Practical Chiral Detectors
There are two basic types of chiral detectors for LC, those that measure optical rotation and those that measure circular dichroism. At the time of writing this book, the only commercially available chiral detectors are those that measure optical rotation. Nevertheless, a detector that measures circular dichroism and utilizes a diode array sensor system is thought to be in the design stage and will be briefly described later.
The successful development of a chiral detector based on optical rotation measurement hinges on the use of the Faraday effect. If a plane polarized beam of light passes through a medium that is subjected to a strong magnetic field, the plane of the polarized beam is rotated a small angle (a), where (a) is given by a = VdH
where (V) qis the Verdet Constant, (d) is the path length and (H) is the magnetic field strength.
This relationship is the Faraday effect. The magnetic field is generated in an air core coil inside of which is a rod made from glass having a high Verdet constant. Now, where (i) is the current through the coil, and (N) is the number of turns in the coil.
Thus (a) can be controlled by varying (i).
The rotational resolution (Aa) can be as little as 10-5 but due to heat losses in the coil the maximum value of (Aa) is about ± 2°. A diagram of the detector is shown in figure 8.
Light from a tungsten lamp passes through two condenser lenses to a polarizer. Plane polarized light from the polarizer passes through a temperature controlled cell to a Faraday modulator and thence through an analyzer and onto a photomultiplier. The modulator is supplied with a crystal controlled AC component. If an optically active sample is present, the intensity of the light falling on the photomultiplier changes. By the use of a phase sensitive amplifier and electrical feed-back, the current though the modulator is automatically adjusted until no AC component appears on the photomultiplier output.
Figure 8
A Chiral Detector Monitoring Optical Rotation Courtsey of JM Science Inc.
Figure 8
A Chiral Detector Monitoring Optical Rotation Courtsey of JM Science Inc.
Unfortunately the sensor has a volume of about 40 |igl, which is extremely large for modern LC columns. However, the urgent need for chiral detection is such that the large cell volume is accepted, and is accommodated by the use of large diameter columns. The device is rigidly supported and due to its stability can measure a rotation (a) of 10-4°. The separation of some carbohydrates monitored by the detector is shown in figure 9. The separation was carried out on a column 12.5 cm long, 4.6 mm in diameter packed with Hypersil APS 1. The mobile phase was acetonitrile/ water (8:2), at a flow rate of 0.5 ml/min. It is seen that the resolution of the mixture is maintained to satisfactory level despite the very large cell volume.
Figure 9
The Separation of Some Carbohydrates Monitored by a Chiral Detector Courtesy of JM Science
Figure 9
The Separation of Some Carbohydrates Monitored by a Chiral Detector Courtesy of JM Science
It is also seen that the direction of rotation is clearly and unambiguously indicated by the direction of the peak. The peaks represent between 20 and 40 |ig of material, which also indicates reasonable sensitivity. From approximate calculations made from the chromatogram the sensitivity for peak (6) appeared to be about 1.4 x 10-7 g/ml, which is only a factor of 2-4 less than that obtainable from the diode array UV detector. However it is extremely difficult to estimate the noise from the chromatogram shown in figure 9. A more accurate estimation of the detector sensitivity can be made from the chromatogram of some essential oil components shown in figure 10.
The separation shown in figure 10 was carried out on a column 20 cm long and 8 mm I.D. packed with Zorbax silica. The mobile phase was n-hexane/chloroform (4:1) and the flow rate was 0.5 ml/min. The width of the second peak((-)-a-terpinyl acetate) is about 0.16 mm and from the flow rate axis, 1.06 cm is equivalent to a volume of 5 ml. Thus the peak width at the base is about 0.75 ml.
Figure 10
The Separation of Three Optically Active Fragrance Compounds Monitored by a Chiral Detector
Figure 10
The Separation of Three Optically Active Fragrance Compounds Monitored by a Chiral Detector
The peak represented 3 |im of material and, taking the concentration at the peak maximum as twice the peak average concentration, the peak maximum concentration was about 8 ^m/ml. The peak height appears to be about three times the noise and so the sensitivity (that concentration that will give a signal equivalent to twice the noise) is about 5.3 |im/ml or in more standard terms 5.3 x 10-6 g/ml. As the chiral detector is a bulk property detector, a sensitivity of 5.3 x 10-6 g/ml seems more realistic. Nevertheless, this sensitivity is a great improvement on many chiral detectors previously described.
The value of a chiral detector in the analysis of physiologically active materials is clear, but the methods so far used have been found somewhat insensitive. A more encouraging procedure would be the measurement of circular dichroism and such instrumentation employing diode array detection is presently under development. Details of the device are difficult to obtain due to patent applications pending and particulars are not available. The basic arrangement, however, is thought to be similar to that depicted in figure 11.
Wide Band Light Source
Wide Band Light Source
Photoelast îc Modulator
Second Order Spettrum
Photoelast îc Modulator
Diode Array 1 First Order Spectrum
Second Order Spettrum
Figure ii
Projected Diode Array Chiral Detector
Light from a wide band source such as a xenon lamp passes through a polarizer and photoelastic modulator producing left and right polarized light of all wavelengths generated by the lamp. The light then passes through the sample cell and the transmitted light falls onto a diffraction grating. The dispersed light is focused onto two diode array sensors. One array receives the zero order spectrum and the second the first order spectrum. It is claimed, that the output from these two diode arrays can be processed to give both the optical rotary dispersion and the circular dichroism for each group of wavelengths monitored by each diode of the arrays. This approach is a great advance on the simpler detector presently available and hopefully more details will be forthcoming in the not too distant future. Fundamentally, the system is a bifunctional detector that provides both chiral and absorption measurements on the column eluent.
It is recommended that those further interested in chiral measurements read a review paper by Drake [7] which gives an excellent survey of the subject and a paper by Drake and Jonas [8] that describes techniques for measuring optical activity.
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