J. Chromatogr. 1986,370,485-493. [91 Haug, A. andLarsen,B.,Acta Chem. Scand. 1962,16.1908- 19 18. 101 Cifonelli, J. A. and Smith, F., Anal. Chem. 1954,26. 1132-1 134. I 1 1 Churms, S., in: Zweig, G. and Sherma, J. (Eds.). Handbook oJ Chromatography, Vol. I, Carbohydrates CRC-Handbook. Boca Raton, Florida, USA 1982, pp. 137-153. 121 Lewis, B. A. and Smith, F., in: Stahl. E. (Eds.), Diinnschichtchromatographie, 2nd Edition, Springer Verlag. Berlin 1966, pp. 769-798. 131 Adachi, S.. Anal. Biochem. 1965,12, 137-142. 141 Ernst, W., Anal. Chim. Acta 1968,40, 161-166. 151 Fey, R. and Mack, F. J., Lebensmittelchem. Gerichtl. Chem. I98 1.35. 50-5 1. [ 161 Gould, G. 0. and Rankin, A. F., Chem. Commun. 1970,489-490. 1171 Gordon, M. J., Huang, X., Pentoney, S., Jr. and Zare. R.. Science 1988,242,224-230. [I81 Honda, S., Iwase, S., Makino, A. and Fujiwara, S.. Anal. Biochem. 1989,176, 72-77.
Electrophoresis 1990, 1 I , 18-22
H. Scherz
5 References [ 11 Cheung, W. Y., Science 1980,207, 19-27.
[21 Wang,J.H. and Waisman.D. M.,Curr. Top. Cell. Regulat. 1979.15, 47-107. 131 Klee, C . B. and Vanaman, T. C., A&. Prolein Chem. 1982. 35. 213-321. 141 Means, A. R., Tash, J . S. and Chafouleas, Jl. G., Physiol. Rev. 1982. 62, 1-39. (51 Kretsinger, R. H., CRC Crit. Rev. Biochem. 1980,8, 119-174. 161 Satyshur, K. A., Rao, S. T., Pyzalska, D., Ilrendel, W., Greaser. M. and Sundaralingam, M., J . B i d . Chem. 1988,263, 1628-1647. 171 Szebenyi, D. M. E. and Moffat, K., J. B i d . Chem. 1986. 261, 8761-8777. 181 Herzberg, 0. and James, M. N. G., Nature 1986,313,653-659. 191 Babu, S. Y., Bugg, C. E. and Cook, W. J., J . Mol. B i d . 1988.204, 191-204.
Heimo Scherz Deutsche Forschungsanstaltfur Lebensmittelchemie, Garching
1101 Plancke, Y. D. and Lazarides, E., Mol. Cell. Biol. 1983. 3. 14 12- 1420. I I 11 Runte, L., Jurgensmeier, H.-L., Unger, C. and Soling, H. D.. FEBS Lett. 1982,147, 125-130. (121 Jorgenson, J . W. and Lukacs, K. D., Anal. Chem. 1981. 53, 1298-1302. I131 Jorgenson, J. W. and Lukacs, K. D., Science 1983.222,266-272. I141 Terabe, S., Otsuka, K., Ichikawa, K., Tsuchiya, A. and Ando. T.. Anal. Chem. 1984,56, 113-1 16. I151 Jorgenson, J. W., Anal. Chem. 1986,58,743A-760A. I161 Lauer,H. H. andMcManigill,D.,Anal. Chem. 1986,58,166-170. I171 Hjerttn, S., Elenbring. K., Kilar, F., Liao, J., Chen, A. J. C. and Zhu, M., J . Chromatogr. 1987, 403,47-61. I181 Cohen,A.S.andKarger,B.L.,J. Chromatogr. 1987,397,409-417. I191 Grossman, P., Colburn, J. and Lauer, H.,Ana/. Biochem. 1988.173, 265-270. l201 Compton, S. W. and Brownlee, R. G., BioTechniques 1988. 6, 432-440.
Thin-layer electrophoretic separation of monosaccharides, oligosaccharides and related compounds on reverse phase silica gel The electrophoretic mobilities of monosaccharides, oligosaccharides, sugar alcohols and sugar acids were determined in 0.3 M borate buffer, pH 10, using thin-layer electrophoresis on silanized silica gel, pretreated with octanol- 1. A rapid separation of a number of sugars, occurring in foods, could be achieved. Using a 0.05-0.1 M neutral solution of barium acetate as electrolyte, thin-layer electrophoresis allowed excellent and rapid separation as well as identification of all common uronic acids which are constituents of many acidic polysaccharides.
1 Introduction Zone electrophoresis is applied in the field of carbohydrate chemistry as an alternative separation method to paper and thin-layer chromatography. Whereas charged molecules such as sugar acids and sugar phosphates migrate in the electric field without any modification, neutral carbohydrates must be converted into ionic forms. Usually, neutral carbohydrates are transformed into borate complexes which in aqueous solution can easily be generated by addition of borate ions at alkaline pH. Under these conditions borate ions react with vicinal hydroxyl groups with resultant formation of complexes, migrating in the electric field. Since the specific chemical structure of carbohydrates strongly influences the formation of such borate complexes, great variations of their mobilities in the electric field are observed, providing a basis for the electrophoretic separation of carbohydrate mixtures. Similar to many other electrophoretic separations, the electrophoresis of carbohydrates also requires an inert solid support for stabilization. In the past, chromatography paper was widely used as a stabilizing matrix and, especially under conditions of ~
~~
Correspondence: Dr. Heimo Scherz, Deutsche Forschungsanstalt fur Lebensmittelchemie, LichtenbergstraRe 4, D-8046 Garching. Federal Republic of Germany
0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
high voltage electrophoresis, good separations were obtained I1,21. Other supports were cellulose acetate 131and glass fiber paper 141, which had been applied preferably to the separation of polysaccharides. However, the above approaches had the following disadvantages, limiting the applicability of electrophoresis to the separation of carbohydrates: (i) need of expensive equipment with an efficient cooling system in the case of high voltage electrophoresis, (ii) limited choice for visualization, especially revelant to chromatography paper and cellulose acetate, excluding all reactions requiring aggressive reagents, and (iii) adsorption effects and strong electroendoosmosis, especially in the case of glass fiber paper, resulting in strongly distorted patterns. Although silanization of glass fiber paper eliminates these undesirable attributes, the currently available procedures result in mechanically unstable sheets [ 5 , 61. Recently, for the separation of polysaccharide -containing thickening agents, a thin-layer electrophoretic procedure was developed, using as support a silanized silica gel, pretreated with octanol-1 [71. The silanized and pretreated silica gel affords a stable inert system on which adsorption and electroendoosmosis effects are strongly diminished and aggressive carbohydrate reagents can be applied for visualization. Good 01 73-0835/90/010 1-0018 $02.50/0
Electrophoresis 1990. I I , 18-22
separation of polysaccharides was obtained 171 and also the successful separation of some low molecular degradation products of the hydrothermolysis of cellulose biomass, e.g. dihydroxyacetone, glyceraldehyde, glucose and aromatic carboxylic acids, has been described [81. In this report electrophoresis on silanized and octanol-1 pretreated silica gel is applied to determine the electrophoretic mobilities of monosaccharides, oligosaccharides, sugar alcohols, sugar acids and sugar phosphates.
2 Materials and methods 2.1 Materials
Thin-layer electrophoresis of monosaccharides and oligosaccharides
19
neutral detergent (Tween 20, Serva). The detergent was added to prevent the formation of a thin film of octanol- 1 on the water surface which would reduce water evaporation and accelerate the desiccation of the plates. A few crystals ofthymol were added to prevent microbial contamination. The side walls of the desiccator were taped with wet filter paper, thus establishing a saturated moist atmosphere with resultant reduced water evaporation from the surface of the plates. After incubation for 3 h the plates could be used for electrophoresis. In most experiments freshly prepared plates were used. Storage under the described conditions should not exceed 48 h.
2.3 Electrophoresis
The following materials were obtained from the indicated sources: silanized silica gel, 60 H, for thin-layer chromatography (Merck, Darmstadt, FRG), octanol- 1 (analytical grade, Merck), glass fiber paper (Whatman GC/F), polyvinylpyrrolidone K-90 ( M , 350 000, Fluka, Buchs, Switzerland), 1,3-dihydroxynaphthalene(naphthoresorcinol; Serva, Heidelberg, FRG), omega-hydroxymethylfurfural (Merck). The following compounds were used as reference substances. Monosaccharides: L-arabinose, D-ribose, D-xylose, D-galactose, D-mannose, D-fructose, L-sorbose were obtained from Merck, L-rhamnose, D-fucose from Serva, and D-lyxose from Sigma (Munchen, FRG). Oligosaccharides: sucrose, lactose, maltose, trehalose, raffinose, melibiose were obtained from Merck, sophorose, gentiobiose, isomaltose, cellobiose from Serva, lactulose, melezitose, palatinose turanose, stachyose, maltotriose from Sigma. Sugar alcohols: xylitol, ribitol, mannitol, sorbitol, erythrol, meso-inositol were from Merck, arabitol from Serva, dulcitol, threitol from Sigma. Sugar acids: galacturonic acid was obtained from Merck, gluconic acid, arabonic acid K salt, ribonic acid, L-y-lactone from Serva, galactonic acid- 1,4-lactone,mannuronic acid-3,6-lactone, glucuronic acid Na-salt from Sigma. Polysaccharides: Naalginate and gum arabic were from Roeper (Hamburg, FRG). All samples were dissolved in the respective buffer. In the case of barium acetate the sugar acid lactones were hydrolyzed prior to separation with a few drops of 0.1 M Ba(OH),. Polysaccharides were hydrolyzed with sulfuric acid, followed by neutralization and removal of sulfate with 0.1 M Ba(OH), 191.
The 0.3 M borate buffer, pH 10,wasprepared according to 171. Crystallized boric acid 18.54 g and Titriplex I11 7.44 g were dissolved in approximately 400-600 m L distilled water, followed by addition of an aqueous solution of 2 M NaOH until a p H of 10 was reached. The solution was then made up to 1000 m L with distilled water. Barium acetate: Equivalent amounts of barium acetate were dissolved in distilled water and made up to 1000 mL (the respective amounts were 12.77 g for 0.05 M, 17.88 g for 0.07 M and 25.54 g for 0.1 M solutions). Electrophoresis was carried out in the Desaga Double Chamber according to [7]. Both electrode vessels were filled with buffer and the silica gel plates on the cooling block were connected with the buffer vessel with strips of glass fiber paper pressed onto both edges of the silica gel plates with 200 x 20 x 1 mm glass plates. On top of these glass plates, second glass plates (200 x 20 x 4 mm) and on these a standard glass plate (200 x 200 x 4 mm) were placed to reduce the evaporation of water during electrophoresis, which might result in irregularities of the electric field. After equilibration for 30 rnin at 350-400 V, 10-15 mmlonggrooves weremadeinthelayerwiththetipofa needle, at a distance of 30 mm from the cathode, and 2-5 pLof the sample, dissolved in the running buffer, were deposited into the grooves with a Hamilton syringe. After replacing the cover plate, electrophoresis was carried out for 60-120 rnin at 350-400 V, with the temperature of the cooling block set at 16- I 8 “C. Electroendoosmosis was determined in each run using omega-hydroxymethylfurfural as uncharged marker.
2.2 Preparation of thin-layer plates
2.4 Visualization
Thin-layer plates were prepared as previously reported [7,81, with some modifications. The improved procedure is briefly described. A slurry of 15 g silanized silica gel in 50 mL dichloromethane or methyl acetate, containing 6 g* octanol- 1 was stirred for 40 rnin using a magnetic stirrer. The solvent was evaporated at 50 “C, using a Rotavapor (Buechi, Flavil, Switzerland), until a lump-free powder was obtained. T o the dry powder in a mortar (diameter: 160 mm) 10 mL of a 2 % solution of polyvinylpyrrolidone in buffer, followed by 35 mL buffer, were added stepwise with intense stirring with a pestle. After stirring for about 10 min, the slurry was spread on 200 x 200 mm glass plates using the Desaga equipment for thinlayer chromatography (Desaga, Heidelberg, FRG). Immediately after coating, the plates were transferred to a desiccator containing, on the bottom, distilled water with a few drops of a
Following electrophoresis the plates were dried at 90 “C for 30 min. Visualization was carried out with the following reagents: Naphthoresorcinol-sulfuric acid prepared according to I 7 I : 0.4 g naphthoresorcinol were dissolved in 50 mLethano1, containing 2.5 mL concentrated sulfuric acid and 1.5 mL octanol1. The reagent must be freshly prepared. The dry, octanol-free plates were sprayed with this reagent and heated at 1 10 “C for 10- 15 min. Monoaldoses, their phosphates and uronic acids appear as deep blue spots, ketoses and their phosphates as red ones. The spots of oligosaccharides are different according to their compositions. The periodic acid-benzidine reagent for sugars and sugar alcohols was prepared according to i101.Solution “a” contained 0.25 g sodium periodate, dissolved in 50 m L distilled water, and solution “b” contained 1.8 g benzidine in 50 mL ethanol, supplemented with 20 mL acetone and 10 mL 0.2 M HCl. The dry plates were sprayed with solution “a” and after 5 rnin with solution “b”. The visualized components appear as light spots on a deep brown background.
*
Valid for new batches of silanized silica gel due to their higher grade of silanization; old batches need only 4 g 171.
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Electrophoresis 199O,Il, 18-22
H. Scherz
3 Results An electropherogram of a mixture ofselected mono- and oligosaccharides is shown in Fig. 1. Most of the carbohydrates, namely glucose, fructose, maltose, lactose, sucrose and raffinose are typical elementsin food, whereas mannose and rhamnose are widespread components of some natural polysaccharides. All these compounds can be well separated in a single run of 120 min. The relative electrophoretic mobilities of monosaccharides, oligosaccharides, sugar alcohols, sugar acids and sugar phosphates were determined in 0.3 M borate buffer, p H 10, using D-ghCOSe as reference substance (Tables 1,2 and 3). The electrophoretic mobilities; of mono- and oligosaccharides are similar to those obtained in high voltage electrophoresis on chromatography paper or glass fiber paper [ 1, 21 (Table 4). The mobilities of sugar acids and sugar phosphates in the same buffer are higher than those of the neutral compounds (Table 3). Hexosephosphates can easily be separated from the neutral compounds as well as from each other. IJsing a neutral medium, this electrophoresis system enables rapid separation ofthe acid compounds without interference from the neutral sugars
Table 1. Relative mobilities (MG)of monosaccharides and sugar alcohols in 0.3 M borate buffer, pH loo) Monosaccharides
MG
Sugar alcohols
D-Glucose D-Mannose D-Galactose D-Fructose L-Sorbose L- Arabinose
1 .oo
0.69 0.92 0.86 0.95
Manriitol Sorbitol Dulcitol Xylitisl Adonitol Arabitol Threitol Erythrol meso-Inositol
D-XylOSe
D-Ribose L-Rhamnose D-Fucose D-LyXOSe
1.oo
1.01 0.70 0.53 0.95 0.67
a) Reference substance: D-ghCOSe.
and sugar alcohols which do not migrate under these conditions. In pure aqueous solutions of barium acetate, an excellent separation of all uronic acids, occurring as monomers in acidic polysaccharides, can be achieved. Separation of sugar phosphates is also possible in the medium (Table 5 ) . The relative electrophoretic mobilities of uronic acids, with D-glucuronic acid as reference substance, were determined at three different concentrations of the electrolyte (Table 6). The mobilities of galacturonic acid and guluronic acid strongly depended on the electrolyte concentration.
Table 2. Relative mobilities (MGofoligosaccharidesin0.3 M borate buffer, pH 10a) Oligosaccharides
*G
1 2 4-Disaccharides Maltose Lactose Cellobiose Lactulose
0.39 0.42 0.32 0.6 1
I 2 6-Disaccharides Isomaltose Gentiobiose Melibiose Palatinose
0.62 0.73 0.79 0.48
1 2 3-Disaccharides Turanose
0.64
1 2 2-Disaccharides
0.90 0.80 0.94 0.79 0.87 0.75 0.87 0.68 0.48
Sophorose Sucrose
0.35 0.20
1 2 I-Disaccharides Trehalose
0.22
Trisaccharides R affinose Melezitose Maltotriose
0.29 0.28 0.26
Tetrasaccharose Stachyose
0.37
a) Reference substance: D-glucose.
origin -
Figure I . Thin-layer electrophoresis of mono- and oligosaccharides on silanized silica gel pretreated with octanol-1, in 0.3 M borate buffer, pH 10. Electrophoresis:400V, 120min, 18 OC.Lane(1) sucrose, (2) rafinose, (3) maltose, (4)lactose, (5) rhamnose, ( 6 ) mannose, (7)fructose, (8) glucose, (M) mixture of all eight compounds. Sample loading: 10 bg of each compound. Visualization with naphthoresorcinol-sulfuric acid. Origin indicated.
21
Thin-layer electrophoresis of monosaccharides and oligosaccharides
ElfctrophorePis 1990, 11, 18-22
Table 3. Relative mobilities (MG) of sugar acids and sugar phosphates in 0.3 M borate buffer, pH 10”)
Table 5. Relative mobilities of sugar phosphates ( M t i ~in) aqueous barium acetate solution, 0.05 M ~ )
Sugar acids
Mti
Sugar phosphate
MtiA
Glucuronic acid Galacturonic acid Mannuronic acid Gluconic acid Galactonic acid Ribonic acid Arabonic acid
1.39 1.28 1.21 1.21 1.23 1.17 1.30
Glucose- 1 -phosphate Glucose-6-phosphate Fructose-6-phosphate Fructose-l,6-diphosphate
0.77 0.86 0.8 1 1.38
Sugar phosphates
Mti
Glucose-6-phosphate Glucose- 1 -phosphate Fructose- 1-phosphate Fructose- l,6-diphosphate
1.30 1.06 1.21 1.44
a) Reference substance: D-glucuronic acid.
Table 6. Relative mobilities (MGA)of uronic acids in pure aqueous barium acetate solutions of different concentrationsa) MGA
a) Reference substance: D-glucose.
Table 4. Comparison of MG-values of some important sugars and sugar alcohols on silanized silica gel-octanol-1 plates with those on chromatography paper and glass fiber paper, in 0.3 M borate buffer, pH loa) Compound
L-Arabinose D-Xylose D-Fucose L-Rhamnose D-Galactose D-Glucose D-Mannose D-Fructose Cellobiose Maltose Gentobiose Saccharose Rafinose Sorbitol Mannitol
Silanized silica gel pretreated with octanol-1
Chromatography Paper
Glass fiber Paper
1.oo 1.01
0.96 1.oo 0.89 0.53 0.93 1.oo 0.72 0.90 0.29 0.34 0.72 0.18 0.28 0.89 0.90
0.94 1 .oo 0.88 0.49 0.9 1 1.00 0.67 0.88 0.26 0.30 0.69 0.15 0.23 0.82 0.95
0.95 0.53 0.92
1 .oo
0.69 0.86 0.32 0.39 0.73 0.20 0.29 0.80 0.90
a) Reference substance: D-glucose.
Glucuronic acid Mannuronic acid Galacturonic acid Guluronic acid
0.05 M
0.07 M
0.1 M
1.oo 0.80 0.42 0.22
1.oo 0.74 0.26 0.06
I .oo 0.68 0.14 -
a) Reference substance: D-glucuronic acid.
The usefulness of this electrophoresis system is demonstrated for a mixture of galacturonic acid, glucuronic acid, mannuronic acid and guluronic acid, along with their individual compounds, and for a mixture of the hydrolysate of sodium alginate and gum arabic as a practical example for the identification of uronic acids in polysaccharides (Fig. 2). In the case ofgum arabic the acid and neutral compounds can be well resolved. The latter are shifted toward the cathode, presumably as a result of a weak interaction between the carbohydrates and the barium ions.
4 Discussion Thin-layer electrophoresis, on a silanized and octanol- I pretreated silica gel, of low molecular weight carbohydrates and some related compounds, affords the following advantages over the preceding procedures: (i) The separation can be car-
Figure 2. Thin-layer electrophoresis of uronic acids on silanized silica gel, pretreated with octanol-1, in 0.07 M barium acetate solution. Electrophoresis: 400 V, 150 min, 16 “C. Lane ( I ) galacturonic acid, (2) mannuronic acid, (3) glucuronic acid, (4) hydrolysate of gum arabic (glucuronic acid), (5) hydrolysate of sodium alginate (mannuronic and guluronic acid), (M) mixture of (1-3) and guluronic acid, (H) omega-hydroxymethylfurfural. Sample loading: 10 kg of each individual compound. Visualization with naphthoresorcinol-sufuric acid. Origin indicated.
22
Electrophoresis 1990,l I , 18-22
H. Scherz
ried out at 350-400 V, instead of the 2000-5000 V necessary in high voltage electrophoresis on chromatography paper, thus obviating the need for expensive equipment and a cooling system. (ii) Silica gel as inert support material permits the application of sulfuric acid containing reagentsfor carbohydrate visualization. These visualization reactions are more sensitive and specific than those used for cellulose supports. (iii) The zones of separated carbohydrates display a better defined geometry than those on other supports such as chromato graphy paper or glass fiber paper. Thin-layer electrophoresis of carbohydrates may be an alternative to thin-layer and paper chromatography, both still the prevailing methods in the field of qualitative analysis of low molecular weight carbohydrate compounds, despite the recent developments of gas chromatography and high pressure liquid chromatography, which are more useful in the field of quantitative analysis. In some important applications, paper and thin-layer chromatography give unsatisfactory results. The identification of mannose in a mixture with glucose and fructose is difficult due to the small differences in Rfvalues of all common development systems [ 11, 121. By contrast the relative electrophoretic mobility of mannose MG 0.69 differs distinctly from that of glucose and fructose (MG 1.00 and 0.86), enabling a clear identification of this compound even in an excess of both latter substances. A similar case is the identification of lactulose ( M ~ 0 . 1) 6 in the prersence of an excess of lactose (MG0.42), which is important for the assay of heated milk 1131. Another important application is the separation of mixtures of common uronic acids, which may prove difficult and time-consuming by paper or thin-layer chromatography due to the small differences in Rfvalues [ 14, 151. Also, in this area thin-layer electrophoresis enables an excellent separation and even partial identification of these substances in a short time. The individual electrophoretic mobilities of the monosaccharide borate complexes strongly depend om the position of the vicinal hydroxyl groups in the molecules and correspond to those on chromatography and glass fiber paper [ 1,2]. For oligosaccharides the mobilities depend pritriarly on the linkages between the sugar units. The relative electrophoretic mobilities of sugars with 1 + 3 and 1 + 6 linkages are generally higher than those with 1 + 1, 1 -+ 2 and 1 + 4 1ink.ages. This is also in good agreement with results obtained on electrophoresis on chromatography paper and glass fiber palper I 1,21. The great differences in mobilities of uronic acids in presence of barium ions can be attributed to the strength ofthe barium-uronic acid complexes. The mobilities of guluronic acid and galacturonic acid strongly depend on the barium-ion concentration (Table 6), with 0.07 M found to be the optimum for the separation of these compounds. As previously shown for Ca-complexes 161, the position of the OH at the C4 of the uronic acid molecule determines the binding strengths - which are higher for guluronic and galacturonic acid, where the OH-groups are in the cis-position to the carboxylic groups at C6. The low
relative electrophoretic mobilities of both compounds can be explained by a partial neutralization of the negative charge of the carboxylic groups by the complex formation with barium ions. Their strong dependence on the barium ion concentration in the electrolyte supports this assumption. It is interesting to compare the application of thin-layer electrophoresis on a pretreated silanized silicagel to the separation of a low molecular weight carbohydrate with capillary zone electrophoresis I 171, recently applied to the separation of reducing monosaccharides as N-pyridylglycamines in 0.1 M borate buffer at pH 10.5 [ 181. Most of the common monosaccharides and uronic acids can be separated and quantitated in about 30 min, which clearly presents an advantage over the prevailing quantitative methods of sugar analysis, such as gas chromatography and high pressure liquid chromatography. In contrast, thin-layer electrophoresis on a pretreated silanized silica gel completes the field of qualitative analysis, especially for the screening of multiple samples, by allowing fast and reliable qualitative and often also semiquantitative determinations. Spot tests and thin-layer chromatography are the methods of choice here. Both methods have their merits and symbolize progress in the area of sugar analysis. Received July 20, 1989
5 Literatur [11 Weigel, H., Adv. Carbohydr. Chem. 1963,18,61-97. 121 Forster, A. B., Adv. Carbohydr. Chem. 1957,12, 81-1 15. 131 Padmoyo, M., and Miserez, A,, Mitt. Geb. Lebensmittelunters. Hyg. 1967,58,3 1-49. [41 Briggs, D. R., Garner, E. F. and Smith, F., Nature 1956. 154-155. 151 Jarvis, M. C.,Threlfall, D. R.and Friend,J.,Phytochemistry 1977.16, 849-852. 161 Schafer, H. and Scherz, H., 2. Lebensm. Unters. Forsch. 1983. 177, 193- 195. [71 Scherz, H., Z. Lebensm. Unters. Forsch. 1985. 181.40-44. 181 Bonn, G., Grunwald, M., Scherz, H. and Bobleter, O..J. Chromatogr. 1986,370,485-493. [91 Haug, A. andLarsen,B.,Acta Chem. Scand. 1962,16.1908- 19 18. 101 Cifonelli, J. A. and Smith, F., Anal. Chem. 1954,26. 1132-1 134. I 1 1 Churms, S., in: Zweig, G. and Sherma, J. (Eds.). Handbook oJ Chromatography, Vol. I, Carbohydrates CRC-Handbook. Boca Raton, Florida, USA 1982, pp. 137-153. 121 Lewis, B. A. and Smith, F., in: Stahl. E. (Eds.), Diinnschichtchromatographie, 2nd Edition, Springer Verlag. Berlin 1966, pp. 769-798. 131 Adachi, S.. Anal. Biochem. 1965,12, 137-142. 141 Ernst, W., Anal. Chim. Acta 1968,40, 161-166. 151 Fey, R. and Mack, F. J., Lebensmittelchem. Gerichtl. Chem. I98 1.35. 50-5 1. [ 161 Gould, G. 0. and Rankin, A. F., Chem. Commun. 1970,489-490. 1171 Gordon, M. J., Huang, X., Pentoney, S., Jr. and Zare. R.. Science 1988,242,224-230. [I81 Honda, S., Iwase, S., Makino, A. and Fujiwara, S.. Anal. Biochem. 1989,176, 72-77.
