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Matrices and stationary phases

Two main forms of matrix/stationary phase material are available, based on a rigid

solid structure. Both forms involve approximately spherical particles of a uniform

size to minimise space for diffusion and hence band broadening to occur. They are

made of chemically modified silica or styrene/divinylbenzene copolymers. The two

forms are:

• Microporous supports: In which micropores ramify through the particles that are
  generally 5 10 mm in diameter.


• Bonded phases: In which the stationary phase is chemically bonded onto an inert
  support such as silica.

11.3.2 Application of sample

The application of the sample onto an HPLC column in the correct way is a particularly

important factor in achieving successful separations. The most common method of

sample introduction is by use of aloop injector(Fig. 11.5). This consists of a metal

loop, of fixed small volume, that can be filled with the sample. The eluent from the

pump is then channelled through the loop by means of a valve switching system and

the sample flushed onto the column via the loop outlet without interruption of the

flow of eluent to the column.

Repeated application of highly impure samples such as sera, urine, plasma or whole

blood, which have preferably been deproteinated, may eventually cause the column to

lose its resolving power. To prevent this occurrence, aguard columnis often installed

between the injector and the column. This guard column is a short (12 cm) column of

the same internal diameter and packed with material similar to that present in the

analytical column. The packing in the guard column preferentially retains contamin-

ating material and can be replaced at regular intervals.

Solvent

reservoir

High pressure

pump

Column

Sample injection

loop

Detector

Recorder and

data system

Sample

collector

Fig. 11.4Components of an isocratic HPLC system. For gradient elution two reservoirs and two pumps

are used with liquid-phase mixing before entry to the sample injection loop.

447 11.3 High-performance liquid chromatography