Capillary Electrophoresis / Electrospray Mass Spectrometry Analysis of Nucleotides Using Nonvolatile Phosphate Buffer
Date Issued
2010
Date
2010
Author(s)
Li, Ming-Shen
Abstract
Nucleotides, composed of a nucleobase, a five-carbon sugar, and one to three phosphate groups, are one kind of very important biomolecules in the living organisms. When analyzing these compounds with capillary electrophoresis (CE), the silanol groups on the capillary wall interact with phosphate groups, thus causing distortion of electrophoretic peaks. Phosphate running buffers can effectively reduce the adsorptive interaction and provide better resolution and peak shape. However, the nonvolatile phosphate buffer would cause severe signal suppressions and chemical interferences in electrospray ionization mass spectrometry spectrum (ESI-MS). In this study, both the low-flow interface and the liquid-junction/low-flow interface were utilized to solve this problem. By using lower concentration phosphate buffer with the addition of isopropanol in CE-MS, we have successfully analyzed nucleotides using a low-flow interface. Under the condition, phosphate ions would migrate to the inlet resulting in less signal suppression. However, due to the addition of isopropanol, the supplement of phosphate ions is needed, and the analysis time also becomes much longer. Liquid-junction/low-flow interface was used to alleviate the problem of long analysis time and re-codition the column. By controlling the electroosmotic flow (EOF) of the connecting column with the addition of isopropanol, phosphate ions would stay in the liquid junction reservoir, whereas the nucleotides would migrate toward the ion source. This approach not only retains the short separation time but also alleviates the signal suppression and interference in MS spectra.
Subjects
nucleotides
CE-MS
phosphate buffer
negative ESI-MS
Type
thesis
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