Mobile Phase Phosphate Research Articles

The analysis of nitrate in environmental samples by reversed-phase HPLC.

A method is presented for the analysis of nitrate in natural waters and waste water by high-performance liquid chromatography (HPLC) with a reversed-phase octadecyl column, aqueous phosphoric acid/dihydrogen phosphate mobile phase, and UV detector. The optimum nitrate concentration is 0.3 to 3 mg/L as N with linear detector response below 3 mg/L. The detection limit is 0.007 mg/L. Relative standard deviations in the optimum range are consistently less than one percent. Several potential interferences have been investigated; nitrite and organic chromophores are resolved from nitrate and do not interfere. Hexavalent chromium and sulfate are slight positive interferences, negligible at typical environmental concentrations. The method produces results in agreement with the accepted chromotropic acid method except in samples from eutrophic lakes, for which evidence is presented indicating that the chromotropic acid is inaccurate.

HPLC-ECD procedure for the measurement of 0-methylation of catechol estrogens by vascular tissue

A precolumn extraction procedure combined with high-performance liquid chromatography with electrochemical detection (HPLC-ECD) has been developed for the identification and quantitation of catechol estrogens, their immediate precursors (estrogens), and their 0-methylated metabolites. These compounds were isolated from Krebs solutions and from perchloric acid tissue extracts or whole tissues by simple solvent extraction prior to quantitation. The optimal conditions for detection by HPLC-ECD were those employing a radial compression module, a C 18 Bondapak cartridge, and an isocratic mobile phase of ammonium phosphate (pH 3.0) combined with 30–70% acetonitrile. A biological application of this procedure includes the investigation of the 0-methylation of catechol estrogens by vascular tissue. In the present study, the 0-methylation of 2-hydroxyestradiol by rabbit aorta has been examined. The 0-methylation in segments of thoracic aorta was significantly less than that in abdominal aortic segments, but was reproducible along the length of the thoracic aorta.

High-performance liquid chromatographic purification of deoxynucleoside 5′-triphosphates and their use in a sensitive electrophoretic assay of misincorporation during DNA synthesis

This paper describes techniques and strategies for semi-preparative high-performance liquid chromatographic (HPLC) purification of 2′-deoxynucleoside 5′-triphosphates (dNTPs). The procedure yields dNTPs that are sufficiently pure for use in a sensitive electrophoretic assay of misincorporation during DNA synthesis. Anion-exchange HPLC was used to purify the four normal dNTPs (dATP,dGTP,dCTP and dTTP), plus the chemically modified analogues, 5-BrdUTP, 5-IodUTP and 1,N 6-etheno-dATP (εdATP). Baseline separations were achieved by isocratic elution of dNTPs with potassium dihydrogen phosphate mobile phase. In general, the resolution of dNTPs was highly dependent on pH, although the influence of mobile phase composition on separation of dNTPs was not the same for all three HPLC packing materials used. A Hewlett-Packard diode array detector was extremely valuable in the identification of contaminating peaks and in the development of optimal mobile phase conditions for dNTP purification. The pur dNTPs were used in the electrophoretic assay of misincorporation, yielding information about the mispairing potential of the modified dNTPs. BrdUMP and IodUMP were misincorporated in place of dCMP during chain elongation catalyzed by purified DNA polymerase I of Escherichia coli. εdAMP was incorporated into DNA in place of dAMP, although at much lower efficiency than dAMP.

Measurement of plant nucleotides by high-performance liquid chromatography

Procedure are described for the chromatographic isolation of nucleotides from plant extracts and fro their separation and measurement by high-performance liquid chromatography (HPLC). A plant extract containing nucleotides plus an array of other plant products and ions was adjusted to pH 2 with hydrochloric acid and passed sequentially through three small low-pressure columns containing: (1) porous polystyrene (Amberlite XAD-2), (2) water-insoluble polyvinylpolypyrrolidone, (3) charcoal—cellulose. At pH 2, the first two columns retained most aromatic compounds but not nucleotides. The latter were retained by the charcoal while inorganic ions and aliphatic compounds passed through all three column. Nucleotides were eluted from the charcoal with ammoniacal alcohol. They were separated on an HPLC anion-exchange column with gradient elution. During the passage of a dilute phosphate mobile phase through the column, large changes in pH occurred because of Donnan equilibrium effects. These effects prevented pH equilibration of the column in a reasonable time, caused irregularities in nucleotide retention time, and even changed elution order. The addition of 0.01 M acetate to a dilute phosphate mobile phase improved pH control so that column pH equilibration was achieved within 50 min. Nucleotide retention times were stabilized and separation improved. At 0.01 M, the acetate showed relatively little tendency to compete with phosphate for exchange sites.

Liquid Chromatographic Determination of Allopurinol in Tablets: Collaborative Study

A liquid chromatographic method for the determination of allopurinol in tablets was collaboratively studied by 7 laboratories. The method uses a C18 reverse phase column, a 0.05M ammonium phosphate mobile phase, hypoxanthine as the internal standard, and photometric detection at 254 nm. Collaborators were supplied with samples of 2 commercial tablets and 1 synthetic tablet powder. The mean recovery value of allopurinol from the synthetic tablet powder was 100.0%. The combined mean coefficient of variation for all 3 types of sample analyzed was less than 2%. The method has been adopted official first action.

High-performance liquid chromatography of biologically important pyrimidine derivatives with ultraviolet—voltammetric—polarographic detection

High-performance liquid chromatographic separation of a number of biologically important pyrimidine derivatives was studied in the reversed-phase system. Good results were obtained using a C 18 alkyl-bonded silica column and an aqueous citrate—phosphate mobile phase of pH 3.5. All eluted components are detected with the UV absorbance detector at 254 nm, whereas the voltammetric detector with a polymeric carbon-paste electrode detects only derivatives containing oxidizable functional groups (amino, mercapto) and the polarographic detector with a mercury electrode only those with reducible groups (nitro, aza). The signal of the electrochemical detectors is proportional to the number of electroactive groups in the solute molecule. The use of two or three detectors in series thus improves the resolution of complex mixtures and facilitates identification.

Measurement of catecholamines in biological fluids by high-performance liquid chromatography : A comparison of fluorimetric with electrochemical detection

An improved method for the determination of catecholamines in biological fluids, by reversed-phase high-performance liquid chromatography (HPLC) with fluorimetric detection is presented. The pH titration previously employed in the alumina extraction was abandoned in favour of the use of a molar excess of pH 8.5 Tris—HCl buffer. A novel lyophilisation step serves to concentrate the catechols and by reconstituting in mobile phase, chromatography disturbances are minimised. The addition of 2 m M octanesulphonic acid to a citrate—phosphate mobile phase at pH 6.0 gave optimal resolution and sensitivity. That HPLC separation can improve the specificity of the trihydroxyindole reaction, to the extent of providing a reliable analytical method, has been demonstrated and validated by the technique of HPLC with electrochemical detection. A correlation coefficient of 0.98 was obtained between the two techniques as applied to the measurement of urinary catecholamines. The HPLC—fluorimetric method was sensitive enough to measure 0.1 ng/ml of noradrenaline or adrenaline at a signal-to-noise ratio of 2.0. Application of the method to the quantitative determination of catecholamines in human urine, plasma and rat brain homogenates is demonstrated.

Analysis of Psilocybin and Psilocin in Mushroom Extracts by Reversed-Phase High Performance Liquid Chromatography

A method has been developed for the analysis of psilocybin and psilocin in dry and preserved mushrooms using reversed-phase high performance liquid chromatography. A mobile phase of phosphate buffered methanol/water/cetrimonium bromide allows good separation of the two hallucinogens.

Determination of pyridoxal 5′-phosphate as the semicarbazone derivative using high-performance liquid chromatography

A high-performance liquid chromatographic (hplc) procedure is described for the determination of pyridoxal 5′-phosphate (PLP) in animal tissues. The procedure is based on extraction with perchloric acid and treatment with semicarbazide to form PLP-semicarbazone. This derivative is quantitatively determined using hplc with an octylsilica column, an acidic phosphate mobile phase buffer, and fluorometric detection. The validity of the method was confirmed by inspection of the fluorescence spectra of the PLP-semicarbazone hplc peaks of semicarbazide-treated standards and tissue extracts. Preparative chromatography for extract purification is not required because of the hplc efficiency and detection specificity. This method provides a simple technique for the rapid, direct assay of PLP in animal tissues.

Quantitative gas-liquid chromatography of TMS nucleic acid constituents

High-performance liquid chromatographic separation of a number of biologically important pyrimidine derivatives was studied in the reversed-phase system. Good results were obtained using a C18 alkyl-bonded silica column and an aqueous citrate—phosphate mobile phase of pH 3.5. All eluted components are detected with the UV absorbance detector at 254 nm, whereas the voltammetric detector with a polymeric carbon-paste electrode detects only derivatives containing oxidizable functional groups (amino, mercapto) and the polarographic detector with a mercury electrode only those with reducible groups (nitro, aza). The signal of the electrochemical detectors is proportional to the number of electroactive groups in the solute molecule. The use of two or three detectors in series thus improves the resolution of complex mixtures and facilitates identification.