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OR versus CD |
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Origin of Response Circular dichroism (CD) is the differential absorption exhibited by a system for left- versus right - circularly polarized light. In order to demonstrate CD activity, a system must contain a dissymmetric center that is coupled in some manner to the chromophore giving rise to the absorption. Thus, in order for a system to be CD active, the term De must be non-zero.i.e. De = el - er 0
In optical rotatory dispersion (ORD), the difference in refractive index ( Dh ) for left- versus right-circularly polarized light exhibited by an optically active system is measured. In order to demonstrate ORD activity, a system must also contain a dissymmetric center.i.e. Dh = h l - h r 0
Since the refractive index of a compound changes very slowly as a function of wavelength, except in the spectral region of an allowed transition, single wavelength optical activity measurements are frequently used. This mode is termed polarimetry. It is experimentally convenient, sensitive, and requires minimal instrumental complexity. Structural Requirements The presence of a chromophore and a chiral asymmetric center in a molecule is not sufficient for CD activity. The two moieties must be in intimate electronic contact in order for the absorbing center to be CD active. This dual requirement means that not all optically active compounds will exhibit a useful CD signal. In order to provide a polarimetric response, a molecule is only required to possess an asymmetric center. The polarimetric response is thus more universal. In general, polarimetry provides a response for virtually all chiral molecules. Wavelength Selection Since CD requires probing at wavelengths corresponding to a region where the molecule absorbs, the probing wavelength chosen is critical. For many molecules which do not possess a chromophore accessible in the UV or visible region, short UV wavelengths (<250 nm) must be used. In static measurements, this is not a problem since small amounts of extremely pure solvents can be used for sample preparation. However, for dynamic detection in HPLC over the far UV range, large quantities of ultra-pure and expensive solvents must be used for both sample elution and to facilitate the CD analysis. At a typical flow rate of 1 mL per minute with a 4.6 mm column, approximately 500 mL of solvent would be used in an 8 hour period. In polarimetric analysis, the probing wavelength is in a region where solvent interference does not affect the measurement. Selectivity Since optical activity is a rare property, usually indicative of biological activity, past or present, chiral-selective measurements are, by their nature, selective. CD, due to the requirement that the chromophore and chiral center must be coupled, does not produce a response for all chiral molecules. Fortunately, polarimetric measurements do produce a response for all chiral molecules. In HPLC, SFC, or CE the use of UV/Vis absorbance detection is popular but actually a limitation. Since the separation system will provide significant discrimination among sample species, a detector that responds only to optically active materials (but not achiral compounds) is often desirable. Universality of Response Polarimetric detection will provide a response for virtually all chiral molecules, while CD, due to the dual requirements of chirality and absorptivity, is more selective. For detection following separation, one could argue that CD's additional selectivity can actually be a detriment and not a benefit since modern high performance separation techniques are extremely efficient in resolving closely related structural analogs. Thus, for a chiral-selective analysis, a technique that responds to virtually all chiral materials would be proferred. Sensitivity In CD measurements, the magnitude of the response is dependent upon the term De, while for polarimetric measurements, the response is proportional to Dh . In general, relative sensitivity for these two techniques will be molecule specific. Another way to address this issue is to see if the technique meets the requirements of modern HPLC. One of the problems with adapting conventional CD or incoherent source-based polarimetric measurements to HPLC has been the low response in sensitivity, which required large sample loading. This in turn led to degradation of the analytical performance of the separation system.The PDR-Chiral Detector has a rotational sensitivity of 25 mdeg. For a weakly optically active compound with Da = 10 (deg (g/mL)-1 dm-1), this translates into a limit of detection of 5 mg/mL (25 mM; MW = 200) for a 5 cm interaction length. For comparison, an analytical scale HPLC column possesses a sample capacity of approximately 0.5 mg. In terms of a 10 mL injection volume, the concentration at which overloading would be a problem is thus 5 x 10-2 g/mL, which is 3 - 4 orders of magnitude larger than the polarimetric LOD. For more strongly optically active molecules, the LOD scales proportionally, and LOD’s of 1 - 2 mM have been determined for various sugars.For a weakly absorbing CD compound (e.g. cholesterol at 300 nm), a De of 2 x 10-4 is typical. Conventionally, a differential absorbance of 50 ppm (5 x 10-5 cm-1) is measurable. For the same 5 cm interaction length, the concentration LOD is estimated to be 1 x 10-2 M, which is on the order of the analytical column limit for sample loading. For CD active molecules such as transition metal complexes, De’s of 2 x 10-2 have been reported and the concentration LOD would decrease proportionally. However, for most organic compounds, a De similar to that of cholesterol is typical. Thus, for CD detection in HPLC, one must be aware of these limits and optimize the measurement for detection rather than separation efficiency, by paying attention to solvent background absorbance, etc.Calibration In polarimetric measurements, the Faraday effect can be used to produce an exact physical standard. In the Faraday effect, a precisely determined current is passed through a coil composed of a specified number of turns of magnet wire. The strength of the resulting magnetic field can be determined to a very high degree of precision. The rotation induced by this magnetic field is therefore also known to the same level of precision. This non-chemical standard is not affected by solvent, ionic strength, other molecules, etc. In circular dichroism, a CD-active compound is used to standardize the instrument (usually d-camphor). Chemical standards are affected by the experimental variables which are difficult to keep constant or to control in a dynamic measurement situation, such as encountered in HPLC. One of the advantages of having an exact physical standard for calibration is that the polarimetric response for an eluting compound can be precisely determined in terms of rotational units. This allows one to accurately, and precisely, determine specific rotations for eluting compounds. Specific rotation has been shown to be sensitive to the arrangement of atoms at, or near, the chiral center. Measurement of specific rotation can thus be used to discriminate between, and identify, closely related structural analogs. Source Modern polarimetric instrumentation suitable for application to HPLC utilizes a coherent, laser source. The spatial profile of the laser allows the light to be efficiently coupled to micro-volume detection cells. The PDR-Chiral Detector can be used in various separation modes from micro-volume HPLC or CE to prep-scale chromatography. Commercially available CD instruments require broadband, non-coherent light sources. These sources are difficult to couple to micro-volume detection situations. Additionally, long interaction path lengths are not possible due to the poor propagational characteristics of these sources. |