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[17] What do Chromatograms tell us? Retention Times are much Shorter then Expected

6 Aug 2013

hromatograms are like fingerprints.  If you can “read” chromatograms well, you often can find a plausible cause. In this series, we will show a series of GC-chromatograms that are obtained from users and discuss some potential causes for the phenomena. Then we can move into some solutions for improvement.

You replace your column for a new one and you find a chromatogram as shown in Fig. 1B.

Your original chromatogram looks like Figure 1A.  The first peak elutes at the same retention time, but the other retention times seem to have decreased significantly.    This is the opposite of the situation we had at the previous blog. What can cause this difference?

Fig. 1 A: original analysis; B: after replacement with a "similar" column. Retention times on new column are much shorter.

First check is the oven temperature. Check if the actual temperature is indeed correct. If the oven is too high it will give this result. Every 15C change of temperature, the retention increase/decrease a factor 2.  Use a separate thermometer to verify the exact oven temperature. Do not automatically “trust” digital readouts.

If the temperature is OK, it must be related to the retention of the separation column.

If the same replacement column is chosen, we have to look systematically what can cause a shorter retention. First secure that we use the same of stationary phase. This can be checked by looking at elution order of test compounds.  You may run the same test as the vendor does to QA the column. If this is shows similar elution, the phase type should be OK. In the example, the chromatogram shows a similar separation, meaning the stationary phase type is pretty similar.

Column diameter can play a bigger role. If a 0.32mm column is used with the same film, the absolute retention will be lower.  Therefore, check column ID. You may have sensed some difference when installing as the 0.32mm column. This column has an OD of 0.45mm and will not easily pass a ferrule that is used for 0.25mm ID columns. A 0.25mm column has an OD of 0.38.  Also 0.25mm columns need a higher pressure to set a certain linear velocity. This may be an indicator for you that the ID is not correct.

For change of Psi to kPa or other unit, you may use this calculator:

If the replacement column has a different film thickness, you also will get the higher retention.  In this case, check the data obtained with the vendor of the column, as they might have delivered a column with a thicker film. Mistakes always can happen.