Polarimeter Evaluation

 

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Polarimeter Evaluation
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Paper 03

 

Evaluation of PDR Laser Polarimeter as HPLC Chiral Detector
Daniel W. Armstrong, Yan-Song Liu, Tim Yu
Department of Chemistry, University of Missouri-Rolla, Rolla, MO 65401

 

Abstract

A recently developed chiral detector, essentially a laser polarimeter, was evaluated for HPLC systems. Various kinds of chiral compounds were detected with considerably different sensitivity. For those compounds which lack a UV chromophore, this detector was much more sensitive, hence it can be used complementarily to the commonly used UV detectors. The detection limits for several chiral compounds were measured and fairly large linear ranges were observed. It will be demonstrated in this poster that this chiral detector has its advantage in identifying chiral separation and the potential of measuring the enantiomeric excess (ee) when there is not a separation. The relationship between the sensitivity with the [a]D value will be discussed.

Introduction

The analysis of chiral compounds has become increasingly important in areas of pharmaceutical, agrochemical and environmental science [1-6]. HPLC methods are among the most effective and widely used techniques for the analysis and preparation of enantiomeric compounds [7-9]. By using chiral stationary phase or chiral mobile phase additives, a large number of racemates have been resolved and the enantiomeric purities of some compounds have been determined in real-world samples.

There are several types of optical detectors for HPLC and each of them has many limitations. Refractive index detectors suffer from a lack of sensitivity and selectivity and gradient elution can hardly be used. Absorption detectors have been the most successful but generally require the presence of convenient absorption bands. Those saturated chiral compounds are not detectable. The far-UV and Fourier transform infrared detectors [10] have many technical problems such as the limitation of chromatographic eluents. By derivatizing the analyte before or after the separation some of those difficulties can be overcome, but convenience and reliability are degraded.

The HPLC detectors based on optical activity are potentially advantageous: there is no limitation in the choice of eluents and it is extremely selective. Chiral detectors are inherently useful in validating HPLC chiral separations. Used in series with a conventional detector, chiral detectors can sometimes be used to determine the enantiomeric composition of an analyte even when there is not a separation.

Currently, chiral detectors are based either on polarimetry or circular dichroism. A CD-based chiral detector was shown far from ideal as a stand-alone LC detector because of its low sensitivity [11]. Comparatively, more efforts have been put in the polarimetry based detectors.

In 1980, Edward Yeung et al [12] reported an HPLC detector based on optical activity. A detection limit of 0.5 mg is demonstrated in the separation of sugars. Since then more improvements have been made to this type of detector and more applications have been demonstrated by several research groups [13]. At certain stages of improvement, a couple of compounds were chosen to evaluate the performance of the detector, such as linear range and detection limits. In addition, the chiral detector's capability of determining the enantiomeric purity without separation was shown with a few amino acid samples [14].

The use of laser light sources made a major contribution to the improvement in performance of chiral detector. An argon ion laser [12] and helium-neon lasers[15,16] have been used. However, many lasers, particularly gas lasers, exhibit a high degree of intensity instability and flicker noise at low frequencies. Diode lasers [17] have inherently low flicker noise and a commercially available diode laser was used in a detector at a near-infrared (820 nm) wavelength.

A polarimetry-based chiral detector was recently commercialized by Product Development Resources (PDR). Bearing a number of pending US and foreign patents, this product has specially custom-built laser diodes and rotators, which are installed to enhance the performance. They are more reproducible in performance, for example, than commercially available products. The operating wavelength from the diode-laser was chosen as 675 nm to avoid certain problems seen in other commercially available polarimeters. Such problems could be poor detection limits, unstable baseline or anomalous results due to absorption and/or refractive index efforts particular to certain compounds.

To evaluate the overall performance of the PDR chiral detector, we have chosen over a hundred chiral compounds with different optical activities to test the applicability of the detector. Other evaluation tests include the linear range and detection limits, comparison with UV detectors and ee determination without chiral separation. Over three months under continuous use the instrument has been consistent and reproducible results.

In addition, for all the tested compounds, the direction of optical rotation at the detection wavelength (675 nm) was found to be identical with that at Sodium D line (589 nm). This can be explained by ORD spectra. Fig. 1 shows two typical ORD spectra. At short wavelengths, the direction of optical rotation may be opposite at different wavelengths. But at longer wavelengths (>500 nm), usually there are few variations in the direction of rotation with wavelength. This observation, that most chiral compounds have the same sign of rotation (+ or -) at 675 nm and 589 nm may be quite convenient in validating chiral separations, because most of the available configuration and specific rotation information in the literature is for the sodium D line. It should be pointed out that optical rotation is a function of solvent, concentration, temperature and wavelength, therefore the detector response at 675 nm doesn't increase linearly with [a]D.

Experimental

Materials
All HPLC columns were obtained from Advanced Separation Technologies, Inc. (Whippany, NJ). The LC column used were Cyclobond I 2000 (native b-Cyclodextrin, 25 cm x 4.6 mm, i.d.), Cyclobond 1 2000 RSP (2-hydroxypropyl-b-Cyclodextrin, 25 cm x 4.6 mm, i.d.) and C18 (5mm spherical ser.# 53-14, 5 cm x 4.6 mm, i.d.). All solvents (methanol, acetonitrile, glacial acetic acid and triethylamine) were purchased from Fisher Scientific (St. Louis, MO). All chiral compounds were purchased from Aldrich, Sigma or Fluka.

Apparatus
The LC enantio-separations were performed using the following equipment: a Shimadzu LC-6A pump, a CR 601 Chromatopac integrator, a SPD-2AM spectrophotometric detector, a RID-10A Refractive Index detector and a PDR-Chiral Laser Polarimeter. The detection wavelength was set at 185 nm for cyclodextrins and sugars or 254 nm for chiral separations. The LC chromatograms for all chiral compounds were obtained by using the following Shimadzu equipment: two LC10AT pumps, a SIL-10A autoinjector, a SLC-10A system controller, a CR 501 chromatopac integrator and a PDR-Chiral Laser Polarimeter. All chromatograms were run at ambient temperature (22 oC). All experimental conditions for over 200 commercially available chiral compounds are given in Table 1.

Results and Discussion

A. Detector Response vs. [

Results and Discussion

A. Detector Response vs. [a]D]D
Over one hundred chiral compounds with different [a]D values were tested for their response with the PDR chiral detector. All the solutions were of the same concentration (3 mg/ mL). The results are shown in Table 1. Compounds with optical activity can be detected with the PDR chiral detector with various response. Generally, the compounds with larger [a]D generate larger response.

B. Detection Limit
The detection limits for several compounds with various [a]D were determined at S/N = 3. Among the six compounds tested, the detection limit can be as low as 0.05 mg. As is shown in Fig. 2, the detection limits decreased with increased [a]D . This is in agreement with other polarimeters using light source at different wavelength.

C. Linear Range
The response linear range was tested for three compounds. (I) (S) - (+) -1,1’- Binaphthyl - 2,2'- diyl hydrogen phosphate, (II) (S) - (+) - 4 - Benzyl - 3 - propionyl -2 - oxazolidinone, (III) (4R)-(+)-isopropyl-2-oxazolidinone. They represent high (I), [a]D=595o, medium (II), [a]D =97 o and low (III), [a]D = 23 o optical rotation at Sodium D line. The linear ranges for the three compounds are in the range of two orders of magnitude. As can be seen in Fig. 3-5, a perfect linearity (R2=0.997-0.999) was observed.

D. Comparision with Other Detectors
In general, a chiral detector can be used in series with a conventional detector, UV or refractive index (RI) detector for example. For the chiral compounds that can not be detected by UV, the PDR-Chiral detector is a good alternative as a stand-alone detector and it can be more sensitive than RI detector. Fig. 6-7 shows the chromatograms of cyclodextrins and sugars detected by PDR chiral detector, refractive index detector and UV detector respectively. For the compounds that have UV chromophores, the chiral detector can conclusively validate the chiral separations as shown in Fig. 8.

E. ee Measurement.
One other important feature of the chiral detector is that if it is used in series with a conventional detector, the ee (enantiomeric excess) can be calculated when there is not a separation. The mixtures of (+)/(-)-4-Benzyl-3-propionyl-2-oxazolidinone at various ratios were analyzed using this method. Each data point is the average of 3-5 chromatograms. Since in a certain range the PDR-Chiral detector responds linearly with optical rotation, the responses should be proportional to ee. Fig. 9 is the calibration curve. It can be used directly for determining enantiomeric excess without a chiral separation. It should be pointed out that at very low ee, the relative error is high. It is reported that even if a high-precision UV detector were used, the relative error constant can still be in the 3% range. [13,14]

Conclusion

A commercialized chiral detector from PDR was evaluated. Compounds with optical activities can be detected with this system. In general, the sensitivity increases with the [a]D and the detection of rotation at detection wavelength (675 nm) is identical with that at Sodium D line. The detection limits are in the submicrogram level and linear range is in the range of two orders of magnitude. This detector is relative stable during over 3 months of continuous use. It's suggested that it can be used as a stand-alone detector for chiral compounds that lack a UV chromophore. Used in series with UV detector, it can conclusively confirm the chiral separation. Also, enantiomeric excess ratios can be calculated even when there is not a separation.