Separation of cellular energetically important compounds by CZE in combination with field enhanced stacking
| Authors | |
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| Year of publication | 2009 |
| Type | Article in Proceedings |
| Conference | Book of Abstracts 15th American symposium on Biotechnology, Biomedical and Pharmaceutical and Industrial Application of CE and Microchip Technology |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | capillary electrophoresis; nucleotides; coenzymes |
| Description | Nucleotides are basic units of nucleic acid and cofactors. They store and transport cellular metabolic energy, participate in processes of biosynthesis and regulate cellular metabolism. Their total concentration and distribution of individual species is highly dynamic with changes in environmental conditions. Monitoring the nucleotides pool enables to verify the physiological state of the cell and predicts the state due to the presence and rations to each other. The main aim of this work was to find appropriate conditions for the selective and rapid determination of purine (AMP, ADP, ATP, GMP, GDP, GTP), pyrimidine (CMP, CDP, CTP, UMP, UDP, UTP) nucleotides, adenine coenzymes (NAD+, NADH, NADP+, NADPH) and Acetyl CoA using capillary zone electrophoresis (CZE). Because of low intra and extracellular concentration of these metabolites, CZE was combined with the online preconcentration technique field enhanced sample stacking to improve the concentration sensitivity. The determination was performed in a bare fused silica capillary using separation voltage 20 kV (positive polarity) and direct detection at 260, 280 and 340 nm. Different concentrations of phosphate buffer (from 50 to 80 mM), pH range from 5 to 7 and temperature of capillary from 16 to 25 C were tested for optimization of the method. The best resolution was found in the pH range from 5.5 to 6 for each buffer concentration; hence this pH was examined in detail. Metabolite samples were dissolved in deionised water and injected into the capillary hydrodynamically (50 mbar, 14 s). |
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