By A. Fallon, R.F.G. Booth and L.D. Bell (Eds.)
This publication is meant to familiarize biochemists with HPLC. Theoretical elements of every mode of chromatography are mentioned in chapters 1-9, supplying an figuring out of a number of the modes of chromatography that are now attainable utilizing commercially on hand columns, from reversed section to affinity. functional elements and instrumentation are coated in bankruptcy 10. the majority of the e-book, which follows, offers examples and purposes of every mode of chromatography in present biochemical perform.
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Additional resources for Applications of HPLC in Biochemistry
Electrochemical detectors are of two classes: ( a ) Bulk property detectors which measure changes in an electrochemical property of the bulk fluid flowing through the detector cell. The most popular detectors of this class are conductivity detectors which apply a potential across the cell so that ions in solution will move towards the attractive electrode with a consequent change in conductivity. Two major disadvantages of conductivity detectors are that the use of buffer salts in the mobile phase should generally be avoided and also that the system is temperature sensitive.
The incorporation of hydro-pneumatic dampeners consisting simply of a length of flattened tubing introduces an undesired extra dead volume between the pump and the column; however, they are still incorporated in some pump designs. The introduction of a fast refill cycle in the piston reduces pulsation APPLICATIONS OF HPLC IN BIOCHEMISTRY 26 Check valve 0 Cam r M otor dri ve I Check valve t solvent flow Fig. 2. Schematic illustration of the mechanism of the reciprocating pump. A piston, which is driven by a cam, alternatively draws in solvent through the inlet check valve (I) during the refill stroke and delivers a pulse in the forward stroke through the outlet The check valves are enlarged to show detail.
The most popular detector cells used in electrochemical detection are thin layer and may be used with either amperometric or coulometric detectors. The most popular design (Bioanalytical Systems) has the mobile phase flow parallel to the 40 APPLICATIONS OF HPLC IN BIOCHEMISTRY electrode surface and a planar auxiliary electrode placed opposite the working electrode with the thin layer channel functioning as a salt bridge to the reference electrode. An alternative design is the wall-jet detector where the flow is perpendicular to the electrode surface (Fleet and Little, 1974); however, although this cell is designed to provide more effective mass transfer to the electrode surface, conventional cells have generally demonstrated superior performances.
Applications of HPLC in Biochemistry by A. Fallon, R.F.G. Booth and L.D. Bell (Eds.)