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Application of capillary electrophoresis in anion binding studies: Complexation and separation of nitrate and nitrite by an azacryptand.

ABSTRACT: Capillary electrophoresis (CE) was employed for studying the complexation of an azacryptand with nitrate and nitrite in aqueous solution. CE separation of a mixture of nitrate and nitrite with 10 mM acetate buffer (pH 3.3) showed two peaks at the retention times of 2.8 and 3.1 min for nitrate and nitrite, respectively. However, when the ligand (2 mM) was added to the running buffer, the peaks emerged in the reverse order and at shorter retention times of 2.7 and 2.5 min for nitrate and nitrite, respectively. The longer retention time for nitrate compared with nitrite indicates a stronger complex formation between the ligand and nitrate, that reduces the migration speed of nitrate as compared with the less strongly bound nitrite. The (1)H NMR titrations of L with these two anions at the pH 3.3, gave the binding constants (log K), 3.75 and 4.23, for nitrite and nitrate, respectively which were in consistence with the results obtained from the CE method.

SUBMITTER: Wu H

PROVIDER: S-EPMC3015164 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Application of capillary electrophoresis in anion binding studies: Complexation and separation of nitrate and nitrite by an azacryptand.

Wu Hao H Saeed Musabbir A MA Hwang Huey-Min HM Zhao Shulin S Liu Yi-Ming YM Hossain Md Alamgir MA

Journal of physical organic chemistry 20110101 1

Capillary electrophoresis (CE) was employed for studying the complexation of an azacryptand with nitrate and nitrite in aqueous solution. CE separation of a mixture of nitrate and nitrite with 10 mM acetate buffer (pH 3.3) showed two peaks at the retention times of 2.8 and 3.1 min for nitrate and nitrite, respectively. However, when the ligand (2 mM) was added to the running buffer, the peaks emerged in the reverse order and at shorter retention times of 2.7 and 2.5 min for nitrate and nitrite, ...[more]

PMID: 21218124

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Project description:Phosphoinositides (PIs) and sphingolipids regulate many aspects of cell behavior and are often involved in disease processes such as oncogenesis. Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) is emerging as an important tool for enzymatic assays of the metabolism of these lipids, particularly in cell-based formats. Previous separations of phosphoinositide lipids by CE required a complex buffer with polymer additives which had the disadvantages of high cost and/or short shelf life. Further a simultaneous separation of these classes of lipids has not been demonstrated in a robust buffer system. In the current work, a simple separation buffer based on NaH(2)PO(4) and 1-propanol was optimized to separate two sphingolipids and multiple phosphoinositides by CE. The NaH(2)PO(4) concentration, pH, 1-propanol fraction, and a surfactant additive to the buffer were individually optimized to achieve simultaneous separation of the sphingolipids and phosphoinositides. Fluorescein-labeled sphingosine (SFL) and sphingosine 1-phosphate (S1PFL), fluorescein-labeled phosphatidyl-inositol 4,5-bisphosphate (PIP2) and phosphatidyl-inositol 3,4,5-trisphosphate (PIP3), and bodipy-fluorescein (BFL)-labeled PIP2 and PIP3 were separated pairwise and in combination to demonstrate the generalizability of the method. Theoretical plate numbers achieved were as high as 2×10(5) in separating fluorophore-labeled PIP2 and PIP3. Detection limits for the 6 analytes were in the range of 10(-18)-10(-20)mol. The method also showed high reproducibility, as the relative standard deviation of the normalized migration time for each analyte in the simultaneous separation of all 6 compounds was less than 1%. The separation of a mixture composed of diacylglycerol (DAG) and multiple phosphoinositides was also demonstrated. As a final test, fluorescent lipid metabolites formed within cells loaded with BFLPIP2 were separated from a cell lysate as well as a single cell. This simple and robust separation method for SFL and S1PFL and various metabolites of phosphoinositide-related signal transduction is expected to enable improved enzymatic assays for biological and clinical applications.

Dataset Information

Application of capillary electrophoresis in anion binding studies: Complexation and separation of nitrate and nitrite by an azacryptand.

Publications

Application of capillary electrophoresis in anion binding studies: Complexation and separation of nitrate and nitrite by an azacryptand.

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