HPLC Pump Research Articles

The measurement of the release of endogenous GABA from rat brain slices by liquid chromatography with electrochemical detection.

A method for the determination of GABA by derivatization with 2,4,6-trinitrobenzenesulphonic acid and subsequent separation and quantitation by HPLC with electrochemical detection was characterized with respect to specificity, reproducibility and sensitivity. No other amino acid occurring in significant amounts in the brain was found to interfere; however, adequate separation of the derivatives of GABA and tryptophan must be carefully checked in each experiment. The sensitivity of the method is essentially determined by baseline noise, which mainly depends on the quality of the HPLC pump; under our conditions, it was about 2 ng/ml analyte. The coefficients of variation determined at two different concentrations relevant for the subsequent experiments were well below 10%. The method proved useful for the assessment of endogenous release of GABA from superfused rat cortical slices by electrical stimulation, which, in contrast to the basal release, was found to be completely calcium-dependent at stimulation frequencies of 5 and 12 Hz, under our conditions. Both stimulated and basal release of GABA was enhanced 4-5-fold by the inhibitor of GABA uptake, SK&F 89976 (10 microM).

Quantitation of biological retinoids by high-pressure liquid chromatography: Primary internal standardization using tritiated retinoids

A single method is described for quantitation of 14 retinoids found in biological material. The method consists of reversed-phase HPLC, internal standardization, and carrier extraction procedures with three synthetic retinoids. Primary standardization of HPLC uv detector is achieved using tritiated all- trans-retinoic acid, all- trans-retinol, all- trans-retinyl palmitate, and all- trans-retinyl acetate. Extraction methods are standardized by correlating the uv absorbance of retinoids at 340 nm with radioactivity of tritiated retinoids of known specific activity. Quantitation of 10 pg of tritiated or 5 ng of nonradioactive retinoid per 0.1 g sample in a polarity range from 4-oxoretinoic acid to retinyl stearate can be achieved in a single, 50-min chromatographic run. A single HPLC pump, a C 18 reversed-phase analytical column, a multistep three-solvent gradient, and inexpensive solvents based on methanol, water, and chloroform comprise this cost-effective chromatographic system. Our primary standardization method allows investigators employing different procedures to compare results between laboratories by standardizing the HPLC uv detector with commercially available tritiated retinoids. With this method we were able to quantitate nanomolar amounts of endogenous retinoic acids and retinyl esters, that “HPLC uv only” conditions usually would not detect in the circulation and liver of rats under physiological conditions.

10] Purification of bacterial luciferase by high-performance liquid chromatography

This chapter discusses the purification of bacterial luciferase by high-performance liquid chromatography. Large quantities of bacterial luciferase (Lase) can be rapidly prepared by HPLC using three chromatography procedures, which have their respective counterparts in low-pressure liquid chromatography: high-performance size exclusion chromatography (HPSEC), high-performance anion exchange chromatography (HPAEC), and high-performance hydrophobic interaction chromatography (HPHIC). In particular, HPHIC and its low-pressure counterpart are most useful and provide purifications of Lase that equal or exceed those obtained by affinity chromatography on diphenylpropylamine-Sepharose. The major concern in the HPLC of biological extracts is that the columns will become clogged by particulates, which possibly could ruin a costly HPLC column. Particulates are removed from the solvents by filtration using a membrane filter with a pore size of 0.45 μm or less (Millipore or Nucleopore). The filtered solvents are stored covered to prevent contamination by dust or other debris. The HPLC pumps and columns are secondarily protected from particulates by solvent filters placed on the solvent intake lines. It is recommended that all solvents be filtered on a daily basis to remove any bacteria and microcrystals of salts that form in concentrated buffer solutions. The steps in the purification of luciferase includes: (1) luciferase from photobacterium leiognathi DD17, (2) preparative high-performance size exclusion chromatography (HPSEC), (3) preparative HPAEC, (4) preparative HPHIC, and (5) luciferase from an aldehyde-requiring mutant of vibrio harveyi.

High-performance liquid chromatography column length designed for submicrogram scale protein isolation

High-performance liquid chromatography of small amounts of protein was readdressed with respect to current gas-phase sequencing technology. Useful primary sequence information can be obtained from as little as 5–100 pmol of material. This corresponds on a mass level to the nanogram to microgram range where, unfortunately, HPLC columns often give low recoveries depending on the size and surface hydrophobicity of the peptide or protein. It was rationalized in this study that reduced column length could have a beneficial effect on recovery without significant loss of resolution. To demonstrate this, six HPLC columns ranging from 0.2 to 25 cm in length were made and evaluated in terms of protein loading and resolution. Column lengths of less than 1 cm were found to increase recovery of surface hydrophobic proteins without loss of resolution, as shown for a standard protein profile. These columns resolve proteins best when loaded with less than 10 μg, with recoveries greater than 90%. All column internal diameters were at least 4.1 mm so that standard HPLC pumps could be used to generate gradients.

Distribution of spinal cord nerve endings containing various neurotransmitters on a continuous density gradient.

Homogenates of rat dorsal or ventral spinal cord were subjected to centrifugation on a continuous density gradient. The gradient was generated according to a new method with the aid of a microprocessor-controlled HPLC pump. The distribution of substance P-like immunoreactivity (SPI) and somatostatin-like immunoreactivity (SRIFI) across the gradient showed two peaks. The SPI peak seen at lower density was found only in dorsal spinal cord tissue. No peak of SPI was seen at this position in homogenates prepared from the spinal cords of capsaicin-pretreated rats. The second peak of SPI, found at a higher density, was accompanied by peaks in the levels of endogenous 5-hydroxytryptamine (5-HT), [14C]glycine, and [3H]norepinephrine uptake. This peak was seen at the same density in the dorsal and the ventral spinal cord. Tissue derived from capsaicin-pretreated rats exhibited one peak of SPI, accompanied by a maximum of [14C]glycine uptake. The uptake of [3H]gamma-aminobutyric acid ( [3H]GABA) was found to have a maximum at a somewhat lower density than that of [14C]glycine. It is concluded that the peak of SPI found at lower density in the dorsal spinal cord is associated with nerve endings belonging to capsaicin-sensitive primary afferents, while other endings, including those also containing 5-HT, are probably associated with the peak of SPI found at higher density.

Quantitative aspects of flow-rate in liquid chromatography and reaction-detection liquid chromatography

Post-column reaction detectors are becoming increasingly popular as specific dedicated detection systems in liquid chromatography. The influence of flow-rate variations on the reproducibility of the peak area is investigated for common LC systems and for LC equipment combined with a reaction detector. It is demonstrated that the flow of modern HPLC pumps is sufficiently stable for quantitative work.