Circular Dichroism Detection Research Articles

A proposal for dichroic experiments in the electron microscope

Building upon the similarities between inelastic electron scattering and X-ray absorption we show that dichroism can be observed in electron energy loss spectrometry (EELS) in the transmission electron microscope (TEM). Natural or magnetic linear dichroism can be studied in electron scattering experiment with definite wave vector transfer in the interaction. The detection of circular dichroism in the TEM relies on interferometric EELS in a particular scattering geometry that allows extraction of the mixed dynamic form factor from energy loss spectra. Similarities between dichroic signals in energy loss near edge structures and X-ray absorption near edge structures are discussed, and a new experimental setup for dichroic measurements in the TEM is proposed.

CD spectrometric methods for the simultaneous determination of ethisterone and its delta5-isomer.

Quick and accurate direct and indirect circular dichroism (CD) spectrometric methods were developed for the simultaneous determination of ethisterone (17alpha-ethinyl-17-hydroxy-4-androstene-3-one) and its delta(5)-isomer (delta(5)-ethisterone). The direct method is based on the selective negative Cotton effect of the delta(4)-3-oxo group in ethisterone (negative maximum at 348 nm in dioxan) and measurement of the ellipticity at 296 nm (positive maximum of delta(5)-ethisterone), where the measured ellipticity is the sum of those of the two isomers. In the indirect procedure delta(5)-ethisterone is transformed to ethisterone by base-catalysed isomerization and the ellipticities are measured at 339 nm in ethanol before and after isomerization. Preliminary experiments show the usefulness of CD detector in the HPLC determination of the mixture of the isomers. A major advantage of the direct CD spectrometric and the HPLC/CD methods is that the delta(5)-isomer with extremely low UV activity can also be directly measured with high sensitivity.

Stereospecific analysis of omeprazole in human plasma as a probe for CYP2C19 phenotype

Omeprazole is a class referred to as proton pump inhibitor; it acts to regulate acid production in the stomach and is used to treat various acid-related gastrointestinal disorders. In the liver, it is metabolized to varying degrees by several cytochrome P-450 (CYP) isoenzymes which are further categorized into subfamilies of related polymorphic gene products. The metabolism of omeprazole is to a large extent dependent on CYP3A4 and CYP2C19. Omeprazole is metabolized to two major metabolites, 5-hydroxyomeprazole (CYP2C19) and omeprazole sulfone (CYP3A4). Minor mutations in CYP2C19 affect its activity in the liver and, in turn, the metabolic and pharmacokinetic profiles of omeprazole. The frequency of CYP2C19 poor metabolizers in population of Asian descent has been reported to range from 10 to 20%. Accordingly, results from population studies indicate that omeprazole can be used as a probe drug for phenotyping CYP2C19. The optical isomers of omeprazole show a clear difference in their metabolism by human liver microsomes. This study demonstrates the stereospecific analysis of omeprazole in human plasma as a probe drug of CYP2C19 phenotyping. The chiral separation of omeprazole was achieved on a chiral column with circular dichroism (CD) detection and LC/MS. A good resolution of enantiomers was obtained. The column used for chiral separation was CHIRALPAK AD-RH column (4.6×150 mm) using phosphate buffer and (or ammonium acetate) acetonitrile as an eluent. After a single oral dose of omeprazole (20 mg), the plasma concentrations of the separate enantiomers of omeprazole were determined for 3.5 h after drug intake. The present study is useful because of the part polymorphism plays in the therapeutic effectiveness of proton pump inhibitors during the treatment of acid-related diseases.

Fluorescence-detected circular dichroism by modulated beam in the wavelength axial direction.

A novel device is described that permits fluorescence-detected circular dichroism (FDCD) detection in a conventional fluorescence spectrophotometer. FDCD is a phenomenon in which the emission intensity of a chiral analyte depends on the polarization state of the excitations. Although FDCD is a sensitive chiral detection technique, measurement of FDCD by using a special CD spectrometer has involved many difficulties. The proposed device consists of a polarizing prism and a retardation plate that fit on the excitation light side in the sample compartment of the fluorescence spectrophotometer. In this method, a FDCD wave superimposed on the fluorescence excitation spectrum of a chiral analyte was detected. We have also demonstrated the possibility of the enantiomeric purity determination of chiral analytes in real samples.

Development of a nonchiral HPLC method with circular dichroism detection for chiral analysis of molybdenum-catalyzed enantioselective synthesis products.

The combination of a circular dichroism detector and nonchiral liquid chromatography was used for the chiral analysis of unresolved enantiomers to determine the enantioselectivity of a molybdenum-catalyzed asymmetric allylic alkylation reaction. The CD/UV peak area ratio of the unresolved enantiomers was calculated and compared with that of a reference standard to determine the enantiomeric purity. The limit of quantitation (LOQ) is 20 ng for the chiral ligand and 1 microg for the branched chiral product. The viability of the system depends on the limit of quantitation of the CD response and the linearity range of the CD and UV response.

Stereospecific analysis of loxoprofen in plasma by chiral column liquid chromatography with a circular dichroism-based detector

The chiral separation of loxoprofen was achieved on a chiral column with UV and circular dichroism (CD) detection. The good resolution of four loxoprofen stereoisomers was obtained. The column used for the chiral separation was Chiralcel OJ column (250×4.6 mm) using hexane–2-propanol–trifluoroacetic acid (95:5:0.1), as an eluent. The flow-rate was 1.0 ml/min and the detection was at 225 nm. In addition, CD and UV spectra were obtained by stopped flow scanning. The method allows the determination of the stereoisomers of loxoprofen in human plasma after the administration of therapeutic dose of the racemic drug, thus HPLC with CD detector is useful for the stereospecific determination of loxoprofen products in biological samples.

Enantiomeric purity determination of malic acid in apple juices by multi-beam circular dichroism detection

A multi-beam circular dichroism (CD) detector which is easily constructed by inserting inexpensive optics into a conventional photo-diode array detector has an advantage of simultaneous detection of the absorbance and CD. The enantiomeric purity determination of malic acid in beverages was performed by this detection system. Malic acid when complexed with Cu(II) was found to have an absorbance maximum at around 750 nm. The l-malic acid–Cu(II) complex showed a positive Cotton effect in its absorbance band and its anisotropy factor (Δ ε/ ε) was relatively large at about 1/170. This complex was retained on a reversed-phase column with the addition of racemic 2-hydroxy-3-methylbutyric acid to the mobile phase as the ligand. A plot of the relative peak areas between the CD and the absorption (Δabs/abs) versus optical purity showed good linearity with a correlation coefficient of 0.999, and the precision expressed as the relative standard deviation of the errors from the regression line was ±2.7% (2 σ). The accuracy of the proposed method was assessed by capillary electrophoresis. Eight commercially available juice products were analyzed using this method. Five of them were thought to be adulterated with synthetic malic acid.

LB 8827: A Variable, Magnetic DBA White Dwarf

We present accurate CCD spectropolarimetry and spectra that show that the hot DB star LB 8827 (PG 0853+164) is a weakly magnetic, variable DBA white dwarf. We confirm Putney's detection of circular dichroism in the He I lines, but on three occasions we measure a weaker field strength and opposite sign of circular polarization, Be 100 kG. The Balmer line strengths appear to vary with time, indicative of changes in the apparent H/He abundance ratio or irregularities over the stellar surface. Fits to the observed energy distribution and to the optical He I line spectrum with model atmospheres yield effective temperatures in the 21,200-27,700 K range, with lower values favored. This suggests that LB 8827 falls within or near the instability strip of the V777 Herculis stars. High time resolution photometric observations presented here rule out white-light brightness variations with amplitudes larger than 1.2 mmag in the period window 20 s to 2 hr. The possibility that the magnetic field might tend to suppress pulsations is discussed.

Chiral analysis of drug substance in clinical plasma extracts using achiral HPLC with circular dichroism detection.

A circular dichroism (CD) detector in series with a UV detector was used with reversed-phase gradient HPLC to do the chiral analysis of unresolved enantiomers of a single stereoisomer drug substance in extracts of clinical plasma samples. The CD/UV peak area ratio of the unresolved enantiomers in the plasma extracts was calculated and compared with pure drug substance standards to show that there was no racemization of the chiral center during pharmacokinetic studies. The CD detector can be used only with chiral compounds with a UV chromophore and the limit of detection was approximately 5 ng with the drug substance and chromatographic conditions used in this study.

Recent advances in chiral detection for high performance liquid chromatography

The unique response characteristics of optical activity detectors make them an important adjunct to high performance liquid chromatography (HPLC) for the analysis of biologically active molecules. The recent commercial introduction of turn-key, circular dichroism (CD) and polarimetric detection systems compatible with the sensitivity and volume requirements of HPLC will allow these detection approaches to be applied to measurement problems in a number of areas including biomedical research and diagnosis, biotechnology, as well as pharmaceutical science. The CD and polarimetric detectors respond directly to the intrinsic optical activity of the analyte, and both the magnitude and sign of the response can be used for quantitative and qualitative determinations. The information available from these detectors is difficult or impossible to obtain by other methods. In this article, the instrumental basis for both the CD and laser-based polarimetric detectors will be reviewed. Several applications will be described in which the unique response characteristics of these detection systems, either alone or in tandem with other detectors, can be used to identify chiral materials, and determine enantiomeric excess. It will be shown that this methodology is effective either without enantiomeric separation, or under conditions of low enantio-selective resolution. With this technology, a real-time assessment of enantiomeric excess can be obtained as a means to optimize and guide enantio-selective separation processes.

Stereospecific analysis of lorazepam in plasma by chiral column chromatography with a circular dichroism-based detector

The chiral separation of lorazepam was achieved on a chiral column with UV and circular dichroism (CD) detection. The good resolution of lorazepam enantiomers was obtained on the column of β-cyclodextrin derivative immobilized silica gel under reversed-phase conditions. The enantiomeric separation and identification of lorazepam were succeeded by CD detector. The method described allows the quantitation of the stereoisomers of lorazepam in human plasma following the administration of a therapeutic dose of the racemic drug. Chiroptical detection is useful for the pharmacokinetic study of chiral drugs.

Multi-beam circular dichroism detector for HPLC

A novel multi-beam circular dichroism (CD) detector for HPLC is described. A unique feature of this detector is the use of a retardation plate to give many quarter-waves in the wavelength region of interest. When a polarizing prism and a thin quartz plate as the retarder are placed in a conventional photodiode array (PDA) detector, the kinetic spectra of an optically active analyte recorded by the instrument contain sinusoidal CD waves in the wavelength axial direction. Extraction of these CD waves superimposed on the absorption spectra was performed with MS Excel. Both the instrumental conditions for the PDA data acquisition and the polynomial data processing were optimized by monitoring the chromatographic elution of camphor. The limit of detection for ajimaline, a chiral alkaloid, was 26 ng injected amount.

Algorithms for the quantitative validation of chiral properties of peptides.

Complexation with Cu(II) ion in strong aqueous base, combined with visible range circular dichroism detection, were used to quantitatively differentiate among the L-enantiomers of the GG, GA, GY, AG, AA, AY, YG, YA, and YY dipeptides and the D-enantiomer of GA. Using ellipticity data at all (n = 1500) wavelengths in the measured spectra, and two novel data reduction procedures, quantitative determinations were made of the compositions of binary mixtures. For mixtures made with the L-GA and D-GA enantiomers, the accuracy of the measured enantiomeric purities was better than 0.17 % over the 1-48 % range for the minor component. The method has considerable potential for use in quality control of peptide and protein biotechnological drug forms.

Characterization of peak purity by fast multiscan circular dichroism detection

A method has been developed to detect inhomogeneity of apparently homogeneous peaks of very similar analytes. The method utilizes the rapid scan feature of state-of-the-art spectrometers/detectors that allow the recording of up to 30 spectra in a single chromatographic peak. Sensitivity and selectivity are enhanced by chiroptical/optical detection. Thus, identification of “front” and “rear” components of the peak can be carried out. The method is exemplified by mixtures of codeine, hydrocodone and oxycodone as analytes.

Instrumentation Developments for X-ray Linear and Circular Dichroism at the ESRF Beamline ID12A.

This paper reports on the performance of the instrumentation developed for the ESRF beamline ID12A, which is dedicated to spectroscopic applications requiring full control of the polarization at energies >/=2.0 keV. Emphasis is placed on the characterization of various optical components of the beamline and on problems associated with either the control or the conversion of the polarization state. A few examples have been selected to illustrate what sort of new spectroscopic information has already been obtained at the beamline. These include the comparison of X-ray magnetic dichroism spectra recorded with linear or circular polarization and the very first detection of X-ray natural circular dichroism in single crystals known to exhibit a very large non-linear susceptibility at optical wavelengths.

Determination of unbound warfarin enantiomers in human plasma and 7-hydroxywarfarin in human urine by chiral stationary-phase liquid chromatography with ultraviolet or fluorescence and on-line circular dichroism detection

Enantiomers of warfarin and 7-hydroxywarfarin in human plasma and urine, respectively, were determined by high-performance liquid chromatography using a cellulose-derivative column with UV or fluorescent detection, and their absolute configuration was determined simultaneously by a circular dichroism spectropolarimeter connected in series. Enantiomers of warfarin and its major metabolites [i.e., ( R)-6-hydroxywarfarin, ( S)-7-hydroxywarfarin and ( RS)-warfarin alcohol] were well resolved. The method was precise and sensitive: within- and between-day coefficients of variation were <9.6% for warfarin enantiomers in plasma and <7.1% for 7-hydroxywarfarin enantiomers in urine, respectively, and the lower detection limits were 20 ng/ml for ( R)-warfarin, 40 ng/ml for ( S)-warfarin, 2.5 ng/ml ( R)-7-hydroxywarfin and 4.5 ng/ml for( S)-7-hydroxywarfarin in 0.5 ml of both plasma and urine. The ultrafiltration technique was used for determining unbound concentrations of warfarin enantiomers in plasma using [ 14C]warfarin enantiomers resolved by the present HPLC system. Clinical applicability of the method was evaluated by determining unbound concentrations of warfarin enantiomers in five consecutive plasma samples obtained from a patient exhibiting an unstable anticoagulant response to warfarin (4 mg/day, p.o.). Results indicated that the present method would be useful in clarifying factors responsible for a large intra- and inter-patient variability in warfarin effects with regard to unbound plasmaenantiomer pharmacokinetics.

Separation of benzodiazepines on a new carbohydrate-based chiral stationary phase for HPLC.

Several benzodiazepines were enantioresolved on a new carbohydrate chiral stationary phase based on maltooligosaccharides. The role of organic modifier, ionic strength, pH and temperature are examined and the results are discussed. In general, selectivity and retention were found to decrease with increasing organic modifier concentration. The appearance of two peaks for diazepam suggests that the reported separations are based on separation of conformers and was supported using circular dichroism detection. Counterintuitively, resolution and enantioselectivity were found to improve with increasing temperatures.

Circular Dichroism Spectroscopy Using Coherent Laser-Induced Thermal Gratings

A new pulsed four-wave mixing technique for the detection and real-time measurement of circular dichroism (CD) in liquid samples is demonstrated. The technique is based on the formation and detection of transient thermal gratings formed by the interference of two laser beams whose polarizations are controlled and modulated using a photoelastic modulator. Through an internal heterodyne process, coherent thermal gratings interfere to greatly enhance a weak circular dichroism signal. By measuring the ratio of the difference of scattered laser light from two different polarizations, ΔS, to the average amount of scattered light, Save, one can determine the value of Δε/ε for the compound under investigation. Samples of chiral camphorquinone are used to demonstrate the technique in the ultraviolet region of the spectrum. These studies of camphorquinone, which has a value of Δε/ε ≈ 10-3 at 266 nm, produce values of ΔS/Save which are orders of magnitude larger than Δε/ε and which approach 2 (the mathematical limit for ΔS/Save). Possible extensions of the technique for measuring CD in very small sample volumes and monitoring time-dependence are discussed.

Electrically and optically active molybdenum impurities in commercial SiC substrates

We report on the identification of optically and electrically active molybdenum impurities in commercial 6H- and 15R-SiC substrates by conventionally and optically detected magnetic resonance (ESR, ODMR) and magnetic circular dichroism (MCD) detection. Two impurity charge states, Mo 4 + (4d 2) and Mo 3 +(4d 3), are reported for both polytypes. We also observed the 3A 2 → 3T 2 crystal field absorptions of Mo 4 + (4d 2) in the 1.0–1.25 eV spectral range. Mo impurities on different inequivalent lattice sites in 6H- and 15R-SiC are resolved for the first time.

Kinetic Analysis of the Folding of Human Growth Hormone: INFLUENCE OF DISULFIDE BONDS

We report the results of a stopped-flow kinetic evaluation of the folding of human growth hormone (hGH). The results are compared with those obtained for a disulfide-modified analog in which the four cysteine residues have been reduced and alkylated to form tetra-S-carbamidomethylated hGH in order to elucidate the role of disulfide bonds in the folding reaction. Multiple detection techniques were applied to monitor both refolding and unfolding processes initiated by guanidine hydrochloride concentration jumps. Using far-UV circular dichroism (CD) detection to monitor folding of hGH, we find that 70% of the secondary structure forms in a burst phase occurring within the stopped-flow dead time. Two slower phases were identified in the observable portion of the CD signal. Multiple kinetic phases were resolved when folding was monitored by intrinsic tryptophan fluorescence or near-UV absorbance as probes of tertiary structure, and the number of time constants required to fit the data depended on the hGH concentration and nature of the denaturant jump. The associated amplitudes also displayed strong dependence on the final denaturant concentration. Results obtained from the tetra-S-carbamidomethylated hGH studies demonstrate that the folding reactions of hGH are remarkably similar in the presence and absence of the disulfide bonds. Disulfide bond reduction in hGH is proposed to affect folding primarily by increasing the population of self-associated intermediate states in the folding pathway.