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Theory of Operation |
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Introduction The PDR-Chiral Inc. chiral detector is based on laser polarimetry. A highly polarized and well-focused laser beam is passed through specially designed flow cells (HPLC, Process Control, SMB, Preparative, SFC, CE, etc.). As the sample passes through the flow cell, the detector measures the subtle rotation of the plane of polarization of the laser beam. The instrument utilizes proprietary low-noise signal extraction techniques to produce a sensitivity of better than 25 microdegrees. The rotation of the polarized light is directly proportional to the net value of optical activity present in the flow cell and is measured by the laser polarimeter in real time. For example, the signal from an enantiopure sample will be directly proportional to its concentration and specific rotation. Also, right-hand rotation will be indicated by a positive value while left-hand rotation produces a negative value. Consequently, a totally racemic sample (50:50 mixture of equal but opposite enantiomers) will produce a net signal of zero because the rotations cancel each other. The most common situation is to have a major and a minor enantiomer. This condition is usually specified as the enantiomeric excess (EE) of the major, presumably preferred, enantiomer. It may also be expressed as enantiomeric purity. In either of these situations, the laser polarimeter can be used to determine EE, provided the concentration and specific rotation are already known. Specific Rotation
By using small, known amounts of enantiopure samples, measurements can be taken with the PDR-Chiral Advanced Laser Polarimeter and combined with the equation shown above to make accurate specific rotation calculations. Here, [a] is specific rotation, a is the measured rotation, l is the path length of the flowcell and c is the concentration of the sample. Enantiopure measurements can generally be obtained by using a chiral separation method based on the wide variety of chiral columns currently available from companies like Chiral Technologies (Exton, PA) and Advanced Separations Technologies (Whippany, NJ). The specific rotation of an enantiomer can be used to differentiate closely related species. As an example, gentamicin, an antibiotic material, is available commercially as a mixture of four structural analogs. These analogs differ in terms of minor structural perturbations, for example, the substitution of a methoxy (OCH3) for a hydroxyl (OH) group. Also, specific rotation can be used to differentiate the pattern of amino acids in peptides and small proteins. In these situations, the molar rotation provides a mass corrected measure of the innate optical activity of the compound, thereby facilitating a comparison between similar, but non-identical, species. The sensitivity of [a] to subtle structural variations at, or near, a chiral center is illustrated by the fact that significant differences are noted between the [a] for gly-ala and ala-gly. While glycine is not optically active, alanine is. Thus, the order with which an optically active amino acid appears in a peptide obviously influences the peptide’s optical activity.Key Points
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