[4]Troubleshooting HPLC- Fronting Peaks

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

Any time you have difficulty with poor peak shape, it is also likely you’re having an issue with loss in response. For this reason, you may also find the previous two posts in this series helpful:

[1]Troubleshooting HPLC-Loss in Response for All Analytes

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

As in the previous posts for this series, these suggestions assume that you are working with an established method and had successful results in the past. As always, it is critical to note when the change occurred and how it might correlate to changes in the system.  Equally important is to change one thing at a time to identify the source(s) of difficulty. The following are examples of things that could contribute to fronting.  I suggest investigating these in this order:

 1. Column phase collapse- This is easy to rule out because it is always accompanied by an obvious shift to shorter retention times.  This is something that can occur in reverse phase LC when using mobile phases that are more than 95% aqueous.  Usually this can be resolved by pumping 100% acetonitrile for several column volumes.  It is best to avoid risking phase collapse by using an appropriate column phase, such as our Ultra Aqueous C18 or Pinnacle DB Aqueous C18 columns, when highly aqueous mobile phases are used.  For further reading on the topic, please see the following:

Which LC Phase Do I Need?

Tech Tip: Why do I Lose Retention on my C18 in Highly Aqueous Mobile Phases?

Can I use a 100% aqueous mobile phase with my LC Column?

 

2. Incompatible solvent composition of sample- As mentioned in the earlier trouble shooting post on Loss in Response for Some, but Not All Analytes, this can impact the shape and sensitivity. This is often more pronounced for the early- eluting peaks.

3. Volume overloading-Injecting too large of a volume can result in fronting, since it broadens the peak.  You can eliminate this possibility by injecting a smaller volume.  Some general guidelines as far as suggested volumes can be found in the FAQ section on our website.

4. Mass overloading- Injecting a solution that is too concentrated in terms of sample material (including matrix) can result in fronting. Often a slight shift to earlier retention times is also observed. Try diluting the solution by a factor of 10 or so and injecting it again. If this is the problem, remember that quantitation standards and samples will need to be injected at a lower concentration to avoid this on an ongoing basis.  In some cases, an improved procedure for extract cleanup, prior to HPLC may be needed.

5. Interfering coelution- As is the case with tailing, an interfering contaminant that coelutes can make a peak look like it is fronting.  If that is the case, you might see the peak shape change from time to time, or there may be a difference between your unknown samples and your quantitation standards. If you think this might be happening, try using a slower gradient or changing your organic/water ratio and see if the peak separates into two or develops a shoulder.

6. Increase in dead volume- In particular, dead volume at the point of sample introduction onto the column can create a fronting peak shape. This will be most obvious for columns with smaller ID and might be more evident with early eluting peaks, although you may see an effect for all analytes.  Make sure the tube fitting at the inlet of the column is fully seated and that you are using the proper fitting and ferrule.  If you are using a guard holder that accommodates a cap frit, such as catalog # 25084, make sure that a cap frit is installed; otherwise, dead volume would result.

7. Other physical damage to column- This phenomenon is described in the earlier post [3]Troubleshooting HPLC- Tailing Peaks. A physical change like this is more likely to cause fronting if the anomaly occurs at or near the column inlet.  This would most likely affect peak shape for all analytes and may or may not be accompanied by a change in column pressure. A column with this type of damage should be replaced.

Be sure to also check out the following videos we have available in our library:

LC Troubleshooting: Minimizing System Volume

LC Troubleshooting: Optimizing Your Injection to Improve Peak Shape

LC Troubleshooting: All of My Peaks Are Tailing! What Should I Do?

Thank you for reading.  This concludes this series of posts.