Reversed phase HPLC advantages

In principle, the analytical results obtained by the GPC spin column/HPLC ESI-MS methodology described in this chapter should be similar to the results obtained using the tandem chromatographic method of GPC/reversed-phase HPLC ESI-MS described in Chapter 3. There are practical advantages for each method. Since each of the chromatographic and mass spectral steps are done serially for the GPC spin column/HPLC ESI-MS methodology, each of the steps can be performed and optimized individually. In the event of mass spectrometer failure, the production of spin column eluate samples can proceed and samples can be stored for future analysis. In contrast, the parallel methodology of tandem GPC/ reversed-phase HPLC ESI-MS requires the simultaneous optimization of multi-... 

Because polyphenolics show chemical complexities and similar structures, isolation and quantification of the individual polyphenolic compounds have been challenging. Many traditional techniques (paper chromatography, thin-layer chromatography, column chromatography) have been used. HPLC, with its merits of exacting resolution, ease of use, and short analysis time, has the further advantage that separation and quantification occur simultaneously. A reversed-phase HPLC apparatus equipped with a diode array detector makes possible the easy isolation and separation of many polyphenolics. For enhanced performance of HPLC separation, the polyphenolics should first be isolated into several fractions to effectively separate the individual polyphenolics (Jaworski and Lee, 1987 Oszmianski and Lee, 1990). 

A number of different approaches, all of which have both advantages and shortcomings, have been proposed for the analysis of water-soluble dyes. The separation of colours has been carried out using ion-exchange resins, reverse-phase HPLC coupled with ion-pair reagents and reverse-phase HPLC at low pH where the ionisation of the dyes is suppressed. The last of these is the technique used at RSSL and is also the method recommended by Wadds (1984). It offers the simplest approach to this type of analysis and a typical HPLC profile of... 

Further purification of the total lipid extract from neutral lipids like triacylglycerols is desirable to increase the separation performance and lifetime of reverse-phase HPLC columns. Several authors have utilized a procedure of base-acid wash for this purpose (Evershed et al., 1988 Seitz, 1989 Hakala et al., 2002), where one takes advantage of the phenolic nature of the steryl femlates. Total lipid extract in acetone or... 

Besides the advantages of short elution times, excellent resolution, and high recoveries, a further benefit of these reversed-phase HPLC methods for peptide mapping is the ease of sample preparation. In many cases the crude digest can be loaded directly. The problems associated with overlapping peptides in an otherwise straightforward application can often be remedied by small changes in the pH of the mobile phase or, alternatively, variation in the polarity and concentration of the counterion. 

Reversed-phase HPLC is by far the most popular mode of chromatography. Almost 90% of all analyses of low-molecular-weight samples are carried out using RP HPLC. One of the main drivers for its enormous popularity is the ability to discriminate very closely related compounds and the ease of variation of retention and selectivity. The origin of these advantages could be... 

The majority of samples dealt with by a typical environmental laboratory are likely to be waters, whether this be groundwaters, rivers, marine or effluents and accordingly the use of reversed phase HPLC has obvious advantages. A look at current literature will show an almost exclusive use of reversed-phase HPLC techniques used for environmental samples. 

This also applies to the separation of isomers, including enantiomers. Reviewing the literature on the isolation of conjugates, reveals that the advantages of reverse phase HPLC has been exploited in nearly all studies reported in recent publications. 

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