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Study Notes: Applications of Chromatography

Introduction
We now know a lot about the theory of chromatography and how things work so what is chromatography used for and what can it do?

Qualitative analysis
The chromatogram of a run provides one piece of information about each species in a sample, the retention time or position, but this information is important and can be used to identify multiple species in a mixture.

With experience it is a relatively simple task to identify species by their position but in initial work the identity of each separated species on the chromatogram should be confirmed by analytical methods such as nuclear magnetic resonance or mass spectroscopy. That is, a chromatogram does not positively identify a species in a mixture.

Of course a chromatogram also provides information of the absence of a species from a sample. For example, lack of steroids from an Olympic weightlifter’s urine sample is seen as a great benefit to the career of the weightlifter!

A graphical representation showing two identical chromatograms, except that the chromatogram for Athlete B has an additional 'steroid' peak as well as the solvent peak.

Quantitative analysis
Chromatography also provides some important qualitative information about the separated species present in a mixture. For column chromatography, comparison of the height of a peak or the area under a peak can be compared with standards to determine the amount of that species present. For planar chromatography the area covered by the spot of the separated species can be measured and again compared to a standard to arrive at an estimation of the amount of the species present.

Standards are usually run separately and it is crucial to ensure conditions do not change between runs. In some circumstances standards may be run with the sample and therefore conditions should not change during the run that will affect the peak heights or widths.

If the run is set up correctly these measured parameters vary linearly with concentration.

Analyses based on peak height
This is measured as shown in the following diagram. Essentially the base lines on either side of the peak are connected with a straight line and the perpendicular distance to the peak is measured. Peak broadening can reduce the accuracy of this approach.

A graphical representation for measuring peak height and area. For example, a peak height is 'a', with a corresponding width Wa and half width Wa divided by 2.

Analyses based on peak areas
Peak areas are independent of peak broadening effects and are therefore a better measure of concentration.

Modern chromatography equipment have digital integrators that will automatically measure areas under the peaks but if these are lacking then manual methods must be used.

These include:

  • cutting out the peak from the chromatogram, weighing on a balance and comparing the mass to a standard area of say 100 cm2
  • for high, narrow, symmetric peaks assume they are triangles and multiply the height of the peak by one half of the width of the base of the peak.

Calibration and standards
The easiest approach is to prepare a series of standard solutions that approximate the composition of the unknown. The chromatograms of the standards are used to plot the peak heights or areas against concentration to obtain a standard curve. This should be a straight line that passes through the origin.

A standard curve used to determine ethyl acetate concentration in beer over the range 0 to 300 mg/L. The line goes through 0 and the point (40, 20.0), where Peak area (x1000) is on the horizontal axis and ethyl acetate concentration (mg/L) is on the vertical axis.

The standard curve is then used to determine the concentration of the unknown sample. Of course the run conditions must be as close to identical as possible and frequent re-standardisation is required for accuracy.

The internal standard method
The greatest accuracy is obtained by the use of an internal standard as uncertainties due to sample injection and changes to the running conditions are eliminated. In this case the internal standard should be of a different species and run close to the peak of interest, but well away from other peaks in the sample. The internal standard is added to the sample at a known concentration.

The ratio of the analyte to the internal standard peak area (or height) is used to calculate the concentration of the analyte.

 

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