HPLC high performance liquid gradient elution

Reversed-phase Cig chromatography column. Keystone Scientific Betasil, 100 x 2.0-mm i.d., 5-pm particle size, 100 A, Part No. 105-701-2-CPF TSQ 7000 LC/MS/MS system with electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) interface and gradient high-performance liquid chromatography (HPLC) unit, or equivalent Vacuum manifold for use with SPE cartridges (Varian Vac Elut 10 or equivalent)... 

High-performance liquid chromatography (HPLC) techniques are widely used for separation of phenolic compounds. Both reverse- and normal-phase HPLC methods have been used to separate and quantify PAs but have enjoyed only limited success. In reverse-phase HPLC, PAs smaller than trimers are well separated, while higher oligomers and polymers are co-eluted as a broad unresolved peak [8,13,37]. For our reverse-phase analyses, HPLC separation was achieved using a reverse phase. Cl8, 5 (Jtm 4.6 X 250 mm column (J. T. Baker, http //www.mallbaker.com/). Samples were eluted with a water/acetonitrile gradient, 95 5 to 30 70 in 65 min, at a flow rate of 0.8 mL/min. The water was adjusted with acetic acid to a final concentration of 0.1%. All mass spectra were acquired using a Bruker Esquire LC-MS equipped with an electrospray ionization source in the positive mode. 

Figure 9.3 Schematic illustration of the electrophoretic transfer of proteins in the chromatophoresis process. After being eluted from the HPLC column, the proteins were reduced with /3-mercaptoethanol in the protein reaction system (PRS), and then deposited onto the polyacrylamide gradient gel. (PRC, protein reaction cocktail). Reprinted from Journal of Chromatography, 443, W. G. Burton et al., Separation of proteins by reversed-phase high-performance liquid chromatography , pp 363-379, copyright 1988, with permission from Elsevier Science.

The concentrates are analysed by High Performance Liquid Chromatography (HPLC) coupled to a fluorescence detector. Elution is performed using a gradient of methanol in a phosphate buffer on a Cj g radial compression column. In order to render the amino acids amenable to fluorescence detection ( X Xgjjj = 455 nm), it is first necessary to derivatize them using orthophtaldialdehyde (O.P.A.) in the presence of mercapto-2 ethanol. Up to 20 amino acids can thus be analysed and quantified, and the complete analysis takes only 35 minutes. Table 2 presents the most commonly determined amino acids during the course of this study. 

Many technical problems can occur with gradient elution, some of which can be avoided through various methods. To begin, gradient elution relies upon the purity of the solvents used. The high-performance liquid chromatography (HPLC) column can collect impurities. 

Problems are often found in many analytical methods due to the complex nature of the mixture and the lack of adequate detection means, thus leading to poor quantitation techniques. For the routine separation of a broad range of surfactants, high-performance liquid chromatography (HPLC) appears to be the most cost-effective [7-18]. Ultraviolet (UV) and fluorescence detectors are commonly used in HPLC analysis of surfactants because of their compatibility with separation techniques requiring gradient elution. However, these detectors have two inherent limitations (a) the detector response is dependent on molecular structure (i.e., degree of aromaticity and type of substitution) and (b) only species with a chromophore can be detected. To overcome those limitations, postcolumn reaction detectors, based on extraction of fluorescent ion pairs, were introduced for on-line detection of alkylsul-... 

Assay Procedures. Tissues, feces, urine samples, and sample fractions were assayed for radioactivity (RA) by liquid scintillation counting essentially as described by Magnussen et al. (9). Tissues were assayed for parent tilmicosin by methanol extraction, purification by liquid-liquid partitioning, and measurement by high performance liquid chromatography (HPLC) on a reversed phase phenyl column with detection by UV absorption at 280 nm. Elution was with a nonlinear gradient of mobile phase consisting of water, acetonitrile, and dibutyl ammonium phosphate at pH 2.5. 

Gradient elution systems for high-performance liquid chromatography have been described. Some HPLC and pump manufacturers have been specifically mentioned and some of their instrumentation described in detail. This does not imply any endorsement from the authors, nor does the omission of any commercially available products imply an unsatisfactory appraisal. 

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