TLC separation and derivative spectrophotometry of some amino acids

Talanta

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Talanta 42 (1995) 1553-1557

Short communication

TLC separation and derivative spectrophotometry of some amino acids Irena Baranowska *, Matgorzata Kozlowska Department of Analytical and General Chemistry, Silesian Technical University, 44-101 Gliwice, Poland

Received 22 November 1994; revised 23 February 1995; accepted 24 February 1995

Abstract

Chromatographic systems for the separation of amino acid mixtures on RP-18 as a stationary phase have been elaborated. The best results were obtained using methanol-water (1:1, v/v; 1:3, v/v; 1:5, v/v) as a mobile phase. The following amino acids have been examined: asparagine, arginine monohydrochloride, cystine, cysteine chloride, glycine, histidine monohydrochloride, hydroxyproline, isoleucine, leucine, lysine monochloride, methionine, ornithine monohydrochloride, phenylalanine, proline, threonine, tryptophan, tyrosine, serine, valine. Histidine (as monohydrochloride) and methionine were determined by first-, second- and thirdderivative spectrophotometry in a mixture of several amino acids. Keywords: Amino acids; Spectrophotometry; TLC

1. Introduction The analysis of amino acids as well as their chromatographic separation is still a current problem, especially in medical analysis. In the literature, several chromatographic systems, based on adsorption TLC, are described for chosen groups of amino acids. Separation has been carried out on silica gel [1-5] and cellulose [6,7]. Bidirectional adsorption chromatography has also been used [8]. Several works are concerned with the method of detection of chromatograms [2-4]; ninhydrin is most often used. In the present work, reversed-phase partition TLC (RP-18) was applied to separation of a chosen group of amino acids. Up to the present time, such separation has not been described. In addition to these investigations, derivative spectrophotometry was used. Spectrophotome* Corresponding author.

S S D ! 0039-9140(95)01569-8

try is also frequently applied to the analysis of amino acids which form coloured compounds with various substances. Cystine and cysteine have been determined spectrophotometrically with Cu(II)-neocuproin reagent [9,10], and also with OsO4 in urine [11]. Amino acids have been analysed as compounds with carbon disulphide [12] and cystein as a compound with OPA [13]. p-Benzoquinoline has also been used for the determination of amino acids [14]. The obtained results were not always satisfactory. The reason for applying derivative spectrophotometry [15] is that it can be more sensitive and selective than zero-order spectrophotometry. A derivative spectrum is a graphical dependence of a value of the differential of absorbance with respect to wavelength on the wavelength. It can be described by the formula d'A d2" - "D~.~.- f ( 2 )

L Baranowska, M. Ko:lowska / Talanta 42 (1995) 1553-1557

1554

Table 1 Rf values of amino acids on bonded phase RP-18 and colour of spots after ninhydrin detection Amino acids

Colour of spot

Rr Methanol/water

Cysteine chloride Cystine Phenylalanine Glycine Hydroxyproline lsoleucine Asparagine acid Leucine Methionine Lysine monochloride Arginine monohydrochloride Histidine monohydrochloride Ornithine monohydrochloride Proline Serine Threonine Tryptophan Tyrosine Valine

Pink Pink Violet Pink Yellow Pink Pink Pink Pink Violet Violet Violet Violet Yellow Pink Pink Violet Pink Pink

Propanol/water

3:1

1:i

1:3

1:5

3:1

I:1

1:3

0.79 0.62 0.68 0.80 0.85 0.73 0.81 0.73 0.77

0.86 0.83 0.53 0.89 0.9 0.69 0.9 0.68 0.76

0.87 0.88 0.48 0.91 0.90 0.71 0.94 0.66 0.75

0.96 0.89 0.48 0.93 0.93 0.68 0.97 0.66 0.78

0.76 0.88 0.84 0.84 0.86 0.85 0.85 0.86 0.85

0.89 0.90 0.74 0.94 0.95 0.79 0.92 0.79 0.82

0.76 0.82 0.78 0.69 0.82 0.78

0.79 0.91 0.91 0.44 0.74 0.81

0.81 0.95 0.94 0.34 0.72 0.83

0.82 0.95 0.93 0.29 0.69 0.84

0.81 0.85 0.86 0.85 0.88 0.86

0.91 0.96 0.95 0.79 0.89 0.91

0.94 0.94 0.63 0.96 0.95 0.80 0.97 0.76 0.82 0.90 0.80 0.84 0.91 0.89 0.96 0.95 0.50 0.77 0.87

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URVELENGTH Fig. I. "Zero-order" spectra of histidine (I), arginine (2), asparagine (3), hydroxyproline (4), isoleucine (5), ornithine (6), proline (7). serine (8) and valine (9). where n is the derivative order, a n d "D~.~ is the value o f the n t h - o r d e r derivative o f the a b s o r p tion spectra o f a s u b s t a n c e x at a certain wavelength a. Derivative s p e c t r o p h o t o m e t r y is especially useful in i m p r o v i n g selectivity. T r y p t o p h a n a n d tyrosine in synthetic peptides [16], a n d aromatic a m i n o acids - - t r y p t o p h a n , tyrosine a n d p h e n y l a l a n i n e - - in proteins [17], were analysed by the use o f s e c o n d - o r d e r derivative spectrophotometry.

In the present paper, the d e t e r m i n a t i o n o f histidine a n d m e t h i o n i n e in the mixtures cont a i n i n g several a m i n o acids was elaborated.

2. Experimental 2.1. Equipment T L C s e p a r a t i o n was d o n e o n 10 x 2 0 ¢ m 2 glass plates covered with a 0.25 m m layer o f

!. Baranowska, M. Ko-lo.'ska / Talanta 42 (1995) 1553-1557

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URVELENGTH

Fig. 2. First-derivativespectra of histidine (I), arginine, ornithine, asparagine, proline, hydroxyproline,isoleucine,serine, valine and mixtures (2). 0.04603-

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URVELENGTH

Fig. 3. "Zero-order" spectra of methionine (I), asparagine (2), hydroxyproline(3), isoleucine(4), orraithine (5), proline (6). serine (7) and valine (8).

RP-18 (E. Merck, Darmstadt) in Shandon's containers. The absorption spectra were recorded on a Hewlett Packard HP8452A UV/vis spectrophotometer with 1 cra cells. 2.2. Reagents

All amino acids were produced by E. Merck, Darmstadt. 1 mg m l - ~ aqueous solutions of amino acids were used, except in the case o f cystine ( 0 . 1 m g m i -~) and tyrosine (0.45mg ml-m), for TLC. Solutions containing 0.1 mg ml -m of amino acids, except for histidine

(0.05 mg ml-~), were used in derivative spectrophotometry. 2.3. Procedure

All chromatograms were developed over a distance o f 15 cm in Shandon's containers. The following developing mixtures were applied: methanol-water (3:1, v/v; 1:1, v/v; !:3, v/v; 1:5, v/v), p r o p a n o l - w a t e r (3:1, v/v; 1:1, v/v; 1:3, v/v) and acetonitrile-water (1:1, v/v). Chromatograms were detected by a 0.25°/'0 solution o f ninhydrin in acetone. Derivative spectra were calculated by the use o f a standard software procedure.

/. Baranowska. M. Ko:lowska / Talanta 42 (1995) 1553-1557

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and mixtures (2). Table 2 Parameters of the straight line dependence and correlation coefficient o f histidine a n d methionine for first-derivative (D), second-derviative (2D) and third-derivative QD) spectrophotometry Histidine

D 2D 3D

Methionine

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b

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a

b

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0.997 0.996 0.997

3. Results and conclusions

3.1. Thin-layer chromatography Some systems suitable for the separation of amino acids have been found (Table 1). The greatest differences between Rf values of investigated compounds were obtained for the systems containing methanol and water. The six amino acids cysteine chloride, isoleucine, phenylalanine, methionine, serine, tryptophan were separated in the mixture methanol-water (1:1, v/v); phenylalanine, methionine, threonine, tryptophan, tyrosine, valine were separated in the mixture methanol-water (1:5, v/v). The best results were obtained for the mixture methanol-water (1:3, v/v). Under these conditions, seven amino acids can be separated: asparagine acid, hydroxyproline, phenylalanine, leucine, methionine, proline, and tryptophan. In spite of the fact that the results obtained in the systems propanoi-water are not so good, monohydrochlorides and monochlorides of chosen amino acids can be separated in

these circumstances. To the best of our knowledge, such separations are not reported in the literature. Spots of all amino acids were of good visibility, with sharp edges against the white backround of the plates.

3.2. Derivative spectrophotometry Spectrophotometric determination of histidine was carried out by the use of derivative spectrophotometry to complete chromatographic separation and analysis of this amino acid in the mixture. The following amino acids were used in this determination: histidine, arginine, ornithine, asparagine, proline, hydroxyproline, isoleucine, serine, valine (Figs. 1 and 2). Zero-order spectrophotometry cannot be applied in this case, while first-, second- and third-order derivative spectrophotometry allows histidine to be determined with a good accuracy at a concentration of 8 ~tg ml- m. Methionine in a mixture with ornithine, asparagine acid, proline, hydroxyproline, iso-

L Baranowska, M. gozlowska / Talanta 42 (1995) 1553-1557

leucine, serine, valine was a n a l y s e d in the s a m e w a y (Figs. 3 a n d 4). In this case it was also p o s s i b l e to d e t e r m i n e a m e t h i o n i n e c o n c e n t r a tion o f 1 0 ~ t g m l - t . S t r a i g h t line d e p e n d e n c e was e v a l u a t e d for a m i n o a c i d s b y the use o f derivative spectrophotometry. Parameters of the e q u a t i o n "D = ac + b

w h e r e "D is the value o f derivative, c is the c o n c e n t r a t i o n , a is the i n t e r c e p t a n d b is the slope, for a p a r t i c u l a r o r d e r o f derivatives a n d suitable c o r r e l a t i o n coefficients for histidine a n d m e t h i o n i n e , a r e g a t h e r e d in T a b l e 2.

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[4] C.G. De Lima, T.C.M. Pastore, C.A. Schwartz, I.S. Cruz and A. Scbben, Talanta, 38 (1991) 1303-1307. [5] M. Alaiz, J.L. Navarro, I. Giron and E. Vioque, J. Planar Chromatogr., 5 (1992) 143-146. [6] L. Sentier, I. Marchal, I. Boudrout and P. Germain, J. Chromatogr., 547 (1991) 531-537. [7] T.K.X. Huynh, A.O. Kuhh and M. Leder, J. Chromatogr., 626 (1992) 301-304. [8] B. Borkowski, Chromatografia cienkowarstwowa w analizie farmacentycznej, PZL, Warszawa, 1973. [9] E. Tutem and R. Apak, Anal. Chim. Acta, 255 (1991) 121-125. [10] M.L. lskander and H.A.A. Medien, Microchem. J., 41 (1990) 172-182. [1 I] I. Bertini, C. Mannucci, R. Noferini, A. Perico and P. Rovero, J. Pharm. Sci., 82 (1993) 179-182. [12] A. Besada, N.B. Tadros and Y.A. Gawargious, Mikrochim. Acta, (1989) 143-146. [13] 1. Chrastil, Analyst, !15 (1990) 1383-1384. [14] H.J.M. Hemandez, R.M. Camanas and M.C.G. AIvarez-Coque, Microchem. J., 40 (1989) 292-296. [15] Z. Marczenko, S. Ku~, and N. Obarski, Chem. Anal., 36 (1991) 879-890. [16] A.W.M. Lee, W.H. Chan and M.F. Ho, Anal. Chim. Acta, 246 (1991) 443-445. [17] Y. Nozaki, Arch. Biochem. Biophys., 277 (1990) 323333.