Although the proteome of each organism is unambiguously coded in its genome, the proteome shows the real biology in action in each particular organism. New powerful tools are being developed for biochemists and biologists to analyze complex biological samples for studying the complete protein supplement of the genome, i. e., the proteome. There are several methods available for proteome analysis including 2-DE and several forms of MS. In recent years, technologies such as microfluidics and array-based systems have appeared in the field of analysis, identification, and quantification of proteins. These novel approaches might help in solving current technical challenges in proteomics. This paper presents a practical application of the first commercially available microfluidic nano-ESI device coupled with nano-LC (i. e., HPLC-chip) for the analysis of samples of some biological protein mixtures.
Whenever a mobile phase contains more than one component, additional signals commonly called system peaks can appear. The origin of these signals is explained through loss of equilibrium in the separation column caused by injection of analyte dissolved in a different solvent than the mobile phase. The system peaks are then generated by a relaxation process started by the non-equilibrium state. An overview of the theory and applications of the system peaks in separation methods, mainly in liquid chromatography, is presented in this paper. Only a brief theoretical discussion of the system peak origin is given as the theoretical aspects of system peak formation have already been published in many papers. The main focus of this review is to summarize applications, in which system peaks were used to measure physical or physicochemical data. Signals of system peaks are often misinterpreted but they offer valuable information about thermodynamics and kinetics of the separation process that takes place in chromatographic column.
The retention behavior of several series of free α- and ω-amino acids and positional isomers of amino pentanoic acid in the hydrophilic interaction chromatography mode (HILIC) was studied. The study was carried out on three stationary phases followed by post-column derivatization with fluorescence detection in order to describe the retention mechanism of the tested amino acids. The effect of chromatographic conditions including acetonitrile content in the mobile phase, mobile phase pH (ranging from 3.5 to 6.5) and concentration of buffer in the mobile phase was investigated. The effect of the number of carbon atoms (nC) in aliphatic chains of the individual homologue of α- and ω-amino acids and the logarithm of the partition coefficient (logD) on retention was also a part of the presented study. A good correlation (r > 0.98) between the logk and logD values of amino acids or nC, respectively, was observed. The described linear relationships were subsequently applied to predict the retention behavior of individual members of the homologous series of amino acids and to optimize the mobile phase composition in HILIC. The obtained results confirmed that the retention mechanism of α-amino acids, ω-amino acids and positional isomers of amino acids was based on the logD values and the number of carbon atoms in the aliphatic chains of amino acids. The elution order of ω-amino acids and positional isomers of amino pentanoic acid was strongly dependent on the mobile phase pH in the investigated range whereas the retention factors of all α-amino acids remained essentially unchanged on all tested stationary phases.
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