Detection systems circular dichroism

The system shown in Scheme 2 is based on the fact that irradiation at 250 and 300 nm yielded photostationary states of slightly different composition. Irradiation of enantiomerically pure (M)-cis-10 or (P)-trans-10 at 250 nm resulted in a photostationary state consisting of 68% (M)-cis-10 and 32% (P)-trans-10, whereas irradiation at 300 nm yielded 64% (M)-cis-10 and 36% (P)-trans-10. Thanks to the thermal stability of these pseudoenantiomers— no (P)-cis or (M)-trans-10 were detected after irradiation—the small difference in composition of the photostationary states could be detected by circular dichroism (CD) spectroscopy. In this case, the photoinduced 180° rotation around the olefin results in a change in the helicity of the tetrahy-drophenanthrene unit. Similar behavior was observed for the structurally related benzoannulated bithioxanthylidene [28]. 

Other IRRAS applications to peptides and proteins. In addition to the pulmonary surfactant system, a variety of other applications employing IRRAS to study peptide and protein conformation and orientation have appeared. The secondary structure conversion of the amyloid (prion)-protein in the normal form into the abnormal form is the main cause of several human and animal diseases, such as Alzheimer s disease [68]. The secondary structure of the first 40 residues of the amyloid protein was detected by circular dichroism (CD) in aqueous solution and with IRRAS at the interface. A stable /1-sheet-enriched state of the amyloid is formed at the air-water interface, in contrast to the initial bulk solution containing high a-helix/random coil and low /l-sheet parts. The change in the pH going from bulk (alkaline pH) to the interface (neutral or slightly acidic pH) can have effects on the conformation at the interface. Another alternative might be the intrinsic hydrophobicity of the air-water interface, which is a hydrophobic-hydrophilic system with air as the hydrophobic part. 

In a suitably chosen mixed solvent system, such as acetic acid and 1-propanol (21), a sharp reversible transition between Form I and Form II can be achieved with a small change in solvent composition. Measurements of optical activity provide a convenient way to follow the transition in dilute solution. There are large changes in optical activity because Forms I and II are helices of opposite handedness. The left panel in Figure 3 depicts the reversible transition that is detected by circular dichroism measurements in mixtures of trifluoroethanol and 1-propanol. Form II is the only conformation present in trifluorethanol. A solution of poly(L-proline) in 35 65 trifluoroethanol 1-propanol exhibits the same circular dichroism pattern as does a solution where the solvent is pure trifluoroethanol. However, further addition of 1-propanol produces a dramatic change in the circular dichroism. At 20 80 trifluoroethanol 1-propanol the circular dichroism pattern is that characteristic of Form I. Data in Figure 3 do not extend beyond 10 90 trifluoroethanol 1-propanol because of the low solubility of poly(L-proline) in 1-propanol. 

A special mention in the field of enantioselective HPLC separations must be made of chiro-optical detection systems, such as circular dichroism (CD) and optical rotation (OR), which can be also used to circumvent the low UV detectability of chromophore-lacking samples [40, 61]. While sensitivity of chiro-optical detection is not always sufficient to perform enantiomeric trace analysis, the stereochemical information contained in the bisignate spectropolarimetric response is useful in establishing elution order for those compounds not available as single enantiomers of known configuration. An example of application of different online detection systems (UV and CD at 254 nm) in the enantioselective separation of a racemic sulfoxide on a commercially available TAG CSP is reported in Figure 2.12, under NP conditions. 

Enantiomeric pnrity assays have also been performed without chromatographic separation being conducted prior to detection, for example, with circular dichroism (CD) and MS. Bertncci et al. [110] developed a chiral assay for pulegone, oxazepam, and warfarin by combining simnltaneons UV, CD, and g factor detection on an achiral separation system with a Hypersil CN colnmn and a mobile phase of hexane 2-PrOH (90 10). The precision (RSD%) of the method ranged from 0.6% to 2.6%, and the LOQs were between 0.1% and 1% (0.2-2.2 j,g). For fnrther information concerning the application of CD and polarometric detection for chiral detection, see the review by Bobbitt and Linder [111]. 

Electronic spectroscopy (see Electronic Spectroscopy), in one form or another, has been the principal method used for the detection of short-lived intermediates. UV-visible absorption was the initial spectroscopic method used with flash photolysis and flow systems, and for each of these methods it remains the most commonly used approach. For species in low-temperature matrices, many varieties of electronic spectroscopy have been used. These include UV-visible absorption and emission, fluorescence, magnetic circular dichroism (MCD) and magnetic linear dichroism, and photoelectron spectroscopy. It is unfortunate, therefore, that in many cases electronic spectroscopy yields little or no stractnral information. The exceptions are high-resolution spectra, where vibrational or rotational flne structure may be seen. 

The crystal structure of both ribonuclease A and S have been determined and residues 2-13 form two-three turns of a-helix (4,). In the dissociated state, S-peptide contains little detectable helix either by circular dichroism (5) or by NMR (6,7). Either the S-protein induces the bound conformation during the association or a conformer too limited in concentration to be detected by spectroscopy is preferentially bound. It has been argued (3) on kinetic grounds that the induced-fit or "zipper" hypothesis is more likely, but no relevant experimental data exists on this or other systems to our knowledge. 

Sensitive Optical Detectors. More sensitive optical techniques that have been used with CE include fluorescence, refractive index, chemiluminescence, Raman spectrophotometry, and circular dichroism. The most sensitive optical detection method used in CE is laser-induced fluorescence (LIE), which is capable of detection limits in the 10 to 10" mol (or better) range. This detection mode is easily accomplished with analytes that are either easily labeled with a fluorescent substrate (e.g., intercalators for double-stranded DNA) or are naturally fluorescent (e.g., proteins or peptides containing tryptophan). CE systems have also been interfaced with mass spectrometers, and electrochemical detection methods have been developed, although such detectors must be isolated electrically from the electrophoretic voltages. 

The large environmental dependence of the ll-(3-hexyl-l-indolyl)undecyltri-methylammonium bromide fluorescence lifetime and fluorescence spectrum has led to its continued use as a fluorescence probe for micellar systems.76 Fluorescence-detected circular dichroism, a technique which appears to have considerable potential, has been applied to the determination of the c.d. spectra of L-tryptophan and L-cystine and two macromolecular systems.78... 

Treatment of a number of covalent polymers substituted with molecular recognition capability with suitable guests leads to chiral induction. The same crown ether-amino acid complementary pair described for the rosettes above was employed in the form of crown-ether pendant cis-transoidai poly(phenylacetylene). When the achiral polymer is treated with amino acids (in the form of their hydroperchlorate salts in acetonitdle) a large induced CD signal is observed in the backbone of the polymer. The polymer is sensitive to small enantiomeric excesses in the amino acid, as little as 0.005% enantiomeric excess of alanine can be detected. In a similar vein, c/5-transoidal poly(carboxyphenylacetylene) shows induced circular dichroism when treated with nonracemic chiral amines In addition, the system displays chiral memory, in that treatment of the complex with achiral amino alcohols results in retention of the chiral polymer backbone. 

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