<1. Selectivity and/or retention time changes>

1. Selectivity and/or retention time changes

Retention time changes can be caused by a wide range of chemical and/or physical problems with the chromatography.

For isocratic separations, the first thing to look at is whether the retention times of all the peaks (including the dead time of the system) changed by the same percentage. If so, this is diagnostic of an incorrect flow rate. We recommend the following steps:

Confirm that your pump flow rate is correctly set.
If the retention times are longer than specified, check the system carefully for evidence of leaks.
If the retention times are longer than specified, check the system for evidence of bubble problems (inadequate degassing) or check valve failure; these will often be accompanied by excessive fluctuations in the baseline or the system pressure.

For isocratic separations, if the dead time meets specification and/or if the retention times of different peaks in the separation have changed by different percentages, then a change in system chemistry should be suspected. We recommend the following steps:

Check that the column temperature is correct (if the column compartment temperature setting is correct, use a thermometer to confirm the actual temperature).
Check to confirm that the mobile phase was correctly prepared (especially if the problem coincided with a new batch of mobile phase!).
Check to make sure that the correct type of column is being used.
If the column is nearing the end of its useful lifetime, replace the column.
If there is a trend to the change (i.e., if retention or selectivity changes in the same fashion with each injection, check to see if the change correlates with the number of injections or with the volume of mobile phase pumped (carry out a series of injections with different delay times between injections, then plot retention time or alpha as a function of time and as a function of number of injections).

If the trend correlates with number of injections, you should suspect that some sample component is building up on the column.
If the trend correlates with time (i.e., with volume of solvent pumped), you should suspect that some mobile phase component is building up on the column or that the column is deteriorating under your separation conditions.

For gradient separations, if the retention times of all the peaks have changed by the same amount, but the dead time of the system is still within specifications, a change in dwell volume is a strong possibility. Check to see whether the instrument plumbing has been changed recently (for example, substitution of a larger or smaller injection loop).

For gradient separations, a change in selectivity can be caused by a number of factors:

Flow rate differences can affect selectivity in gradient separations (unlike isocratic separations).
A change in instrument dwell volume can affect selectivity, particularly for early-eluting peaks. Check your dwell volume to confirm that it meets specifications.
A change in operating temperature of the column is always a possibility. Check that the column temperature is correct (if the column compartment temperature setting is correct, use a thermometer to confirm the actual temperature).
Differences in column equilibration at the beginning of the run can affect retention and selectivity. Check to make sure that the column equilibration time is exactly the same before each run. Alternatively, confirm that the equilibration time has been sufficient to completely equilibrate the column.
A malfunction in the pumping system or proportioning valve may result in retention and/or selectivity changes. This is very likely if the problems are intermittent.
Check to confirm that the mobile phase was correctly prepared (especially if the problem coincided with a new batch of mobile phase!).
Check to make sure that the correct type of column is being used.

Ref: LC-GC, 15(9) 826 (1997)
Ref: LC-GC, 17(10) 908 (1999)