Getting Started in HPLC

Section 3D. Other Factors

In this final section of Getting the Separation we will consider some "odds & ends": the effect of column temperature, sample size in relation to detector settings, and other LC methods besides reversed-phase chromatography.

Consider first the importance of temperature in affecting the LC separation. The general result of an increase in temperature is to decrease the retention times of all peaks in the chromatogram as shown at the right. Often this is accompanied by some decrease in sample resolution just as in the case of increasing solvent strength. In other cases, peaks within the chromatogram may change their relative positions as temperature is varied, and this leads to more complex alterations in the separation. Peaks may move together and become severely overlapped, and in some cases two peaks may simply change places.

Because of these temperature-related effects, it is advantageous to hold column temperature constant, while the temperature of the room changes. That is, keeping column temperature constant is more likely to maintain the same separation from day to day, and within a day. The column can be thermostatted, using either ovens with circulating air (air bath), or metal contact heaters. Ovens are usually more convenient. If the LC system is not thermostatted, variations in room temperature can be compensated for in various ways. Changes in column temperature can be minimized in this case by protecting the column against drafts, and by wrapping the column with insulating material. Severe changes in room temperature during the working shift can nevertheless cause problems, even with this expedient. For example, a common situation in the summer is for a laboratory to heat up during the day - if the lab is not air-conditioned. This leads to a reduction in separation time, and possibly some loss in resolution. If the separation is barely adequate at lower temperatures, it may not be acceptable at higher temperatures.


Changing temperature affects retention and may also affect selectivity

More serious is the case of a lowering of the temperature, especially for samples that are processed automatically using an autosampler. In this case, as the temperature decreases, retention time increases, and there may no longer be enough time between sample injections to get all the bands off the column. Then peaks from a previous sample may overlap the chromatogram for the next sample. Changes in retention of this kind can often be compensated for by increasing the flow rate; the separation time can then be readjusted to the original (higher temperature) value, and often the effects of changing temperature and flow rate together cancel in terms of resolution or separation. However, this kind of adjustment (change in flow rate) should only be done if the LC procedure specifically allows it.

Consider next the effect of a change in sample size on the resulting chromatogram. We talked in Unit 2 about the use of detector settings to adjust the size of peaks in the chromatogram (seen on the data system display). Specifically, a change in attenuation adjusts the relative heights of all peaks by the same factor. For the case of very small peaks, which are hard to see and to measure accurately, a decrease in attenuation (increase in sensitivity) at the detector can make the peak larger and easier to work with. This is illustrated at the right for two different attenuations (in terms of Absorbance Units Full Scale or AUFS).

However the precision of peak size for very small bands is not just a function of how tall the band is on the recorder strip chart. It also depends on the NOISE in the baseline. If the baseline is erratic or drifts, as in the case of the 0.05 AUFS chromatogram, the peak height is limited by our inability to precisely measure the baseline. We say that the SIGNAL-TO-NOISE RATIO is small. Decreasing the detector attenuation does not by itself improve the signal-to-noise, because baseline noise increases in proportion to peak height. However bigger peaks are easier to measure manually (a data system does not care what attenuation you use, but it is affected by attentuation changes at the detector).

If we increase the size of the sample we inject, while decreasing attenuation by the same ratio, the chromatogram will normally show no change; that is, an increase in the amount of sample injected is just cancelled out by decrease in the detector sensitivity. However this situation only holds for moderate sample sizes. If we increase the sample size too much, eventually the column becomes overloaded (too much sample), retention times decrease and the separation degrades (poorer resolution). We generally want to avoid samples that are this large, and to carry out separations only on non-overloaded columns.



2000, LC Resources Inc. All rights reserved.
Last revised: April 02, 2001.