If you have experienced retention time shifts with LC, you know it can be complicated and can cause problems with quantitation if not resolved. Hopefully this post will help you navigate through troubleshooting this situation. For the purposes of this discussion, we will be discussing changes in retention on one individual column, not differences from one column to another. Also we are assuming that the analyst is following an established method that worked for a significant period of time prior to seeing changes in retention.
It is always useful to define the specific problem to help narrow down the possible causes. An important factor is whether all of your peaks have shifted or whether it is only certain ones.
If all of the peaks are shifted by about the same time interval, the variations are likely caused by variation in flow rate in the instrument. This can be a shift in either direction, but is often toward longer retention times/slower flow rate. This can be verified by collecting eluent from the column into a graduated cylinder and using a stopwatch to measure the time taken to fill the container to the mark. Flow rate variations can be caused by any of the following:
- Leaks in the flow path- Check to make sure all fittings are tight and in good condition.
- Worn or faulty pump seals- Replacing the seals is often a good precaution to prevent this.
- Faulty check valves- Get help from an instrument service person if needed to verify performance of check valves and other parts in the flow path.
- Bubbles in the pump or tubing- Make sure your system has a degasser built into the system that degasses solvent prior to pulling it into the solvent pump. If a degasser is not available, mobile phase can be degassed manually before using for LC. Please note that any mobile phases prepared to contain buffer salts or any modifiers in solid form should be filtered prior to use for LC. We have a membrane microfiltration system available for this if needed. Filtration systems such as this also serve to degas mobile phases because the solvent is pulled through by vacuum.
- Changes in instrument settings- This may seem obvious, but one should always confirm that program parameters have not changed from time to time. When multiple users are performing the same analysis on shared equipment, this can often be an issue.
- Changes in column temperature- this can affect the column in a way similar to flow rate and can move the retention in either direction. Make sure you have the column in a thermostatic column compartment to avoid this.
If all peaks are dramatically shifted to an early retention time, coming out together at or near the void volume, the issue is likely phase dewetting, or what we used to call phase collapse. This is caused by an attempt to use a mobile phase that is more than 95% water/aqueous with a column that is not aqueous compatible. Please see the following post to find out which columns are compatible and which are not.
If early eluting peaks are shifted, but late eluting peaks are relatively unaffected, the issue is usually related to differences in the solvent ratio with sample and/or mobile phase. This could be caused by any of the following:
- Solvent composition of sample is not compatible with the mobile phase or gradient- This is usually accompanied by poor peak shape. Try making your sample in your mobile phase solvents at the same ratio as the starting conditions of your gradient (or same ratio as your mobile phases if using isocratic conditions).
- Sample volume too high, overloaded- Peaks usually are fronting under these conditions, but sometimes can tail. Try injecting a smaller volume.
- Error in preparation of mobile phases- It is always helpful to carefully remake the mobile phases to rule this out.
- Changes in instrument settings- see discussion above for this.
- Guard cartridge is saturated with matrix- This should be replaced any time a change in peak shape or retention is noticed. Preferably it should be replaced as a regular preventative maintenance procedure before adverse effects are noticed.
- Column may be damaged or contaminated with matrix at the inlet- If changing the guard doesn’t help (or if you are not using a guard), try flushing the column to clean. Instructions are located here:
If only some of the peaks are shifted, seemingly random, or if all peaks are shifted in varying degrees, several different things could be happening:
- Sample matrix is interfering with elution of some compounds- Try making injections of those compounds in mobile phase with no matrix to rule this out. Additional cleanup of the sample extract may be needed.
- Sample pH is different from the mobile phase- Sometimes this explains why some peaks are affected, but others are not. Try matching the pH with that of the mobile phase and check it regularly.
- Column has a buildup of sample matrix- Often this is accompanied by an increase in pressure, noisy baseline, and/or ghost peaks. Try flushing the column to clean (see link above).
- Column has reached the end of its lifetime- If flushing does not correct the problem, the column needs to be replaced.
Posted by Ayodeji Ogunlowo