Page 250 - Vogel's TEXTBOOK OF QUANTITATIVE CHEMICAL ANALYSIS

8 COLUMN AN0 THIN4AVER LlîlUlO CHROMATOCRAPHV
using a pulse dampener; dual- and triple-head reciprocating pumps can be
operated without a pulse dampener since they minimise pulsation, but are more
expensive than single-head pumps.
The choice of a suitable mobile phase is vital in HPLC and it is appropriate
to refer to the factors influencing this choice. Thus, the eluting power of the
mobile phase is determined by its overa!! polarity, the polarity of the stationary
phase and the nature of the sample components. For 'normal-phase' separations
eluting power increases with increasing polarity of the solvent, while for
'reverse-phase' separations eluting power decreases with increasing solvent
polarity. Optimum separating conditions can be often achieved by using a
mixture of two solvents, and gradient elution is frequently used where sample
components Vary widely in polarity. For gradient elution using a low-pressure
mixing system, the solvents from separate reservoirs are fed to a mixing chamber
and the mixed solvent is then pumped to the column; in modern instruments
delivery of solvent to the pump is controlled by time-proportioning electrovalves,
regulated by a microprocessor.52
Other properties of solvents which need to be considered are boiling point,
viscosity (lower viscosity generally gives greater chromatographic efficiency),
detector compatibility, flammability, and toxicity. Many of the common solvents
used in HPLC are flammable and some are toxic and it is therefore advisable
for HPLC instrumentation to be used in a well-ventilated laboratory, if possible
under an extraction duct or hood.
Special grades of solvents are available for HPLC which have been carefully
purified to remove UV-absorbing impurities and any particulate matter. If,
however, other grades of solvent are used, purification may be required since
impurities present would, if strongly UV-absorbing, affect the detector or, if of
higher polarity than the solvent (e.g. traces of water or ethanol, commonly
added as a stabiliser, in chloroform), influence the separation. It is also important
to remove dissolved air or suspended air bubbles which can be a major cause
of practical problems in HPLC, particularly affecting the operation of the pump
and detector. These problems may be avoided by de-gassing the mobile phase
before use; this can be accomplished by placing the mobile phase under vacuum,
or by heating and ultrasonic stirring. Compilations of useful solvents for HPLC
are a~ailable.~~

Sample injection system. Introduction of the sample is generally achieved in
one of two ways, either by using syringe injection or through a sampling valve.
Septum injectors allow sample introduction by a high-pressure syringe
through a self-sealing elastomer septum. One of the problems associated with
septum injectors is the leaching effect of the mobile phase in contact with the
septum, which may give rise to ghost peaks. In general, syringe injection for
HPLC is more troublesome than in gas chromatography.
Although the problems associated with septum injectors can be eliminated
by using stop-flow septumless injection, currently the most widely used devices
in commercial chromatographs are the microvolume sampling valves (Fig. 8.3)
which enable samples to be introduced reproducibly into pressurised columns
without significant interruption of the mobile phase flow. The sample is loaded
at atmospheric pressure into an external loop in the valve and introduced into
the mobile phase by an appropriate rotation of the valve. The volume of sample
introduced, ranging from 2pL to over 100pL, may be varied by changing