Optimizing chromatographic separations
In an ideal chromatographic analysis the sample molecules will be completely
separated, and detection of components will result in a series of discrete
individual peaks corresponding to each type of molecule. However, to
minimize the possibility of overlapping peaks, or of peaks composed of more
than one substance, it is important to maximize the separation efficiency of
the technique, which depends on:
- the selectivity, as measured by the relative retention times of the two
components, or by the volume of the mobile phase between the
peak maxima of the two components after they have passed through the
column; this depends on the ability of the chromatographic method to
separate two components with similar properties;
- the band-broadening properties of the chromatographic system, which
influence the width of the peaks; these are mainly due to the effects of
diffusion.
The resolution of two adjacent components can be defined in terms of
k',
α and N, using eqn [31.6]. In practical terms, good resolution is achieved when
there is a large 'distance' (either time or volume) between peak maxima, and
the peaks are as narrow as possible. The resolution of components is also
affected by the relative amount of each substance: for systems showing low
resolution, it can be difficult to resolve small amounts of a particular
component in the presence of larger amounts of a second component. If you
cannot obtain the desired results from a poorly resolved chromatogram,
other chromatographic conditions, or even different methods, should be tried
in an attempt to improve resolution. For liquid chromatography, changes in
the following factors may improve resolution:
- Stationary-phase particle size - the smaller the particle, the greater the
area available for partitioning between the mobile phase and the
stationary phase. This partly accounts for the high resolution observed
with HPLC compared with low-pressure methods.
- The slope of the salt gradient in eluting IEC columns, e.g. using
computer-controlled adapted gradients.
- In low-pressure liquid chromatography, the flow rate of the mobile phase
must be optimized because this influences two band-broadening effects
which are dependent on diffusion of sample molecules:
- the flow rate
must be slow enough to allow effective partitioning between the mobile
phase and the stationary phase: and
- it must be fast enough to ensure
that there is minimal diffusion along the column once the molecules have
been separated. To allow for these opposing influences, a compromise
flow rate must be used.
- If you prepare your own columns, they must be packed correctly, with no
channels present that might result in uneven flow and eddy diffusion.
