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Study Notes: Optimisation of Column Performance

Introduction
Optimisation of chromatographic separations is achieved by varying the experimental conditions of the run until the components of the mixture are separated cleanly in a reasonable amount of time. There are two aspects to achieving good separation:

  • The components in the mixture need to migrate or travel down the column at sufficiently different rates.
  • The peaks for the components need to be relatively sharp and uniform (as components migrate, they tend to broaden or spread out such that they can overlap each other, thereby compromising detection/accuracy).

Obviously just altering conditions with no knowledge of the underlying theory of chromatographic separations would be very time consuming, wasteful and unlikely to lead to good separations.

Optimisation has two aims:

  • Reduction of zone broadening (the component moves through the column as a zone which is detected by the detector and translated into a peak).
  • Altering the migration rates of the components.
A poor separation of components A and B. Graph shows peaks a and b are overlapping at resolution of 0.75.
   
An improvement in resolution leads to a partial separation of the peaks. Graph shows peaks a and b have separated slightly at resolution of 1.0.
   
A further improvement in resolution has led to separation of the peaks. Graph shows peaks a and b are resolved from each other at resolution of 1.5.

Resolution refers to the extent of separation of the peaks of interest on a chromatogram.

Column resolution
Resolution RS is a quantitative measure of the ability of the column under specific operating conditions to separate (or resolve) two analyte peaks. The resolution for a given stationary phase can be improved by lengthening of the column thus increasing the number of plates, but a negative effect of this approach is the increased time required for separation and the band broadening that will occur.

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Optimising column performance - Reduction of peak broadening
Variables that may be adjusted to reduce peak broadening are those that lead to an improvement in column efficiency. Making adjustments to increase the number of theoretical plates (N) or decreasing plate height (H) will therefore reduce the tendency of peaks to broaden.

The variables to be adjusted include:

  • linear velocity of the mobile phase (too low and too high reduces efficiency)
  • diameter of packing particle (smaller is generally better)
  • thickness of the liquid coating of the stationary phase (HPLC)
  • column temperature (GC)
  • diameter of the column (narrower is better).

Optimising column performance – Altering migration rates
Migration rates are altered with the aim of ensuring that the components of interest all elute from the column within a reasonable time and are sufficiently separated from each other. The extent of separation in this regard indicates how effective the chromatographic column is.

The variables to be adjusted that influence migration rates include:

  • column temperature (GC)
  • mobile phase composition (HPLC) eg 70% methanol/30% water arrow right 50%/50%, gradient elution
  • chemical composition of the stationary phase – use a different type of column
  • special chemical effects – incorporating a species in the stationary phase that interacts with one or more of the sample components to enhance resolution.

You should be aware that there are a numbers of specific parameters relating to the study of migration rates:

  • Capacity factor
  • Selectivity factor.

If you are working on the development or optimisation of chromatographic techniques you may need to find out about these terms.

The general elution problem
This is best illustrated in the following diagram of a six-component mixture - three pairs of closely migrating peaks.Skip flash movie

Graphs

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Look at the three panels (a), (b) and (c), which represent separations under three different conditions. Note that in each panel only two of the peaks are well resolved with minimal broadening. The other two pairs are either not resolved (overlap) or are broad and diffuse.

This is a common problem in chromatography and may be overcome by the following two approaches:

  • Set up the run so that only the peaks of interest are well resolved and not worry about the others. For example, if peaks 3 and 4 are those of interest then set up the run with the same conditions as panel (c).
  • Change conditions during the separation to achieve the required resolution of all 6 peaks. That is, use a gradient or stepwise elution. For HPLC, change the composition of the mobile phase and for GC use temperature increases (temperature programming) to induce the appropriate changes. These will be discussed in the HPLC and GC sections later.

 

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