Determining Sugar Composition of Food Gum Polysaccharides by HPTLC 2001, 53, 579-581 L. W. Doner U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, 600 E. Mermaid Lane, Wyndmoor, PA 19038, USA Key Wards Thin-layer chromatography of sugars Si 50000 plates Polysaccharides Food gums Summary Most commercially important food gums, including gum arabic, guar gum, and carrageenan are polysaccharides which consist of multiple sugars, including uronic acids. For the first time, the sugars in these and other gums were determined by comparison of R F values with stan- dards by HPTLC on Si 50000 plates after acid hydrolysis of the polysaccharides. The solvent system consisted of n propanol:water:triethylamine:30% NH3 (80:20:0.2:4). Analysis was rapid, as the separations were accomplished using a single plate development and the sugars were located by simple charring. Sugar separations on Si 50000 with this solvent system are more efficient than others and provide the additional advantage over bonded-phase silicas in that sugars can be detected using aggressive methods. Introduction A novel synthetic porous silica, Si 50000, has proven very useful for separating po- lar analytes such as amino acids [1] and sugars [1 3] by HPTLC. Si 50000 has an extremely large pore diameter (5000 nm) and a low surface area of about 0.5 m2/g [1]. Other HPTLC quality silica gels have a pore diameter of about 6 nm and a sur- face area of about 500 m2/g [4]. The low surface activity [1] of Si 50000 results in minimal spot tailing of polar compounds such as carbohydrates. Optimal HPTLC resolution of mono- saccharides on Si 50000 was obtained using a single development with a solvent sys- tem consisting of n-propanol/water/25% NH3 [1]. When other silica gels were used [5 8] as stationary phases, multiple plate developments were required in order to achieve comparable resolution of sugars. Impressive separations of branched cyclo- dextrins [9] and of homologous glucans up to DP 30 [10] have also been achieved. These separations [9, 10] used multiple plate developments with n-butanol/pyri- dine/water mixtures. A rapid procedure was required to moni- tor sugar composition of potentially useful polysaccharides isolated from underutilized and abundant agricultural processing wastes. Corn fiber gum hemicellulose [11, 12] for example, contains D-xylose, Lara- binose, D,L-galactose, and D-galacturo- nic acid. The presence of glucose in corn fiber gum hydrolyzates would indicate contamination with starch. Also, a proce- dure to monitor sugar composition of polysaccharides isolated by various meth- ods from flaxseed meal was required. In the present study I applied separations on Si 50000 plates to monitor these processes, and also to conveniently assay the sugar composition of several important indus- trial polysaccharides. Experimental Materials Si 50000 HPTLC plates, 10 H 20 cm (E. Merck, Darmstadt, Germany, cat. no. 15135) without fluorescent indicator, were purchased from EM Science (Gibbstown, NJ, USA). The plates were cut to appro- priate widths as needed. Sugars, the poly- saccharides pectin, gum arabic, xanthan gum, gellan gum, guar gum and carragee- nan, and triethylamine and N-(1- naphthyl)ethylenediamine dihydrochlor- ide were purchased from Sigma (St. Louis, MO, USA). Corn fiber gum [12] and flax- seed mucilage [13] were prepared as de- scribed earlier. Short Communication Chromatographia 2001, 53, May (No. 9/10) 0009-5893/00/02 579- 03 $ 03.00/0 (cid:14)9 2001 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH 579
`
`MYLAN Ex 1051, Page 1
`
`

`

`the sugar-containing filtrate was dried un- der a stream of nitrogen. The syrups were dissolved in water (1 mL), and the solu- tion was filtered through a 0.2 ix Anotop 10 plus membrane filter (Whatman, Maidstone, England, cat. no. 6809 3022). Corn fiber gum, flaxseed mucilage, guar gum and carrageenan (10 mg) were hydro- lyzed with N H2SO 4 (1 mL) at 100 ~ for 1.5 hr. After cooling, solutions were neu- tralized by addition of BaCO3 and then treated as above. Figure 1. A) HPTLC chromatogram of sugars in food gum polysaccharide hydrolyzates using single development with n-propanol:water:triethylamine:30% NH3 (80:20:0.2:4). 1 = guar gum; 2 = xanthan gum; 3 = gum arabic; 4 = gellan gum; 5 = standard mixture of rhamnose, fucose, xylose, arabinose, glucose, galactose, glucuronic acid, and galacturonic acid (listed in order of decreasing RF value); 6 = corn fiber gum; 7 = flax mucilage; 8 = pectin; 9 = carrageenen. B) HPTLC chromato- gram of standard di- and tri- and tetrasaccharides using two developments with n-propanol:water:- triethylamine:30% NH 3 (85:15:0.2:4). 1 = mixture of maltose and maltotriose; 2 = sucrose; 3 = tre- halose; 4 = raffinose; 5 = stachyose. HPTLC Separations of Sugars Polysaccharide Hydrolysis For HPTLC, solutions of sugars and polysaccharide hydrolyzates were pre- pared at levels of about 10mgmL 1. About 0.5IXL were spotted, and plates were typically developed to about 8 cm, which required about 60 min. The solvent system consisted of n-propanol:water:- triethylamine: 30% NH3 (80:20:0.2:4) un- less otherwise specified. Sugars were de- tected after spraying with a solution of 5% H2SO 4 and 6.5 mM N 1 (1 naphthyl)ethyle - nediamine dihydrochloride in methanol and heating on a hot plate or placing in a 100 ~ oven [14]. Pectin, gum arabic, xanthan gum, and gel- lan required relatively strong acid condi- tions for hydrolysis to constituent sugars. These polysaccharides (10 mg) were weighed into screw-cap vials and mixed with 150 IXL 12N H2SO4, vortexing peri- odically over 45 min at room temperature. The acid was then diluted to 1N H2SO4 by adding water (1.65mL) and the mixture heated in an oven at 100 ~ for 1.5hr. After cooling to room temperature, BaCO3 was gradually added until solution pH was neutral by pH paper. The BaSO4 was removed by vacuum filtration, and Results and Discussion The sugars most commonly found in food gum polysaccharides are L-arabinose, D- xylose, D-glucose, D- and L-galactose, D- galacturonic acid, D-glucuronic acid, L- rhamnose, and L-fucose. Conditions for their separation on Si 50000 HPTLC plates were optimized, using a solvent sys- tem similar to that used earlier [1], consist- ing of n-propanol/water/25% NH3. It was observed that the addition of a small quantity of triethylamine to the mixture resulted in chromatographic mobility and highly efficient resolution of the two uro- nic acids. Many combinations were tested and optimal results for the separation of the eight sugars was achieved using n-pro- panol:water:triethylamine:30% NH3 (85: 15:0.2:4) as solvent system. Their separa- tion using a single development is shown in Figure 1A, lane 5, and the Rf values of these and other mono-, di-, tri-, and tetra- Table I. RF values of standard sugars on Si 50000 HPTLC plates using single elution with n-propanol: water: triethylamine : 30% NH3 (80: 20:0.2 : 4). Sugar Rv Sugar RF Sugar RF Sugar Rv Arabinose .497 Gulose .471 Sorbose .491 Sucrose .474 Ribose .566 Mannose .490 Psicose .531 Laminaribiose .484 Xylose .591 Fucose .686 Isomaltose .237 Turanose .486 Lyxose .604 Rhamnose .784 Gentiobiose .259 Lactitol .270 2-deoxy-Ribose .850 2-deoxy-Glucose .800 Lactose .267 Raffinose .225 Galactose .361 Galacturonic Acid .111 Cellobiose .367 Melezitose .277 Glucose .435 Glucuronic Acid .200 Maltose .387 Maltotriose .344 Talose .460 Fructose .467 Trehalose .391 Stachyose .082 Table II. Proportions of sugars in food gums, calculated from previously published structural information on corn fiber gum [12] and other gums [15]. Ar = L-arabinose; Xy = D-xylose; Ga = D or D,L-galactose; G1 = D-glucose; GaA = D-galacturonic acid; G1A = D-glucuronic acid; Ma = D-mannose; Rh = L-rhamnose; Fu = L-fucose; 3,6-anhydro-D-Ga = 3,6-anhydro-D-Galactose. Ar Xy Ga G1 GaA G1A Ma Rh Fu 3,6-anhydro- D-Ga Guar gum 1 Xanthan gum Gum arabic 2 4 Gellan gum Corn fiber gum 7 10 1 Flaxseed mucilage 3 6 4 Pectin Carrageenan 1 2 2 1 2 1 1 2' 1 1 1 2 5 20 1 580 Chromatographia 2001, 53, May (No. 9/10) Short Communication
`
`MYLAN Ex 1051, Page 2
`
`

`

`saccharides are listed in Table I. Figure 1B is a chromatogram indicating possible se- parations among the disaccharides mal- tose, trehalose, and lactose, the trisacchar- ides maltotriose and raffinose and the tet- rasaccharide stachyose. These higher sac- charides were effectively mobilized by using two developments with the solvent system n-propanol:water:triethylamine: 30% NH3 (85:15:0.2:4). The sugar composition of corn fiber gum [12] and the other food gum polysac- charides [15] that were evaluated are listed in Table II. All the expected sugars were resolved and all but one were detected, as shown in Figure 1A. In corn fiber gum, the relatively low level of glucuronic acid was not detected (Figure 1A, lane 6). Flax mucilage (Figure 1A, lane 7) contained all but D-glucuronic acid of the eight sugars in the standard mixture (Figure 1A, lane 5). We found that uronic acids undetected by simple charring were revealed when plates were heated at 100 ~ after spraying with 6.5mM N(lnaphthyl)ethylenedia- mine dihydrochloride in methanol con- taining 5% H2SO4 [14]. Similarly, L-rham- nose was then detected in pectin, along with traces of xylose, arabinose, glucose, and galactose. Use of this spray reagent also allows for sensitive quantitation of sugars by densitometry [14]. In the flax mucilage (Figure 1, lane 7) hydrolyzate, a component with a mobility less than ga- lactose was detected, whose Rf did not correspond to any of the monosacchar- ides tested (Table I). Work is underway to characterized this material. In conclusion, we have modified a mo- bile phase described earlier [1] for separat- ing sugars by HPTLC on Si 50000 plates by addition of triethylamine. This allows efficient resolution of the sugars, includ- ing uronic acids, which constitute the im- portant food gum polysaccharides. The use of our solvent system and Si 50000 plates is the most effective means by which to separate sugars by HPTLC. References [1] Hauck, H.E.; Halpaap, H. Chromatogra- phia 1980,13, 538. [2] Patzsch, K.; Netz, S.; Funk, W. J. Planar Chromatogr. 1988, 1, 39. [3] Doner, L.W. In Metho&' in Enzymology: Wood, W.A.; Kellogg, S.T., Eds, Aca- demic Press, New York, vol 160, 1988, p 176. [4] Hahn-Deinstroop, E. J. Planar Chroma- togr. 1992, 5, 57. [5] Batisse, C.; Daurade, M.-H.; Bounias, M. J. Planar Chromatogr. 1992, 5, 131. [6] Mantovani, G.; Vaccari, G.; Dosi, E.; Lodi, G. Carbohydr. Polym. 1998, 37, 263. [7] Han, N.S; Robyt, J.F. Carbohydr. Res. 1998, 313, 135. [8] Simonovska, B. J. Assoc. Ojf Anal. Chem. 2000, 83,675. [9] Koizumi, K; Utamura, T; Kuroyanagi, T.; Hizukuri, S.; Abe, J.-I. J. Chromatogr. 1986, 360, 397. [10] Koizumi, K; Utamura, T; Okada, Y. J. Chromatogr. 1985, 321,145. [11] Doner, L.W.; Hicks, K.B. Cereal Chem. 1997, 74, 176. [12] Doner, L.W.; Chau, H.K.; Fishman, M. L.; Hicks, K. B. Cereal Chem. 1998, 75, 408. [13] Anderson, E.; Lowe, H. J. Biol. Chem. 1947, 168, 289. [14] Bounias, M. Anal. Biochem. 1980, 106, 291. [15] BeMiller, J.N.; Whistler, R.L.: Barkalow, D.G. In industrial Gums: Whistler, R.L.; BeMiller, J.N. Eds, Academic Press, New York, 1988, p 227. Received: Jan 3, 2001 Accepted: Feb 13, 2001 Short Communication Chromatographia 2001, 53, May (No. 9/10) 581
`
`MYLAN Ex 1051, Page 3
`
`