Enantiomeric Excess

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Phenylalanine
Enantiomeric Excess

Bimodal Response Potential

Since the laser polarimeter's bimodal response is well understood, it was proposed that a mathematical model of the response could be developed in order to facilitate data analysis. Although adequate quantitative results were obtained using either the polarimetric peak height or area, with a properly defined model, the entire data set could be used to quantitate each enantiomer.

Experimental versus Simulated Response

For this experiment, a chromatographic separation was compared with a gaussian model used to simulate the polarimetric response. The figure above shows the laser polarimeter's response for D-DNS-phenylalanine. (A) and (B) represent the experimental and simulated responses, respectively. The experimental results were obtained from a separation of 15 m g of injected material using a b -cyclodextrin column and a mobile phase system consisting of 70/30 ACN/NH₄NO₃ at a flow rate of 1.0 mL/min. The excellent agreement between the simulated and experimental response shows the validity of this model.

Given the previous results, a simulated bimodal response was then modeled as the difference between two gaussian functions. Again, the agreement between the simulated and experimental results is excellent. This is illustrated in next figure. Thus, this model can be used to fit polarimetric data for an incompletely resolved enantiomeric pair allowing more precise and accurate quantitation.

In addition to a 50/50 (racemic) mixture of the enantiomeric pair, the entire range of enantiomeric excess was evaluated including both very low (5%) ee and very high (95%) ee.

It is important to note that these results, obtained under conditions of incomplete chromatographic resolution (R_s = 1.00), demonstrate excellent correlation (r~0.999) between the calculated and actual enantiomeric content of the sample (curves below). For this experiment, the eluent was 80% ACN and 20% 0.025M NH₄NO₃, pH 4.0. These results, obtained using the unique bimodal response of the laser polarimeter and the Gaussian data analysis model, are as good as those previously reported for analyses achieving a baseline separation of the enantiomers.

In many situations, and in spite of a substantial commitment of resources, a baseline separation can not be achieved. In other circumstances, a poor separation will rapidly deteriorate to an unacceptable level with column use. This is even more of a problem when the sample contains compounds which, although achiral, can irreversibly adsorb to the column altering its chromatographic properties. Previously, such situations required column replacement followed by re-validation of the method. In the worst case scenario, it would require the development of a new separation protocol incurring additional time and expense.

With the bimodal data analysis model and the PDR-Chiral laser polarimeter, excellent quantitative information can still be obtained on a sample even as the peak resolution degrades. The methodology works equally well for both enantiomers.

Minimum Measurable Quantities of DNS-Phenylalanine Enantiomers
Eluent^a	Resolution	Parameter	+40% EE^b	0% EE^c	+60% EE^d
80/20	Rs = 1.00	L peak	0.8%	1.5%	2.4%
		D peak	0.8%	1.4%	2.1%
70/30	Rs = 0.66	L peak	0.6%	1.0%	1.6%
		D peak	0.6%	1.0%	1.6%
50/50	Rs = 0.34	L peak	0.6%	0.9%	1.4%
		D peak	0.6%	1.0%	1.4%
40/60	Rs = 0.32	L peak	3.4%	5.5%	8.9%
		D peak	3.4%	5.5%	8.9%
^a ACN/NH₄NO₃ (0.025 M, pH 4.0) except the 40/60 eluent (0.10 M, pH 4.0) ^b + 40% EE: 30% L & 70% D; - 40% EE: 70% L & 30% D ^c 50% L & 50% D ^d + 60% EE: 80% L & 20% D; - 60% EE: 20% L & 80% D

The table above provides a detailed summary of minimum measurable change in enantiomeric excess obtained for DNS-phenylalanine as a function of chromatographic resolution and enantiomeric excess. Several important points can be made concerning these results.

Results

First, the quantitative capabilities of the laser polarimeter coupled to an enantio-selective separation system are excellent, even under conditions of poor chromatographic resolution. For example, if we look at the system when R_s = 0.66, the minimum measurable enantiomeric excess is at the 1% level in spite of the fact that the two peaks are not baseline resolved. This level of performance can be achieved throughout the range from very low, to very high ee.

Second, for resolutions of ~0.3, which would be problematic for other detection systems, the polarimetric response provides a clear crossing point for the partially separated enantiomers, and this unique response allows quantitative information to be obtained even under these conditions. At low ee, the minimum measurable %ee is at the 2-3 % level, and at high ee, the MMQ is below 9% ee. Again, these results are obtained from a separation that would produce only a single peak in a unimodal detection system such as the UV/Vis.

Finally, similar results were obtained for two other amino acids, threonine and valine, showing that the methodology is independent of the sign of the rotation.