Troubleshooting HPLC- Loss in Response for Some, but Not All Analytes

Patterns in your HPLC chromatography can exhibit telltale signs that point toward probable sources of error.  In this brief series of posts, we will look at possible scenarios that you may encounter in the laboratory and how you might approach resolving those difficulties.

When some, but not all of your peaks are low, and you are analyzing multiple analytes, the number one rule is to look for trends. As mentioned in the previous post for this series, when you have only one analyte, or a handful of very similar analytes, the following may also apply. Again in this post, the assumption is that you are using an established working method and that you see a loss in response for every sample or standard that is injected. If you are only seeing the problem with samples and not your quantitation, please the earlier post on Method Troubleshooting.

If you have a lower response for some but not all of your peaks, here are examples of some things that could account for this:response_cgram (2)

1. Incompatible solvent composition- Double-check the solvent composition of your prepared samples and standards and make sure nothing has changed. Also, if solubility is marginal, some of the other causes that follow may be pushing it over the edge. If your sample solvent is stronger than your starting mobile phase, you may see a “smearing” effect, which means some of your analyte is traveling through the column prematurely while the rest is retained somewhat normally. This will appear more pronounced for early eluting peaks. Of course, this type of peak distortion also creates a decreased overall peak height and apparent response. Also, check out previous blog posts such as the one entitled “Solvent mismatch isn’t only a GC issue:…” and the FAQ section on our website for further discussion of this phenomenon.

2. Incorrect detector settings- Make sure that a setting such as wavelength has not changed on your detector program.  If you started out with a good method, it is usually just a matter of making sure you’re still following it properly. Depending on the functional groups of various analytes, some chromophores may not be active at the wavelength you are monitoring, which would explain why response is still adequate for some of your analytes. This would be similar to monitoring an incorrect Mass/Charge ratio for LCMS.

3. Activity (pH-related)- Are the compounds that lack response characteristically acidic or basic? (Hint: Look up their pKa’s to see.) If they are, please see the earlier posts regarding buffers (Links shown below.) You might also notice poor peaks shape if buffer is needed or if your buffer isn’t prepared correctly.

When should you use a buffer for HPLC….

How much buffer to weigh…..

4. Matrix effects (short term)- Certain substances in sample matrices can affect analyte response in surprising ways and biological samples can vary significantly. These can suppress detector response, and/or ionization in mass spec. They can affect peak shape and, on rare occasions, they may form complexes with analytes of interest. This type of effects can usually be seen immediately and can be identified easily by comparison to injections made without matrix. Don’t forget that your quantitation standard might contain sample matrix, so you may need to make a new solution in clean solvent for troubleshooting purposes. To read more about these effects and their impact on LCMSMS analyses, check out the following links:

Matrix Effects in Multi-Residue Pesticide Analysis When Using Liquid Chromatography-Tandem Mass Spectrometry

Mitigating Matrix Effects: Examination of Dilution, QuEChERS, and Calibration Strategies for LC-MS/MS Analysis of Pesticide Residues in Diverse Food Types

Performance and Matrix Effect Observed in QuEChERS (Journal of Chromatographic Science, Vol. 49, Oct 2011)

5. Matrix effects (long term)- More insidious effects can occur over time, where the sample matrix can actually change the nature of the stationary phase inside the column, sometimes making it more prone to adsorption of analytes. This is harder to identify because once this happens, it tends to affect all subsequent injections, whether they contain matrix or not. While particulates can be filtered out prior to injection, it may take more effort to remove active components from the sample matrix in order to protect the column. Examples of these include salts and proteins from blood, plasma or urine samples.  This can also be caused by remnants of derivatization reagent that might be present. A guard column cartridge is the first line of defense in protection against this. However, it is more ideal to remove the offending components prior to injection by using sample/extract cleanup such as SPE. The earlier post Live Long and Prosper gives more suggestions for avoiding these issues. Sample matrix components can also coat the inside of tubing and valves and create similar symptoms. (Yes, I have seen this happen.) Usually the easiest fix is to replace tubing and/or fittings associated with that portion of the flowpath.  If you find that your column has been affected by repeated injections of sample, you might benefit from reading our technical note on LC Cleaning Recommendations.

6. Incompatibility with column phase- This is not encountered frequently, but should be mentioned, nonetheless. Please double check your Column Care Instructionsto make sure your analysis is compatible with the column phase. An example of an inappropriate solvent or sample would be an aldehyde or ketone solvent used with amino phase.

7. Column age- At some point a column needs to be replaced, although its life span depends on the application and frequency of usage. Matrix effects are usually the number one contributor to shortening of column lifetime, although changes back and forth between solvents, pH ranges, pressures, etc. contribute also. When in doubt, it is always best to keep a backup column on hand.