Page 245 - Vogel's TEXTBOOK OF QUANTITATIVE CHEMICAL ANALYSIS

TYPES OF LlilUlO CHROMATOCRAPHV 8.2

of these being the most widely used. A practical consideration is that highly
active adsorbents rnay give rise to irreversible solute adsorption; silica gel, which
is slightly acidic, rnay strongly retain basic compounds, whilst alumina (non-acid
washed) is basic and should not be used for the chromatography of base-sensitive
compounds. Adsorbents of varying particle size, e.g. 20-40pm for TLC and
down to 5 pm for HPLC, rnay be purchased commercially.
The role of the solvent in LSC is clearly vital since mobile-phase (solvent)
molecules compete with solute molecules for polar adsorption sites. The stronger
the interaction between the mobile phase and the stationary phase, the weaker
will be solute adsorption, and vice versa. The classification of solvents according
to their strength of adsorption is referred to as an eluotropic se rie^,^^ which
rnay be used as a guide to find the optimum solvent strength for a particular
separation; a trial-and-error approach may, however, be required and this is
done more rapidly by TLC than by using a column technique. Solvent purity
is very important in LSC since water and other polar impurities rnay significantly
affect column performance, whilst the presence of UV-active impurities is
undesirable when using UV-type detectors.
In general, the compounds best separated by LSC are those which are soluble
in organic solvents and are non-ionic. Water soluble non-ionic compounds are
better separated using either reverse-phase or bonded-phase chrornatography.
2. Liquid-liquid (partition) chromatography (LLC). This type of chromatography
is similar in principle to solvent extraction (see Chapter 6), being based upon
the distribution of solute molecules between two immiscible liquid phases
according to their relative solubilities. The separating medium consists of a
finely divided inert support (e.g. silica gel, kieselguhr, etc.) holding a fixed
(stationary) liquid phase, and separation is achieved by passing a mobile phase
over the stationary phase. The latter rnay be in the form of a packed column,
a thin layer on glass, or a paper strip.
It is convenient to divide LLC into two categories, based on the relative
polarities of the stationary and mobile phases. The term 'normal LLC' is used
when the stationary phase is polar and the mobile phase is non-polar. In this
case the solute elution order is based on the principle that non-polar solutes
prefer the mobile phase and elute first, while polar solutes prefer the stationary
phase and elute later. In reverse-phase chromatography (RPC), however, the
stationary phase is non-polar and the mobile phase is polar; the solute elution
order is commonly the reverse of that observed in normal LLC, i.e. with polar
compounds eluting first and non-polar ones later. This is a popular mode of
operation due to its versatility and scope, the almost universal application of
RPC arising from the fact that nearly al1 organic molecules have hydrophobic
regions in their structure and are therefore capable of interacting with the
non-polar stationary phase.* Since the mobile phase in RPC is polar, and
commonly contains water, the method is particularly suited to the separation
of polar substances which are either insoluble in organic solvents or bind too
strongly to solid adsorbents (LSC) for successful elution. Table 8.1 shows some
typical stationary and mobile phases which are used in normal and reverse
phase chrornatography.

*The reverse-phase technique is used less, however, with the advent of hydrophobic bonded phases
(Section 8.2(3)).