PubMed · 11721949
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A Felinger, M C Pietrogrande. 2001-11-01. Decoding complex multicomponent chromatograms.. https://doi.org/10.1021/ac012531r
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It has been found that the interconversion of enantiomers on a chromatographic column during the separation process can be studied by the first-order kinetic equations derived both for reversible and irreversible reactions in a stationary system if the extent of interconversion is not too high. The equation derived for irreversible reactions gives, however, results also for higher degrees of enantiomerization while that derived for reversible interconversion failed. The irreversible equation was used to determine the enantiomerization barrier of N-(p-methoxybenzyl)-l,3,2-benzodithiazol-l-oxide enantiomers by supercritical fluid chromatography. The racemate of N-(p-methoxybenzyl)-l,3,2-benzodithiazol-l-oxide was separated by supercritical fluid chromatography on the (R,R)-Whelk-Ol column with supercritical carbon dioxide containing 20% methanol as a mobile phase. Peak areas of enantiomers prior to and after the separation used for the calculation of the enantiomerization barrier were determined by computer-assisted peak deconvolution of peak clusters registered on chromatograms using commercial software.
Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry coupled with capillary reverse-phase liquid chromatography was used to characterize intact proteins from the large subunit of the yeast ribosome. High mass measurement accuracy, achieved by "mass locking" with an internal standard from a dual electrospray ionization source, allowed identification of ribosomal proteins. Analyses of the intact proteins revealed information on cotranslational and posttranslational modifications of the ribosomal proteins that included loss of the initiating methionine, acetylation, methylation, and proteolytic maturation. High-resolution separations permitted differentiation of protein isoforms having high structural similarity as well as proteins from their modified forms, facilitating unequivocal assignments. The study identified 42 of the 43 core large ribosomal subunit proteins and 58 (of 64 possible) core large subunit protein isoforms having unique masses in a single analysis. These results demonstrate the basis for the high-throughput analyses of complex mixtures of intact proteins, which we believe will be an important complement to other approaches for defining protein modifications and their changes resulting from physiological processes or environmental perturbations.
Non-equilibrium chromatography (NEC) is a chromatographic mode for the rapid separation of polymers. The retention behavior of various proteins (human, chicken, bovine serum albumin) and supercoiled circular double-stranded DNA (plasmids) was investigated using a phosphate buffer as a mobile phase at different velocities and column temperatures with a C1 column with very low-packing particle diameter as a stationary phase. It was shown that the two factors (temperature and velocity) constituted important parameters in the retention mechanism of plasmids and proteins in NEC. The protein was retained more than the plasmid. At all the temperatures (5, 10, 15, 20, 25 degrees C) the plasmid retention increased over the entire flow-rate range (0.02-1.8 ml/min). For the protein, the retention curve presented a decrease in the relative retention time until a critical value of the mobile phase flow-rate, followed by an increase. The transition between the two well known NEC methods, slalom chromatography and hydrodynamic chromatography was clearly visualized for proteins at the lowest temperature, but did not appear for plasmids due to their strong compact structure.