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phases have been developed where the liquid phase has been covalently bonded to the
supporting material which may be silica or a silicone polymer. The silicone polymer bonded
phases have the particular advantage that as well as not being eluted by the developing
solvent, they are chemically, hydrolytically and thermally stable. In normal phase liquid
liquid chromatography, the stationary phase is a polar compound such as alloys nitride or
alloylamine derivatives and the mobile phase a non polar solvent such as hexane. For
reverse phase chromatography, the stationary phase is non polar compound such as a C8
or C18 hydrocarbon and the mobile phase a polar solvent such as water / acetonitrile or
water / methanol mixtures.
Many different types of ion exchangers are available of which the cross linked
microporous polystyrene resins are widely used. Pellicular resin forms are also available, as
are bonded phase exchangers covalently bonded to a cross linked silicone network. These
resins are classed as hard and readily withstand the pressures required during analysis.
The stationary phases for exclusion separations are generally porous silica, beads,
polystyrene or polyvinyl acetate the eluting solvent is an organic system, and the beads are
available in a range of pere sizes. Semi rigid gels such as sephedex or bio gel P and non
rigid gels such as sepharese and bio gel A are only of limited use in HPLC since they can
withstand only low pressure. The supports for affinity separations are similar to those for
exclusion separations. The spacer arm and ligand are attached to these supports by similar
chemical means to those used in conventional low pressure affinity chromatography. Table
30.1 lists some examples of commonly used stationary phases and their applications.
The column:
The columns used for HPLC are generally made of stainless steel and are
manufactured so that they can withstand pressure of up to 5.5 × 107 Pa (8000psi). Straight
column of 20 to 50 cm in length and 1 to 4 mm in diameter are generally used though
smaller capillary columns are available. The best columns are precision bored with an
internal mirror finish which allows efficient packing of the column. Porous plugs of stainless
steel or Teflon are used in the ends of the columns to retain the packing material. The plugs
must be homogeneous to ensure the uniform flow of solvent through the column. It is
important in some separations involving liquid partition and ion exchange that the column
temperature us thermostatically controlled during the analysis.
Column packing procedure:
Columns may be purchased already packed from commercial companies with
specified packing material structure and dimensions. Many workers, however, prefer to pack
their own columns since this is cheaper than purchasing pre packed columns. Several
methods are available for packing columns and the method used will depend on the nature
of the packing material and the dimensions of the particles. The major priority in the packing
of a column is to obtain a uniform bed of material with no cracks or channels. Rigid solids
103
and hard gels should be packed as densely as possible, but without fracturing the particles
the packing process. The most widely used technique for column packing is the high
pressure slurring technique. A suspension of the packing is made in a solvent of equal
density to the packing material. The slurry is then rapidly pumped at high pressure into a
column with a porous plug at its outlet. The resulting bed of packed material within the
column can then be prepared for use by running the developing solvent through the column,
hence equilibrating the packing with the developing solvent. When hard gels are packed, it is
necessary for them to be allowed to swell first in the solvent to be used in the
chromatographic process before packing under pressure. Soft gels cannot be packed under
pressure and have to be allowed to pack from slurry in the column under gravitational
sedimentation only, in a similar way to the packing of columns for conventional column
chromatography.
Chromatographic solvent (mobile phase):
The choice of mobile phase to be used in any separation will depend on the type
separation to be achieved. Isocratic separations may be more made with a single solvent, or
two or more solvents mixed in fixed proportions. Alternatively a gradient elution system may
be used where the composition of the developing solvent is continuously changed by use of
suitable gradient elution system may be used where the composition of the developing
solvent is continuously changed by use of a suitable gradient programmer. In the majority of
cases this involves the use of two pumps. All solvents for use in HPLC systems must be
specially purified since traces of impurities can affect the column and interfere with the
detection system. This is particularly the case if the detection system is measuring
absorbance at below 200 nm. Purified solvents for use in HPLC systems are available
commercially, but even with these solvents a 1 to 5 µm micro filter is generally introduced
into the system prior to the pump. It is also essential that all solvents are degassed before use
otherwise gassing tends to occur in most pumps. It tends to be particularly bad for aqueous
methanol and ethanol solvents, gassing (the presence of air bubbles in the solvent) can alter
column resolution and interface with the continuous monitoring of the column effluent.
Degassing may be carried out in several ways; by warming the solvent, by stirring it
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