High performance liquid chromatography chromatographic conditions

Liquid chromatography (LC) and, in particular, high performance liquid chromatography (HPLC), is at present the most popular and widely used separation procedure based on a quasi-equilibrium -type of molecular distribution between two phases. Officially, LC is defined as a physical method... in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction [ 1 ]. In other words, all chromatographic methods have one thing in common and that is the dynamic separation of a substance mixture in a flow system. Since the interphase molecular distribution of the respective substances is the main condition of the separation layer functionality in this method, chromatography can be considered as an excellent model of other methods based on similar distributions and carried out at dynamic conditions. 

Mulholland, M., and Waterhouse, J. (1987). Development and evaluation of an automated procedure for the ruggedness testing of chromatographic conditions in high-performance liquid chromatography./. Chromatogr. 395, 539-551. 

Certain classes of lipids are susceptible to degradation under specific conditions. For example, all ester-linked fatty acids in triacylglycerols, phospholipids, and sterol esters are released by mild acid or alkaline treatment, and somewhat harsher hydrolysis conditions release amide-bound fatty acids from sphingolipids. Enzymes that specifically hydrolyze certain lipids are also useful in the determination of lipid structure. Phospholipases A, C, and D (Fig. 10-15) each split particular bonds in phospholipids and yield products with characteristic solubilities and chromatographic behaviors. Phospholipase C, for example, releases a water-soluble phosphoryl alcohol (such as phosphocholine from phosphatidylcholine) and a chloroform-soluble diacylglycerol, each of which can be characterized separately to determine the structure of the intact phospholipid. The combination of specific hydrolysis with characterization of the products by thin-layer, gas-liquid, or high-performance liquid chromatography often allows determination of a lipid structure. 

We have followed the pathway of TNT degradation in the consortium using reverse phase high performance liquid chromatography (HPLC) and mass spectroscopy (MS). Analysis of the supernatant in the consortium showed sequential reduction of TNT to TAT however, TAT only occasionally accumulated, and even then only in very low concentrations. In addition, methylphloroglucinol (MPG) and />-cresol transiently appeared in the supernatant. All compounds were identified using HPLC by UV spectra and retention times as compared to authentic standards chromatographed under the same conditions. The mono- and diamino intermediates were also confirmed by the MS results. 

Fig. 30 Silver ion high-performance liquid chromatography (Ag-HPLC-FID) with flame ionization detector (FID) analysis of the triacylglycerols of chromatographed Crepis alpina seed oil. Ag-HPLC-FID conditions 0.5-mg sample 5-micron Chromspher Lipids column (Chrompack International, Middelburg, The Netherlands) (4.6 X 250 mm) mobile phase 0.5% acetonitrile in hexane (v/v) flow rate 1.0 ml/min FID. Chromatogram peak triacylglycerol fatty acid abbreviations S, saturated (palmitic and stearic) O, oleic L, linoleic and Cr, crepenynoic fatty acids.

An overview and discussion is given of literature methods published after 1989 devoted to the ion-interaction chromatographic determination of inorganic anions. Seventy references are quoted. Ion-interaction chromatography makes use of commercial reversed-phase stationary phase and conventional high-performance liquid chromatography instrumentation. The basis of the technique, the modification of the stationary phase surface, the choice of the ion-interaction reagent as well as the dependence of retention on the different variables involved are discussed. Examples of application in the fields of environmental, clinical and food chemistry are presented. The experimental conditions of stationary phase, of mobile phase composition as well as detection mode, detection limit and application are also summarized in tables. 1997 Elsevier Science B.V. 

Due to high water solubility of monosaccharides, the use of the most routine high-performance liquid chromatography (HPLC) reversed-phase columns is also not suitable for their analysis. Extremely pure solvents have to be used if ultraviolet (UV) detection is applied. If a refractometer is used as the detector (RD), extremely steady chromatographic conditions are necessary. Nevertheless, HPLC is applied in the practice. The modern approach involves the use of propylamino columns (e.g.. Refs. 3 and 4). 

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