By A. Fallon, R.F.G. Booth and L.D. Bell (Eds.)
This booklet is meant to familiarize biochemists with HPLC. Theoretical points of every mode of chromatography are mentioned in chapters 1-9, delivering an knowing of a number of the modes of chromatography that are now attainable utilizing commercially to be had columns, from reversed section to affinity. functional points and instrumentation are lined in bankruptcy 10. the majority of the e-book, which follows, provides examples and purposes of every mode of chromatography in present biochemical perform.
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Additional info for Applications of HPLC in Biochemistry
Secondly, the solute must interact with, and diffuse through, the stationary phase. On ion-exchange resins the rate of diffusion through the stationary phase, to and from the ion-exchange site, is relatively slow. T h s slow mass transfer results in a large contribution towards band broadening. Pellicular materials with only a thin layer of resin were evolved to 46 APPLICATIONS OF HPLC IN BIOCHEMISTRY I I I I 20 40 60 Temperature, *c Fig. 3. Effect of temperature on column efficiency for the resolution of fumaric acid.
This machine allows either selected peaks or time-windows to be collected and the sample volume which can be collected is virtually unlimited. g. with OPA) and then automatically inject derivatised samples. 9. 1. Autosampler The most obvious step in the automation of a chromatographic system is to automate sample injection. The ability to perform re- APPLICATIONS OF HPLC IN BIOCHEMISTRY 42 peated functions without operator attendance is a labour-saving method available to all chromatographers. Machines such as the Waters Intelligent Sample Processor (WISP) and Kontron MS1, which are accurate down to less than 10 pl with negligible cross-contamination, are suitable for most applications.
3. Mobile phase effects Many biochemical compounds which are charged at a specific pH can be separated by ion-exchange chromatography. 2) The degree of solute retention can be finely controlled by the pH of the mobile phase. 1) to the left and only low concentrations of A- will compete for anion-exchangesites. Consequently, a drop in pH will cause acidic solutes to elute rapidly from an anion-exchange column. 2) to the left and only low concentrations of BH+ will compete for cation-exchange sites.