Temperature Effects
The choice of operating temperature can have a profound effect on a chromatographic separation due to
the temperature dependence of the distribution ratio D of each solute or to be strict, of the distribution
coefficient KD (cf. solvent extraction, p. 56). The relation is an exponential one,
and a change in temperature of 20 K can result in a twofold change in KD. As , the enthalpy of
sorption, is usually negative increasing the temperature decreases KD, or D, which leads to a
corresponding decrease in tR, the retention time, or an increase in Rf, the retardation factor. Therefore, in
the interests of speed, a higher temperature may be considered desirable but this can be at the expense
of resolution, which will be affected by the increased rates of diffusion and mass transfer. At lower
temperatures resolution may be better, but the time required may be unacceptably long and a
compromise is usually sought. Whatever the ultimate choice of operating temperature it is important
that it is reproducible if valid comparisons are to be made.
Isolation of Separated Components
One aspect in which column chromatography differs from counter-current distribution is in the recovery
of the separated components. In Tswett's original method, the individual pigments were recovered from
the column itself rather as they would be collected from the glass separation units of the Craig
apparatus. Nowadays, the usual procedure is to remove the components of a mixture from the column
sequentially by sweeping them through with the mobile phase, a process known as elution. A sample is
introduced onto the top of the column and the pure mobile phase passed through it continuously until all
the components have been eluted. The order of elution depends on the individual distribution ratios for
each solute, and incomplete separation may occur where these are not sufficiently different. A plot of
volume of eluting agent or time against concentrations of the eluted species provides an elution profile
from which both qualitative and quantitative information can be obtained (Figure 4.16).
Gradient elution is a procedure whereby the conditions under which the sample is eluted are
progressively varied throughout the separation so as to speed up the process. This can be achieved by
altering the composition of the mobile phase or increasing the temperature or flow rate. The effect is to
elute components more rapidly in the latter stages and sharpen their elution profiles. Stepwise elution is
a similar procedure in which elution conditions are changed at predetermined times rather than
continuously.
Two other means of separating and removing components from a column are frontal analysis and
displacement development, but these are of secondary importance. In frontal analysis sample is
continuously applied to the top of the column. Eventually, as the stationary phase becomes