Page 251 - Vogel's TEXTBOOK OF QUANTITATIVE CHEMICAL ANALYSIS

EilUlPMENT FOR HPLC 8.3

Mobile phase To column Mobile phase To column

Adjustable -
length
sample loop

(a) Sampling mode (b) Injection mode
Fig. 8.3 Operation of a sample loop.

the volume of the sample loop or by using special variable-volume sample
valves. Automatic sample injectors are also available which allow unattended
(e.g. overnight) operation of the instrument. Valve injection is preferred for
quantitative work because of its higher precision compared to syringe injection.
The column. The columns most commonly used are made from precision-bore
polished stainless steel tubing, typical dimensions being 10-30cm long and
4 or 5 mm interna1 diameter. The stationary phase or packing is retained at
each end by thin stainless steel frits with a mesh of 2 pm or less.
The packings used in modern HPLC consist of small, rigid particles having
a narrow particle-size distribution. The types of packing may conveniently be
divided into the following three general categories.

(a) Porous, polymeric beads based on styrene-divinylbenzene copolymers.
These are used for ion exchange (see Chapter 7) and size exclusion
chromatography (Section 8.2), but have been replaced for many analytical
applications by silica-based packings which are more efficient and
mechanically stable.
(b) Porous-layer beads (diameter 30-55 pm) consisting of a thin shell(1-3 pm)
of silica, or modified silica or other material, on an inert spherical core (e.g.
glass beads). These pellicular-type packings are still used for some ion
exchange applications, but their general use in HPLC has declined with
the development of totally porous microparticulate packings.
(c) Totally porous silica particles (diameter < 10 pm, with narrow particle size
range) are now the basis of the most commercially important column
packings for analytical HPLC. Compared with the porous-layer beads,
totally porous silica particles give considerable improvements in column
efficiency, sample capacity, and speed of analysis.

The development of bonded phases (Section 8.2) for liquid-liquid chromato-
graphy on silica-gel columns is of major importance. For example, the widely
used C-18 type permits the separation of moderately polar mixtures and is used
for the analysis of pharmaceuticals, drugs and pesticides.
The procedure chosen for column packing depends chiefly on the mechanical
strength of the packing and its particle size. Particles of diameter >20 pm can
usually be dry-packed, whereas for particles with diameters < 20 pm slurry