Reversed-phase HPLC chromatographic modes

If simple sample pretreatment procedures are insufficient to simplify the complex matrix often observed in process mixtures, multidimensional chromatography may be required. Manual fraction collection from one separation mode and re-injection into a second mode are impractical, so automatic collection and reinjection techniques are preferred. For example, a programmed temperature vaporizer has been used to transfer fractions of sterols such as cholesterol and stigmasterol from a reversed phase HPLC system to a gas chromatographic system.11 Interfacing gel permeation HPLC and supercritical fluid chromatography is useful for nonvolatile or thermally unstable analytes and was demonstrated to be extremely useful for separation of compounds such as pentaerythritol tetrastearate and a C36 hydrocarbon standard.12... 

Sulfosalicylic acid has most commonly been used to precipitate proteins prior to ion-exchange amino acid analysis (11). In this mode, SSA allows for a very simple sample preparation that requires only centrifugation of the precipitated sample and then direct injection of the resulting supernatant solution. The supernatant solution is already at an appropriate pH for direct injection. Also, the SSA does not interfere chromatographically since it elutes essentially in the void volume of the column. It has been noted that, if an excessive amount of SSA is employed, resolution of the serine/threonine critical pair can suffer (12). The use of SSA prior to reversed-phase HPLC can be more problematic, since its presence can interfere with precolumn deriva-tization. For example, Cohen and Strydom (13) recommend the separation of the amino acids from the SSA solution on a cation-exchange resin prior to derivatization with phenylisothiocya-nate (PITC). 

Chromatographic method The determination of silicate based upon the adsorption of preconcentrated phosphomolybdic and silicomo-lybdic heteropoly acid (HPA) in the dynamic mode on a microcolumn packed with an Amberlite XAD-8 polyacrylate adsorbent is reported [49]. The method is based on the adsorption of HPAs followed by desorption with acetonitrile and determination by reversed-phase HPLC. Another highly sensitive HPLC method for the simultaneous determination of soluble silicate and phosphate in environmental waters is used in ion-pair liquid... 

As in other areas of analysis, HPLC is a useful complementary technique to GLC in analyzing pollutants in the environment. An advantage of HPLC in environmental analysis is that molecules of varying polarity (e.g. pesticide + metabolite mixtures) can be analyzed in one chromatographic run. Since aqueous mobile phases can be used in reversed-phase HPLC (including ion-pair partition modes) sample preparation is often less extensive than in GLC. The number of clean-up steps can also be reduced by the use of a precolumn to protect the analytical column or by a preliminary size separation of a crude extract on an exclusion column. 

Of all the chromatographic techniques used, reversed phase HPLC is by far the most popular with at least 60% of all analytical separations carried out in this chromatographic mode. The term reversed phase chromatography was originally coined by Howard and Martin (1950) who carried out liquid-liquid chromatography on a stationary phase of paraffin oil and -octane with aqueous eluents. In such a partition system the conventional methodology, which used a polar stationary phase and a less polar mobile phase, was reversed, with the mobile phase being more polar than the stationary phase. 

This class of vitamins comprises a group of molecules which are also known as retinoids and also includes certain carotenes which display vitamin A-like activity (particularly a-, and y-carotene from plants). Many derivatives of these compounds are found naturally and include the alcohol, the acetate and the palmitate. The best chromatographic mode for the resolution of the vitamin A class is often determined by the conditions used for the extraction. For example, saponification to remove lipids is often followed by extraction into solvents, such as hexane, which are compatible with aqueous reversed phase HPLC using either a Cg or C,g siUca-based stationary phase (Fig. 11.8.1). An alternative system has been reported using a Cjg support and a mobile phase of 0.5% acetic acid in acetonitrile (Annesley et al., 1984). 

For LC-MS, the chemical ionization mode for the mass spectrometry is the most readily used since the large amounts of mobile-phase solvents naturally act as the chemical ionization reagent. This can limit the utility of LC-MS because the mobile-phase solvent choices are determined by the HPLC conditions. There is usually little flexibility in the choice of solvents. For example, the use of acetonitrile or methanol is a common variable in reversed-phase HPLC, but most other solvent switches lead to gross changes in the chromatographic separation. The most ideal chemical ionization reagents are very likely not usable for this reason. Ro-sele-Mele et al. used this approached to identify the porphyrins, both free and bound to metals, in a shale oil. 

The PRISMA model was developed by Nyiredy for solvent optimization in TLC and HPLC [142,168-171]. The PRISMA model consists of three parts the selection of the chromatographic system, optimization of the selected mobile phases, and the selection of the development method. Since silica is the most widely used stationary phase in TLC, the optimization procedure always starts with this phase, although the method is equally applicable to all chemically bonded phases in the normal or reversed-phase mode. For the selection of suitable solvents the first experiments are carried out on TLC plates in unsaturated... 

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