Applications of HPLC in Biochemistry by A. Fallon, R.F.G. Booth and L.D. Bell (Eds.)

By A. Fallon, R.F.G. Booth and L.D. Bell (Eds.)

This e-book is meant to familiarize biochemists with HPLC. Theoretical points of every mode of chromatography are mentioned in chapters 1-9, delivering an knowing of some of the modes of chromatography that are now attainable utilizing commercially on hand columns, from reversed section to affinity. functional features and instrumentation are coated in bankruptcy 10. the majority of the e-book, which follows, provides examples and functions of every mode of chromatography in present biochemical perform.

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A popular use of fluorescence detection is in peptide chemistry where no convenient intrinsic chromophore is present. Derivatising agents such as orthophthalaldehyde and fluorescamine are used extensively in both pre- and post-column systems allowing detection of low picomole quantities (Chapter 11). In addition, detection can be performed using the intrinsic fluorescence of many compounds such as steroids, vitamins, and nucleotides. The conventional fluorescence detector consists of the following components: (1) A light source, either xenon or mercury arc, or quartz halogen.

This will be accentuated by high flow rates and can be best minimised by coating the stationary phase thinly over a non-porous particle. However, such pellicular paclungs suffer from the disadvantage that there is a relatively large, chromatographically inactive core which reduces the loading capacity of the column. g. large internal volume fittings, tubing, detector cells) or alternatively by an inefficient method of sample introduction. It is therefore important to use low, or better A . 1 1' 15 THE THEORY OF HPLC Ch.

Injectors The early design of HPLC injector systems was strongly influenced by those used in gas chromatography (GC). This is obvious when one considers that, although septum injection is still popular in GC, it has been almost totally supplanted in HPLC by high pressure injection Vents Load t + + inject Fig. 3. Illustration of the arrangement of a high pressure injection valve. In the load position the sample is introduced via the sample loop (positions 1,4). Upon injection the solvent flow from the pump (2) is directed through the sample loop to the column (3).

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