Green analytical method for identification of amino acids, vitamins and sugars with preliminary separation on HPTLC plates

Indian Journal of Chemical Technology Vol. 20, May 2013, pp. 180-184 Green analytical method for identification of amino acids, vitamins and sugars with preliminary separation on HPTLC plates Ali Mohammad 1, *, Asma Siddiq 1, Abdul Moheman 1 & Gaber E El-Desoky 2 1 Department of Applied Chemistry, Faculty of Engineering & Technology, Aligarh Muslim University, Aligarh 202 002, India 2 Department of Chemistry, College of Science, King Saud University, Riyadh, Saudi Arabia Received 19 July 2012; accepted 23 October 2012 High performance thin layer chromatography (HPTLC) has been used as a green analytical method for the identification and separation of amino acids, vitamins and sugars using silica static flat bed in contact of n-butyl alcohol, ethylene glycol or ethyl acetate and their mixtures. From the point of view of chromatographic performance, a mixture of n-butyl alcohol 70% aqueous ethylene glycol ethyl acetate in the ratio of 5:2:3 by volume proves to be more efficient than the individual components for providing useful separations of amino acids, vitamins and sugars from their multi-component mixtures. The effect of presence of inorganic ions (cations and anions) as impurities in the sample has been examined for the separation of amino acids, vitamins, and sugars. The limits of detection for different analytes are found to be 7.5 µg spot -1 for proline, thiamine, ascorbic acid, mannose dextrose and fructose; 1.5 µg spot -1 for pyridoxine and xylose; 0.75 µg spot -1 for leucine, methionine, histidine, maltose and galactose; and 0.15 µg spot -1 for glutamic acid. The worked out HPTLC procedure has been tested for its applicability in the identification and separation of amino acids and vitamins present in the commercially available drug and beverage samples respectively. The proposed method is simple, rapid and free from the use of volatile organic solvents and is therefore environmentally acceptable. Keywords: Amino acid, Green eluent, High performance thin layer chromatography, Sugar, Vitamin The term green analytical chemistry (GAC) was first introduced by Namieśnikis 1 with the main objective of developing green analytical technologies by modifying the existing methods in order to reduce the use of hazardous chemicals. High performance thin layer chromatography (HPTLC) is widely used for the identification and separation of organic compounds including amino acids, vitamins and sugars in a variety of matrices 2-11. Normal phase thin layer chromatography (NP-TLC) with silica gel as stationary phase has been most favourable analytical tool in separation science 11-15. Compared to TLC plates, HPTLC plates have thinner layers containing finer particles with a smaller size distribution, providing shorter migration distances, faster separations and lower reagent and solvent consumption 16. The modern high performance thin layer chromatography (HPTLC) with densitometry scanning makes this technique highly reliable for the analysis of a variety of product 7,15. A number of TLC systems effective for separating biomolecules (amino acids, vitamins and sugars) have *Corresponding author. E-mail:alimohammad08@gmail.com; mohammadali4u@rediffmail.com been reported in literature where the solvents used were mostly organic, aqueous-organic, mixed aqueous-organic in nature 7-10,15. Due to the volatile nature of some of these organic solvents they are capable to contribute to the formation of smog in the environment through free radical air oxidation processes. Also they are often highly flammable and can cause a number of adverse health effects including eye irritation, headaches and allergic skin reactions and are carcinogenic. Thus, there is an urgent need to use alternative solvents to keep the environment pollution free. These solvents are known as green solvents. These are environmentally benign as compared to petrochemical solvents 17. The low solubility of organic molecules in water has been the main reason for a restricted use of water as a green solvent in chemical analysis. On the other hand, aqueous ethylene glycol has been a better alternative for use as green solvent due to its miscibility in water in all proportions 18-19. The aim of this work is to identify a versatile green solvent for on-plate identification of biomolecules belonging to different classes of organic compounds with preliminary separation by TLC. This study will provide an eco- friendly chromatographic system

MOHAMMAD et al.: GREEN ANALYTICAL METHOD FOR IDENTIFICATION OF AMINO ACIDS, VITAMINS & SUGARS 181 to replace existing analytical methods involving the use of toxic organic solvents (viz, dichloromethane, pyridine, chloroform, cyclohexane, hexane, ether, benzene, etc.) 20-25. Experimental Procedure Chemicals and reagents Silica gel 60 HPTLC plates (Merck Germany, #1.05721.0001); n-butyl alcohol, ethanol, ethylene glycol, ethyl acetate, iodine and resorcinol (Merck India); ninhydrin, amino acids, vitamins and sugars (CDH India) were used. Test solutions Test solutions (1.0%, w/v) of amino acids, vitamins and sugars were prepared in demineralized double distilled water. In case of pyridoxine and riboflavin few drops of NaOH were added to make clear solution. Detection All the amino acids were detected by spraying ninhydrin (0.3%) solution in ethanol. Purple spots appeared on heating the HPTLC plates at 60 C for few min. All vitamins were visualized as yellow spots by keeping the plate in chamber containing iodine vapors. Sugars were detected by orcinol solution prepared by mixing 250 mg orcinol, 7.5 ml concentrated sulfuric acid, 200 ml ethanol and 50 ml demineralized double distilled water. Color was developed by spraying this solution and heating HPTLC plates at 110 C for 5-10 min in an electrically controlled oven. The detected spots appeared as brown-purple for all sugars except fructose and xylose which appeared as orange and blue spots respectively. Stationary and mobile phases Silica gel G 60 HPTLC plates were used as stationary phase in whole experiment. The solvent systems used as mobile phase were n-butyl alcohol (M 1 ), ethylene glycol (M 2 ), ethyl acetate (M 3 ), n-butyl alcohol 70% aqueous ethylene glycol ethyl acetate (5:3:2) (M 4 ), n-butyl alcohol 70% aqueous ethylene glycol ethyl acetate (5:2:3) (M 5 ). Procedure Precoated silica plates were activated at 50ºC ± 1ºC for 30 min in an electrically controlled oven, cooled to room temperature (25ºC ± 1ºC) and stored in a closed chamber. Test solutions (0.10 µl) of all analytes were applied on silica gel 60 HPTLC plates (10 cm 10 cm) with the help of micropipette (Tripette, Germany) at about 1.0 cm above the lower edge of the plates. The spots were completely dried and then developed in the chosen mobile phases by the ascending technique in a rectangular twin-trough chamber. Solvent ascent was fixed to 8.0 cm from the origin in all cases. After development, the plates were dried at 50ºC in an oven and the spots were detected by using appropriate detector for desired analyte. The R L (R F of leading front) and R T (R F of trailing front) values for each spot were determined and the R F value was calculated using the following equation: R F = 0.5 (R L + R T ) Separation For the separation, equal volumes of the analytes to be separated were mixed and an aliquot (0.10 µl) of the resulting mixture was loaded on the activated HPTLC plates. The plates were developed to 8.0 cm using mobile phase M 5 (n-butyl alcohol 70% aqueous ethylene glycol ethyl acetate), the spots were detected and the R F values of the separated amino acids were calculated. The separation time was 20 min. Densitometry TLC-scanning densitometric evaluation of amino acids, vitamins and sugars were carried out by spotting 0.10 µl of analytes mixture on silica HPTLC glass plates (10 cm 10 cm). The plates were developed with selected mobile phase (M 5 ). Trial plates for each samples loaded were run simultaneously to locate the exact position of analyte spots. After chromatographic development, the spots were detected by spraying respective chromogenic reagent. The plates were subjected for densitometric scanning in the wave length range 350-700 nm with plate height run 8 cm. Interference To investigate the effect of inorganic cations (K +, Mg 2+, Co 2+, Cu 2+, Th 4+ and Ba 2+ ) and anions (CO 3 2-, CH 3 COO -, Br -, NO 3 - ) on the mutual separation of amino acids (Hisidine + Glutamic acid +Proline + Methionine + Leucine), an aliquot (0.10 µl) of the mixture of amino acids was spotted on the HPTLC plate at the origin. After complete drying of spot an aliquot (0.10 µl) of impurity solution (cations or anions of 1.0%) was spotted on the same spot. The spot was redried and the chromatography was performed using mobile phase M 5. The resolved spots were detected and the R F values of separated amino acids were determined.

182 INDIAN J. CHEM. TECHNOL., MAY 2013 Similar procedure was applied for investigating the effect of inorganic ions (cations and anions) on the resolution of vitamins and sugars. Limit of detection The detection limits of separated analytes were determined by spotting 0.10 µl of analyte solutions of different concentrations on the HPTLC plates which were developed with mobile phase M 5 and the spots were visualized using the appropriate detection reagent. This process was repeated with successive reduction in the concentration of analyte. The amount of analyte just detectable was taken as the detection limit. Application The practical utility of this method was examined by analysis of a commercially available drug (Beplex fort) and a beverage sample (orange juice). Results and Discussion The experimental findings are shown in Tables 1 and 2 and Figs 1 and 2. Table 1 shows that the mobility of all thirteen amino acids on silica gel 60 HPTLC layer is influenced by the nature and composition of mobile phase systems. The following trends have been observed: (i) In n-butyl alcohol (M 1 ), lysine, proline, serine, arginine, and tryptophan remain close to the point of application. Other amino acids in this mobile phase show low or medium R F values. (ii) With ethylene glycol (M 2 ), all amino acids migrate with solvent front showing R F in the range 0.86-0.96. The high mobility is due to the presence of two hydroxyl groups which make this solvent polar, which makes the interaction of amino acids with ethylene glycol significant as compared to silica. Thus, ethylene glycol is not suitable for separation purposes. (iii) In case of ethyl acetate (M 3 ), all amino acids show marginal difference in their R F values (~0.20-0.23) and thus it is also not useful as mobile phase for the separation of amino acids. (iv) The detected spots of amino acids are found to be more compact in case of ethyl acetate compared to n-butyl alcohol and ethylene glycol. Table 1 Mobility (in terms of R F values) a of amino acids on silica gel 60 HPTLC plates using different mobile phases(m 1 M 5 ) Amino acids M 1 M 2 M 3 M 4 M 5 Leucine 0.14 ± 0.007 0.21 ± 0.011 0.96 ± 0.048 0.56 ± 0.028 0.51 ± 0.026 Isoleucine 0.15 ± 0.007 0.23 ± 0.012 0.94 ± 0.047 0.58 ± 0.029 0.46 ± 0.023 Norleucine 0.40 ± 0.020 0.21 ± 0.011 0.94 ± 0.047 0.56 ± 0.028 0.46 ± 0.023 Phenylalanine 0.32 ± 0.016 0.22 ± 0.011 0.96 ± 0.048 0.56 ± 0.028 0.51 ± 0.025 Tyrosine 0.26 ± 0.013 0.23 ± 0.012 0.92 ± 0.046 0.55 ± 0.028 0.42 ± 0.021 Lysine 0.04 ± 0.002 0.22 ± 0.011 0.86 ± 0.043 0.02 ± 0.001 0.01 ± 0.001 Proline 0.06 ± 0.003 0.23 ± 0.012 0.88 ± 0.044 0.25 ± 0.013 0.20 ± 0.010 Serine 0.05 ± 0.003 0.21 ± 0.011 0.93 ± 0.047 0.08 ± 0.004 0.16 ± 0.008 Glutamic acid 0.36 ± 0.018 0.24 ± 0.012 0.91 ± 0.046 0.27 ± 0.014 0.11 ± 0.006 Methionine 0.16 ± 0.008 0.20 ± 0.010 0.94 ± 0.047 0.47 ± 0.024 0.35 ± 0.018 Arginine 0.04 ± 0.002 0.22 ± 0.011 0.90 ± 0.045 0.04 ± 0.002 0.01 ± 0.001 Histidine 0.25 ± 0.013 0.21 ± 0.011 0.92 ± 0.046 0.06 ± 0.003 0.02 ± 0.001 Tryptophan 0.04 ± 0.002 0.23 ± 0.012 0.91 ± 0.046 0.58 ± 0.029 0.48 ± 0.024 a Each value is an average of four measurements. Table 2 R F values a calculated for the separation of amino acids in spiked drug and vitamin in spiked juice samples Sample Spiked Separation Resolved analytes R F Amino acid Biplex fort Histidine, glutamic acid, praline and leucine Pentnary separation Histidine glutamicacid Glutamic acid proline Proline methionine Methionine leucine Vitamin Orange juice Pyridoxine Ternary separation Thiamine ascorbic acid Ascorbic acid pyridoxine a Each value is an average of four measurements. 0.23 ± 0.012 0.25 ± 0.013 0.37 ± 0.019

MOHAMMAD et al.: GREEN ANALYTICAL METHOD FOR IDENTIFICATION OF AMINO ACIDS, VITAMINS & SUGARS 183 Fig. 1 Separation of maltose from other sugars on silica gel 60 HPTLC plates using mobile phase M 5, Bars represent standard deviation Fig. 2 Densitogram of resolved amino acids (A) histidine, (B) glutamic acid, (C) praline, (D) methionine, (E) leucine; vitamins (F) thiamine, (G) ascorbic acid, and (H) pyridoxine; and sugars (I) maltose, (J) fructose + galactose + mannose + dextrose and (K) xylose Hence, based on above observation, it is clear that the separation of amino acids from their mixture is not possible by any one component in mobile phases tested. Hence, mixtures of these solvents (n-butyl alcohol, 70% aqueous ethylene glycol, and ethyl acetate in 5:3:3 and 5:2:3 ratios (M 4 and M 5 ) have been used to obtain differential mobility pattern of analytes which will results in their separation from multicomponent mixtures. On the basis of different mobility pattern of amino acids, and maximum possibilities of separation, n-butyl alcohol-70% aqueous ethylene glycol-ethyl acetate (5:2:3, M 5 ) was selected as the best possible combination of components of mobile phase. By using mobile phase M 5, following observations are made. (i) Vitamins such as thiamine (R F 0.03), chlorine chloride (R F 0.02), folic acid (R F 0.03) and cobalamin (R F 0.05) remain at/near the point of application showing strong retention towards silica layer. Vitamins (ascorbic acid, pyridoxine, and riboflavin) show differential R F values (0.21, 0.59 and 0.38 respectively) and hence they can be separated from other vitamins. (ii) All sugars except maltose (R F 0.26) show marginal difference in their R F values (range from 0.37-0.58). Thus, maltose can be separated from other sugars. (iii) Separation of five components mixture of amino acids is achieved. The resolved amino acids are found to be histidine (R F 0.02), glutamic acid (R F 0.11), proline (R F 0.20), methionine (R F 0.35) and leucine (R F 0.51). (iv) Separation of vitamins from their ternary mixtures is also observed. The resolved vitamins are thiamine (R F 0.01), ascorbic acid (R F 0.18), and pyridoxine (R F 0.64) or cobalamine (R F 0.03), ascorbic acid (R F 0.18), and pyridoxine (R F 0.59). (v) Selective separation of maltose from other sugars that are fructose, galactose, mannose, dextrose and xylose is obtained (Fig. 1). Figure 2 shows the HPTLC chromatograms of the resolved amino acids, vitamins and sugars. Effects of foreign substances such as cations (K +, Mg 2+, Co 2+, Cu 2+, Th 4+ and Ba 2+ ) and anions as impurities (CO 2-3, CH 3 COO -, Br -, NO - 3 ) on chromatographic parameters ( R F, α, and Rs) have been studied for representative separations of amino acids, and vitamins. From the results, it is clear that the presence of impurities has slight influence on the magnitude of the chromatographic parameters of amino acids in most cases. The separation is always possible except 2- in case of CO 3. However, in case of vitamins, the foreign substances also have slight effect but separation is not hampered except in case of Cu 2+. The marginal increase or decrease in values of these parameters is due to the increase in spot size of the analyte as a result of various interactions with these additives. In case of sugars, cations and anions affect the separation (R F values) in most of the cases. Improved separations are achieved in the presence of impurities.

184 INDIAN J. CHEM. TECHNOL., MAY 2013 The lowest possible detectable amounts of different analytes are: 7.5 µg spot -1 for proline, thiamine, ascorbic acid, mannose, dextrose, and fructose; 1.5 µg spot -1 for pyridoxine and xylose; 0.75 µg spot -1 for leucine, methionine, histidine, maltose, and galactose; and 0.15 µg spot -1 for glutamic acid. The applicability of the proposed method (silica gel 60 as stationary phase and M 5 as mobile phase) for the identification of methionine (R F = 0.43) in pharmaceutical formulation and thiamine (R F = 0.01) and ascorbic acid (R F = 0.26) in real juice sample has been tested. It is clear that the proposed method is satisfactorily applicable to the identification of methionine in Beplex Fort and vitamins (thiamine, and ascorbic acid) in orange juice with preliminary separation from other components on HPTLC plate (Table 2). Conclusion The chromatographic system comprising silica gel 60 HPTLC as stationary phase and n-butyl alcohol- 70% aqueous ethylene glycol-ethyl acetate (5:2:3, v/v) as mobile phase has been identified more efficient for separations of amino acids, vitamins and sugars from their multi-component mixtures. The proposed method could be applied for the identification of methionine, and vitamins (thiamine and ascorbic acid) in drug and juice samples. Acknowledgement The authors are thankful to the Dean of Scientific Research at King Saud University for funding the work (project No RGP-VPP-130). References 1 Namieśnik J, Environ Sci Pollut Res, 6 (1999) 243. 2 Mohammad A & Zehra A, Colloid Surf A: Physicochem Eng Aspects, 301(2007) 404. 3 Chomicki A, Kloc K & Dzido T H, J Planar Chromatogr, 24 (2011) 6. 4 Buhl F & Galkowska M, J Planar Chromatogr, 19 (2006) 401. 5 Mohammad A & Zehra A, Acta Chromatogr, 20 (2008) 637. 6 Kartsova L A & Koroleva O A, J Anal Chem, 62 (2007) 255. 7 Mohammad A, Moheman A & El-Desoky G E, Cent Eur J Chem, 10 (2012) 731. 8 Mohammad A & Laeeq S, J Planar Chromatogr, 24 (2011) 491. 9 Wagner S D, Pachuski J, Fried B & Sherma J, Acta Chromatogr, 12 (2002) 159. 10 Kim Y, Fried B & Sherma J, J Planar Chromatogr, 14 (2001) 61. 11 Sherma J, Anal Chem, 72 (2000) 9R-25R. 12 Grinberg N, Modern Thin-Layer Chromatography (Dekker, New York), 1990. 13 Bhushan R & Martens J, in Handbook of Thin-Layer Chromatography, edited by J Sherma B Fried, 3 rd edn (Marcel Dekker, Inc., New York), 2003. 14 Spangenberg B, Poole C F & Weins Ch, Quantitative Thin- Layer Chromatography (Springer-Verlag Berlin Heidelberg), 2011. 15 Mohammad A & Moheman A, in High-performance Thin- Layer Chromatography (HPTLC), edited by M M Srivastava (Springer-Verlag Berlin Heidelberg), 2011. 16 Patel R B & Patel M R, An introduction to analytical method development for pharmaceutical formulations, 2008. http://www. pharmainfo.net/reviews/introduction-analyticalmethoddevelopment-pharmaceutical-formulations. 17 Sharma S K & Mudhoo A, Green Chemistry for Environmental Sustainability (Taylor and Francis, CRC press), 2010. 18 Mohammad A, Haq N & Siddiq A, J Sep Sci, 33 (2010) 3619. 19 Mohammad A, Gupta R, Naushad Mu & El-Desoky G E, J Disp Sci Technol, 32 (2011) 1179. 20 Postaire E, Cisse M, Le Hoang M D & Pradeau D, J Pharm Sci, 80 (1991) 368. 21 Chavan J D & Khatri J M, J Planar Chromatogr, 5 (1992) 280. 22 Hess B & Sherma J, Acta Chromatogr, 14 (2004) 60. 23 Buhl F, Szpikowska-Sroka B & Galkowska M, J Planar Chromatogr, 18 (2005) 368. 24 Hossu A M, Radulescu C, Ilie M, Balalau D & Magearu V, Revista De Chimie, 57 (2006) 1188. 25 Vasta J D, Cicchi M, Sherma J & Fried B, Acta Chromatogr, 21 (2009) 29.