Molar absorptivity chromatography

For the SAXS studies a CBH II sample was prepared by affinity chromatography from r. reesei QM 9414 to give the enzyme in a homogeneous form 27. In SDS-PAGE the protein had a size of 58 kDa and the isoelectric point was 4.9. Glycosy-lation was estimated as 8 to 18 % 36. The molar absorptivity at 280 nm was 75 000 M xm To obtain the core protein partial proteolytic hydrolysis with papain was per-... 

The molar absorptivity (e) of a known molecule is constant under identical conditions of solvent, concentration and path length, and can be used to quantify the amount of a particular pharmaceutical in a tablet. Such assays form the basis of many quality assurance procedures in the pharmaceutical industry, and have been extensively used by the British Pharmacopoeia (B.P.). More recently, however, high-performance liquid chromatography (HPLC) has replaced UV analysis in many B.P. assays, as most industrial analyses routinely use HPLC. 

The use of benzoylation for the formation of UV-absorbing derivatives of non-UV absorbing hydroxy steroids has been reported for the analysis of these compounds by liquid chromatography [33]. This reaction may prove useful for the separation of steroids which cannot be separated as their DNPH derivatives. Molar absorptivities of many steroid benzoates are normally greater than 100001/mole cm at 230 nm. 

The detector signal is much more dependent on specific compound properties in liquid chromatography than it is in gas chromatography. For example, the UV signal is a function of the molar absorptivity, which varies between 0 and 10 0001 mol cm depending on the compound used. The molar absorptivity and absorbance maximum also vary between homologues. Hence at least one calibration chromatogram must be obtained for each quantitative analysis. ... 

The ultraviolet-visible spectra of most compounds are of limited value for qualitative analysis and have been largely superseded by the more definitive infrared and mass spectroscopies. Qualitative analytical use of ultraviolet-visible spectra has largely involved describing compounds in terms of the positions and molar absorptivities of their absorption maxima, occasionally including their absorption minima. Indeed, some organic compounds are still characterized in terms of the number of peaks in the UV-visible spectrum and their absorbance ratios. This is usually the case in phytochemistry and photodiode array chromatography and when the analyst has a limited range of compounds to work with whose spectra are known to differ. In the pharmacopeias, however, absorbance ratios have found use in identity tests, and are referred to as Q-values in the U.S. Pharmacopia (USP). 

A major limitation in the increased application of modern liquid chromatography is the sensitivity of the detector system (70,71). For example, although a few UV detectors have been described which are usable at low nanogram levels, these involve special operating conditions or compounds with higher molar absorptivities (70,72,73). With ordinary UV absorbing species, quantitative LC is usually limited to a few tenths of micrograms (7],). 

The isolated conjugate was identified as pentachlorophenyl-sulfate by precipitation with BaCl2, by column and thin-layer chromatography, by UV-absorption spectra, and by determination of the molar ratio of PCP to SO4. PCP-glucuronide which is excreted in bile was not detected(j0. 

A solution of 20 g of desoxycorticosterone in 190 ml of absolute ethanol was stirred in an atmosphere of hydrogen in the presence of 1.68 g of 25% palladium on calcium carbonate catalyst. After 20 hours, approximately 1 molar equivalent of hydrogen had been absorbed and hydrogen uptake had ceased. The catalyst was removed by filtration and the filtrate evaporated in vacuo to yield 20 g of nearly pure product, MP 135°C to 140°C. The crude product was demonstrated to be free of starting material by paper chromatography. A highly purified product was obtained by recrystallization from acetone-water with cooling in an ice bath, yield 14.5 g, MP 152°C to 154°C. The product was characterized by analysis and by absence of ultraviolet absorption. 

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