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Phenylalanine |
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Bimodal Response The most important and unique aspect of polarimetric detection is related to the sign of the signal one obtains for a particular enantiomer. While one enantiomer rotates light in such a direction as to produce a positive signal, the other enantiomer rotates light in the opposite direction and produces a negative signal. This bimodal response can be used to show the degree (no pun intended) to which a separation has been achieved, or to otherwise assess the quality of separation with a chiral-selective method. This information is difficult, or impossible, to obtain using other, unimodal, detection systems. Confirmation of Elution Order Polarimetric detection of a chiral separation of D-DNS-phenylalanine (A) and L-DNS-phenylalanine (B) obtained using a b -cyclodextrin column is shown in the figure below. The signal from an internal calibration standard occurs at about 1-minute and can be used to normalize peak heights verifying that the two enantiomers produce equal but opposite responses.
For a unimodal detector, one would have to change the relative amounts of the two enantiomers to ensure that the observed peaks are produced by the enantiomeric pair and not by impurities in the sample. This would require that pure preparations of the two enantiomers be available, a situation that may not exist, especially in the early stages of drug development. Bimodal Quantitation In the next figure, we show UV absorbtion (A) and laser polarimetric detection (B) of a mixture of the DNS-phenylalanine enantiomers separated on a b -cyclodextrin column. In this separation, the chromatographic resolution is approximately 0.5. As we shall see, the bimodal response of the laser-based polarimeter allows quantitation of enantiomeric mixtures at chromatographic resolutions that are much less than 1.0. From the UV detector signal trace, it would be very difficult, and perhaps impossible, to provide accurate quantitative information on the enantiomeric ratio of this sample. However, with the laser polarimeter's bimodal response we can achieve reasonable quantitation at resolutions below 0.5. Quantitation with Varying EE A systematic study was conducted in which the laser polarimeter’s capability to provide accurate quantitative information on enantiomeric mixtures was evaluated as a function of two parameters. These parameters were chromatographic resolution and enantiomeric excess. First, a series of samples with increasing enantiomeric ratios was injected and the peak heights measured for the D- form. A calibration curve was constructed, correlating the peak height obtained from the laser polarimeter with the enantiomeric fraction of D-DNS-phenylalanine. This curve is shown below. Note that the chromatographic resolution between the two enantiomers was less than 0.7. 
Good correlation (r ~0.999) was noted between the peak heights of each enantiomer and the actual fraction in the enantiomeric mixture. The quality of the correlation was the same for both D- and L- forms of the analyte even though the L- form actually elutes earlier than the D- form. The detector’s unique bimodal response provides a distinct differentiation point for apportionment of either peak height or peak area between the two partially separated enantiomers. Again, it should be noted that this level of quantitation would not be possible with a unimodal detector such as a UV/Vis absorbance detector. Even at the lowest resolution tested, 0.3, where the UV/Vis detector shows only a single peak, the polarimetric detector allows quantitation down to the 5-10% ee level. For applications, such as the generation of combinatorial libraries of chiral drug candidates, this level of accuracy is adequate, allowing more structural permutations to be analyzed in less time. |
