Limits of Detection

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Introduction

In the section on Chemical Effects we learned that optical activity is affected by concentration, solvent, pH and temperature. Thus, proper method optimization can have a pronounced effect on the Limits of Detection (LOD). There are at least two approaches to detecting " other enantiomeric" impurities with the PDR-Chiral Detector:

Use a chiral separation method to compare the relative peak heights or peak areas of the two enantiomers.

Use an achiral separation method and measure the net rotation of mixed-enantiomer peaks.

The second approach assumes that specific rotation is known or has been previously measured using our instrument. To achieve good accuracy it is necessary to make the specific rotation measurement at the same concentration, solvent, pH and temperature to be used in the trace measurement method. Any reduction from the expected value of rotation is then due to the presence of the other enantiomer. Variations in overall HPLC system performance, such as uncertainty in the exact amount of sample injected, often limit this approach to � 0.5% accuracy.

The First Approach

The detection limit data collected by Dr. Daniel Armstrong and graduate students early in 1998 at the University of Missouri at Rolla are plotted below as the upper trace (diamonds labeled "Armstrong").

The other two traces (squares labeled "Armstrong/3" and triangles labeled "Armstrong/30") are plotted as reference to potential improvements in overall system performance, as compared to the methods, columns, HPLC, and PDR-Chiral Detector system they used. Their objective was to characterize the relative response of a large number of chiral molecules, not optimize detection limit.

The "Armstrong/3" is a 3 times improvement and the "Armstrong/30" is a 30 times improvement. From the chart in figure 44, it is clear that, assuming no improvement in overall system performance, as compared to Armstrong, and accepting an LOD of 800 nanograms,then we could comfortably operate over a wide range of specific rotations.

The next chart plots LOD, in nanograms, versus injected mass in micrograms for a 0.1% impurity. Thus an LOD of 800 nanograms requires an injected mass of 800 micrograms.

If a factor of 3 is realized by optimizing the HPLC system and method, then the LOD is reduced to 267 nanograms and the injected mass is reduced to 267 micrograms. This is the middle trace ("Armstrong/3") in the previous chart.

The most optimistic trace on the previous chart ("Armstrong/30") shows an additional improvement factor of 10. This brings the injected mass down to 27 micrograms.

The Second Approach

The chart below plots the range of possible values for the net rotation of a peak that contains both enantiomers. In order to detect a 0.1% enantiomeric impurity, the system must have a limit of quantitation (LOQ) better than 0.1% of the injected mass.