One of the topics that comes up regularly in this column is the importance of controlling the column temperature. Retention
times can change by 1–2% for each 1 °C change in the column temperature, and peak spacing can change, as well. One of the
illustrations I like to use to emphasize the importance of using the column oven rather than ambient temperature relates to
a couple trips I took in 2008. In January I was in central China in a laboratory that was not heated, and the ambient temperature
was 10 °C; six months later I was in a temporary laboratory in Israel where, if the temperature wasn't 35 °C, it certainly
felt like it! These temperatures are drastically different from the 20–25 °C that most of us would quote when asked to put
a number on "ambient" temperature. Imagine trying to transfer a method between those two laboratories. In addition to control
of the column temperature, it is important that the mobile phase be preheated sufficiently so that there is no more than a
maximum of ≈6 °C difference between the column inlet and outlet.
Following this discussion in one of the classes, an attendee asked about the influence of the temperature of the solvent reservoir
and of the autosampler. My feeling is that the temperature of the solvent in the reservoir is not important, because by the
time the solvent travels through the pump and autosampler and all the associated connecting tubing, any preheating or cooling
provided in the reservoir will be canceled out by the thermal mass of the system. The control of the temperature of the autosampler
is most commonly accomplished by heating or, more commonly, cooling the sample tray. The goal with autosampler cooling is
to prevent sample degradation before injection, and because it only cools the sample it will have little or no influence on
the mobile-phase temperature.
So the conclusion of the discussion is that you should always use the column oven to control the column temperature, even
if it is just to set it a few degrees above room temperature — for example at 30–35 °C — to ensure a constant column temperature
and thus more stable retention times. You should cool the autosampler tray if your samples are likely to degrade at room temperature
before they are injected. Heating or cooling the mobile-phase reservoir has little, if any, effect on the chromatography.
Broad Peaks with a New Column
Another class attendee wondered why he observed broad peaks for his sample after installing a new column. This is a good example
of a time to apply the divide-and-conquer principle of troubleshooting — do a physical or mental experiment to divide the
problem up into smaller parts so that you can eliminate as many possible causes as you can. Before doing anything, I would
reinject the sample, and if the peaks were still broad, I would reinject a reference standard. If both of these injections
are still bad, it is not a problem with the particular sample.
My first question would be to determine if the problem occurred because the column was replaced. Were the peaks of normal
width before column replacement, but broader after? If so, then either the new column is bad, a nonequivalent replacement
was used, or something happened in connecting the column to cause the problem. The connections at each end of the column may
be the source of the problem, especially if polyether ether ketone (PEEK) tubing and fittings are used. If these are not seated
properly, a small gap in the connection can result in extracolumn volume and corresponding peak broadening. When adjusting
PEEK fittings, I recommend turning the pump flow off, loosening the fitting, pushing the tubing into the connection to ensure
it hits the bottom, and then tightening the nut. If you try to tighten the fitting with the flow on, it can slip during the
tightening process, creating exactly the problem you are trying to correct. Once again, reinject a reference standard to see
if the problem is now corrected. If the problem persists, the next easy step would be to replace the column with another new
column, double-checking that the manufacturer and part number is the same as the original column. While it is rare to receive
a bad column today, it can happen. No harm is done by double-checking with another new column, and if it is good, you can
just put the spare column in stock for later use.
Another check that should be made is to examine the chromatogram (or chromatograms) for other changes. Has the retention time
of one or more peaks changed? Has the peak spacing (relative retention) changed? If either of these situations has occurred,
it suggests a change in chemistry of the system. We've already eliminated the column as the source of problems through substitution
of another new column. The remaining chemical possibilities are the mobile phase and the column temperature. If the problem
origination coincided with a change in mobile phase, this is a likely source of the problem. For example, if acids or bases
are present, an error in pH adjustment of the mobile phase might be the problem source. Because it is easy to check by substitution,
make up a new batch of mobile phase and see if the problem is corrected. The final chemical possibility is a change in column
temperature. Make sure the column oven is on and set to the proper temperature.