Quantitative Analysis of TLCSeparated Enantiomers

TLC is generally coupled with spectrophotometric methods for quantitative analysis. Quantification can be achieved by in situ densitometry or after extraction of solutes from the scraped layer. The evaluation of detection limits for separated enantiomers is essential because precise determinations of trace levels of d- or l-enantiomer in an excess of the other is becoming more and more important.

On Chiralplates and HPTLC-CHIR layers, densito metry can be performed after postchromato-graphic derivatization of compounds with ninhy-drin or vanadium pentoxide. Successful separation of amino acids on Chiralplates depends on the hydrochloric acid content of the applied solution (usually a methanol-0.1 mol HCl 1 : 1 (v/v) mixture).

Remission-location curves of dl-a-hydroxycar-boxylic acids, achieved in reflectance mode with a Shimadzu CS930 scanner or a Desaga CD60 densitometer, show that only enantiomers with high ARF values *0.10) can be baseline resolved on 10 cm x 10 cm HPTLC-CHIR plates (Figure 3). On such plates, l-2-hydroxy-3-phenylpropionic acid spiked with 1% d enantiomer (ARF = 0.12) gives rise to partially resolved peaks but the d isomer is still visible. With respect to small particle size HPTLC-CHIR layers, higher Rs values have been obtained on 20 cm x 20 cm Chiralplates owing to migration distances being twice as long (a values being equal).

The remission-location curves of Figure 4 and the calibration line for l-phenylalanine (Figure 5) demonstrate that quantitative determinations of l-isomer in d-phenylalanine on Chiralplates (ARF = 0.10) are possible in a working range of 0.04-0.4 ^g/spot, that is 0.1-1%. Further determinations include 0.1% d-t-Leu in l-t-Leu (ARF = 0.11), 0.1% l-5,5-dimethyl-thiazolidine-4-carboxylic acid in the d-enantiomer (ARf = 0.14) and 1% d-hydroxyphenylalanine in the l-enantiomer.

The peak of 1% Dns-d-Glu in l-enantiomer is visible on 20 cm x 20 cm RP-18 plates (Merck) impregnated with a solution of 8 mol L_1 N,N-di-n-propyl-l-alanine and 4 mmol L_1 cupric acetate.

On 10 cm x 20 cm cellulose plates l-tryptophan spiked with 5% d-enantiomer gives rise to partially resolved peaks owing to the small ARF value (0.06).

The use of MCTA allows the determination of enantiomeric mixtures in the ratios 100 : 1 and 200 : 1. (S)-2,2,2-Trifluoro-1-(9-anthryl)ethanol can be detected at 1% level in (R) enantiomer on OPTI-TAC F254 plates eluted with ethanol-water 80 : 20 (ARf = 0.17; length of run 10 cm). Baseline-resolved b c

Figure 2 Structure of acidic drugs.

peaks (ARF = 0.10) were obtained for the two atropisomers of 1,1'-binaphthyl-2,2'-diamine on 20 cm x 20 cm home-made MCTA plates at 100 : 1 ratio. Partial resolution only was observed at a ratio of 200 : 1, but the S isomer is still visible (Figure 6).

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