What is a good chromatograph?...

In order to obtain accurate measurements, it is necessary to have an understanding of what factors in our chromatography experiments affect the shape of chromatograms and thus our ability to characterize and separate mixtures. This is where science becomes art, as continued lab experience and practice with chromatography is how best to learn to finesse results into accuracy. However, we will discuss two main topics, resolution and band broadening, and how flow rate and stationary/mobile phase interaction can affect them and our ability to obtain a good chromatogram.

Resolution

Resolution is a measure of our ability to distinguish between two different peaks. If two eluents have similar retention times, then their chromatograms may blend together and appear as one curve. Thus we can easily lose track of mixture components and inaccurately compute retention times and concentrations for false peaks.

The above chromatogram appears to be have only one peak, but we can detect a slight anomaly -- the notch in the top -- which might suggest that there is more there than we thought. We require a way to increase the time between these retention times. Recall that retention time is a function of the mobile phase-stationary phase interaction. The nature of our mobile phase and stationary phase and the equibrium of our analytes between the two phases determines their retention times. Each chromatography type has its own methods for adjusting these interactions, such as temperature and concentration control, that directly affect the quality of the phases themselves.

Increased resolution demonstrates the presence of two analytes.

Band Broadening

While two peaks might have adequately space retention times, precision problems can arise if the peaks themselves are not sharp enough. Band broadening is an increase in width in our peaks. If we refer back to our discussion of Gaussian distributions, recall that chromatograms are useful because they graphically afford us a mean retention time. The standard deviation is a graphical measure of the relative width of these curves and mathematically an indicator of the total data's proximity to the mean value. We might have an average retention time, but if the standard deviation is too great, then the mean is not a precise representation of the entire data collection and therefore is not as useful.

Band broadening arises from the same mobile/stationary phase interaction as described above. There is a greater variation in the type of interactions that analyte molecules are having with the phases, causing a wider distribution of retention times. Careful choice of mobile and stationary phases will also help in curtailing this phenomena. Another possible solution, which could also assist with the previous resolution problem, would be to alter the flow rate of the mobile phase. Adjusting the flow rate changes the ability of the analyte to interact with the stationary phase. Increasing the carrier flow will cause a faster elution because the analyte will have less time to spend interacting with the stationary phase and thus less time IN the stationary phase. Decreasing the carrier flow allows more time for these interactions to occur and will increase band broadening while increasing resolution.

Methods for achieving better separations are discussed in more depth in our separate treatments of chromatography types.