Anal. Chem. 1987, 59, 2735-2736
2735
AIDS FOR ANALYTICAL CHEMISTS Thin-Layer Chromatographic Separation of Diastereomeric Amino Acid Derivatives Prepared with Marfey's Reagent Kenneth J. Ruterbories and David Nurok* Department of Chemistry, Indiana Uniuersity-Purdue University a t Indianapolis, Indianapolis, Indiana 46223
l-Fluoro-2,4-dinitrophenyl-5-~-alanine amide (FDAA) has been introduced by Marfey (1) for the preparation of diastereomeric derivatives of amino acids. The original report described the high-performance liquid chromatography (HPLC) separation of these diastereomers for each of five amino acids and included a chromatogram showing 11 peaks; 10 due to the derivatives and the eleventh due to the hydrolyzed reagent. The structure of a distereoisomeric pair of FDAA derivatives is shown in Figure 1. Marfey pointed out that the reaction was quantitative, that the only byprodud formed was that of the hydrolyzed reagent, that the derivative contained a highly absorbing chromophore which allowed detection in the nanomole range, and that the method of resolution is flexible in that amino acids other than L-alanine amide can be used for preparing a corresponding reagent. We have been able to locate only one other report on the use of FDAA for preparing diastereomersfor chromatography. Aberhert and co-workers (2)reported that the diastereomeric FDAA derivatives of 0-leucine were very well resolved. We report below on the separation of the diastereomeric FDAA derivatives of 22 amino acids by thin-layer chromatography (TLC).
EXPERIMENTAL SECTION FDAA was purchased from the Pierce Chemical Co. (Rockford, IL) and was used to prepare derivatives according to the procedures of Marfey (I). TLC was performed in the SB/CD chamber (Regis Chemical Co., Morton Grove, IL) in position 5 which corresponds to an effective plate length of 83 111111. Twenty-five milliliters of solvent was used for each development. The SB/CD chamber was used to allow for the possibility of continuous development TLC. However, this was not necessary because of the relatively large pRf values obtained. TLC was performed on Whatman (Clifton, NJ) plates that incorporated a fluorescent indicator. The silica gel plates were catalog no. 4851-820and the bonded CI8plates were catalog no. 4803-800. Reverse-phase TLC was performed with a binary solvent consisting of methanol and 0.3M aqueous sodium acetate adjusted to pH 4 by the addition of 2 N hydrochloric acid.
RESULTS AND DISCUSSION FDAA derivatives were prepared for 22 D , L - = ~ o acids and for 18 L-amino acids. It was assumed that the reagent reacted only with the a-amino group. These derivatives were chromatographed either on silica gel TLC plates using the binary solvent system acetic acidltert-butyl methyl ether or on bonded ClS TLC plates using the binary solvent system methanol/0.3 M sodium acetate. When such binaries are used, it is possible to compute how ARf,the difference in Rf values for a given solute pair, varies with the solvent composition (3). The separation profile of diastereomers can differ substantially as is illustrated in Figure 2 for the FDAA derivative of D,L-arginine (Figure 2a) and D,L-phenyldanine(Figure 2b). 0003-2700/87/0359-2735$0 1.50/0
Table I. (ARf)==for Diastereomeric FDAA Amino Acid Derivatives on Bonded CISLayers parent amino acid
(Mf)ma.
XP
DL-alanine DL-arginine DL-aSparagine DL-aSpartiC acid DL-citrulline DL-cystine DL-ethionine DL-glUtamiC acid DL-histidine DL-iSOleUCine DL-leucine DL-lySine DL-methionine DL-nOrVahe DL-norleucine DL-phenylalanine
0.17 0.06 0.13 0.22 0.12 0.08 0.20 0.11 0.08 0.21 0.20 0.15 0.20 0.20 0.20 0.18 0.13 0.11 0.21 0.15 0.22 0.21
0.29 0.24 0.19 0.07 0.23 0.35 0.40 0.12 0.23 0.42 0.43 0.43 0.36 0.38 0.43 0.41 0.30 0.19 0.24 0.36 0.53 0.37
-
DL pr 01in e
DL-serine DL-threonine DL-tryptophan DL-tyrosine DL-Valine
"Mole fraction of methanol/aqueous buffer (pH 4) at which
(af)occurs.
The maximum value of ARf is referred to as (ARf)mm. Table I lists the values of and the mole fractions at which these occur for the FDAA amino acids chromatographed on bonded CI8layers. Table I1 lists the corresponding values on silica gel layers. It is seen that the diastereomeric pairs of all 22 amino acids are separated on C18layers with values in the range of 0.06-0.22 whereas only 14 can be separated on the silica gel layer. The (ARf)mar for these is in the range of 0.05 to 0.14. With the exception of citrulline, (ARf)ma is greater in the reverse-phase system than in the normal-phase system. In the case of citrulline the difference in the value of (ARJmm for the two systems is small enough (0.02)to be possibly due to experimental error. Marfey reported that the diastereomeric derivative prepared from the L-amino acid had the lower retention in a reversed-phase system. This was ascribed to greater intramolecular hydrogen bonding in the D than in the L diastereomer; this would result in the former being more hydrophobic and interacting more strongly with the nonpolar stationary phase. A similar chromatographic behavior was found for the reverse-phase TLC where the L diastereomer has the higher R,. For those pairs of diastereomers that are separated on a silica gel layer in the normal-phase mode, it is the D diastereomer that has the higher Rp This is consistent with the above model. The diastereomer with the greater degree of intramolecular hydrogen bonding would be expected to have lesser interaction with the silica surface and would thus have the higher R,. 0 1987 American Chemical Society
2738
ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987 H
H
'C-NH,
O2N
II
NH
0
I
H-C-R
R-C-H
I
I
C-OH
II
0
C-OH
I1
0
a
a.
Figure 1.
c
b. L,L-
(a) and D,L-FDAA(b) amino acid derivatives.
0 25
0.25
0 20
0.20
-
015
a
0.10
005 0 0
02
04
06
08
10
0
02
04
06
08
M O L E FRACTION
MOLE FRACTION
a
b
10
Flgure 2. Plots of AR, vs the mole fraction of methanol for the diastereomeric pairs FDAA arginine (a) and FDAA phenylalanine (b).
Table 11. (AR,),, for Diastereomeric FDAA Amino Acid Derivatives on Silica Gel Layers parent amino acid DL-alanine DL-arginine DL-asparagine DL-aEpartiC acid DL-CitrUlline DL-CyStine DL-ethionine DL-glUtamiC acid DL- histidine DL-isoleucine DL-leUCine DL-lySine DL-methionine DL-nOrValine DL-norleucine DL-phenylalanine DL-prOline DL-Serine DL-threonine DL-tr yptophan DL- tyrosine DL-Valine
C
d
0.15
-3 0 10
b
Figure 3. Computer-simulated(a) and experimental (b) chromatograms for the FDAA amino acid derivatives of isoleucine (I), leucine (11), and phenylalanine (I II).Computer-simulated (c) and experimental (d) chromatograms for the FDAA derivatives of arginine (IV). IA, IIA, I I I A , and IVA are for the derivatives prepared from L-amino acid; IB, IIB, IIIB. and IVB are for the derivatives prepared from o,L-amino acid.
Wf)m&U
x,"
0.06
0.26
0.00 0.00 0.00
-b
0.14
0.75
0.00 0.09 0.00 0.00 0.12 0.10 0.09 0.08 0.11
0.14 0.07 0.06 0.00 0.00 0.09 0.05 0.10
A -b -b
0.20 -b
-b
0.18 0.19
0.40 0.23 0.22 0.21 0.22 0.58 A -0
0.22 0.24 0.22
"Mole fraction of acetic acidltert-butyl methyl ether at which (ARf)ma.occurs. * N o separation was detected. The X , value at which (AR,)m8x occurs cannot be determined. The following are a few tentative correlations between the structure of the diastereomers and the corresponding value of in Table I: (1)When R in Figure 1 represents a three- or four-carbon alkyl group (valine, norvaline; leucine, norleucine, isoleucine) then the value of (AI?,) is independent of structure being either 0.20 or 0.21; the difference
of 0.01 most probably representing experimental error. (2) A value of 0.20 is found also where R represents a thioether group (methionine, ethionine). (3) The mole fraction of occurs increases with the size of methanol a t which (AI?,)the alkyl group (CH3 = 0.29, C3H7= 0.37, C4H9= 0.43) but not with the isomeric identity of the group. (4)The value of varies widely (0.06-0.22) where R contains a functionality that is either a strong proton acceptor or donor. If separation is influenced by the relative degree of intramolecular hydrogen bonding in a pair of diastereomers, then it is not surprising that the presence of such a group can alter the value of (ARf),=. It should be noted that the above correlations would not be evident if chromatography was performed a t only one solvent composition and AR,, rather was used as a separation parameter. than There is a good agreement between computer prediction and actual experimental separation as illustrated in Figure 3 for the separation of the diastereomers of FDAA isoleucine, leucine, phenylalanine, and arginine, the latter representing the pair of diastereomers that is most difficult to separate in this solvent system. Detection was by fluoresence shadowing which may somewhat exaggerate the actual resolution of the arginine diastereomers. In conclusion it should be noted that while FDAA derivatives of each of the 22 amino acids in Table I can be separated by reverse-phase TLC, it is nevertheless impossible to separate all 22 amino acid derivatives (i.e. 44 individual compounds) in a single conventional run due to a large number of overlapping Rf values. It may be possible to solve this problem by two-dimensional TLC or by a technique such as parallel development TLC ( 4 ) .
ACKNOWLEDGMENT The TLC plates were a gift from Whatman Chemical Separations, Inc. LITERATURE CITED (1) Marfey, P. CarlsbergRes. Commun. 1984, 4 9 , 591. (2) Aberhart, D. J.; Coning, J. A.: Lin, ti. J. Anal. Biochem. 1985, 751, 80. (3) Nurok, D.: Richard, M. J. Anal. Chem. 1981, 53, 563. (4) Nurok, D.: Tecklenburg, R. E., Jr : Maidak, B. L. Anal. Chem. 1984,
56, 293.
RECEIVED for review March 18, 1987. Accepted July 20, 1987. This work was supported by a grant from the Dow Analytical Laboratories University Support Program.
