What are some tips for troubleshooting carryover or ghost peaks on my LC column? - WKB246120

ENVIRONMENT

• Troubleshooting
• Carryover
• Ghost peaks

ANSWER

If you see carryover when injecting a blank, there are a few things to try to isolate the source of the carryover or ghost peak:

1. If a gradient method, perform a blank injection using a double gradient.*
   • This can help isolate the issue to either the mobile phase (something in the mobile phase or leeching into the mobile phase) or the sample/injector.
2. If an isocratic method, collect data for twice as long as the end time of the method to see if peaks elute after the end time of the method.
   • If peaks elute after the run time, these peaks will appear during the next sample injection.
3. Perform an injection volume study to see if unknown peak areas change as you change injection volume.
   • If peak areas increase with more volume, the source may be in the vials.
   • If peak area does not increase with more volume, the source may be in the injector, the vial septa, or the mobile phase.
4. Perform a zero volume injection.
   • This test is performed to check if the vial septa might be the source of the unknown peak(s).
5. For samples that require derivatization:
   • Perform a blank injection where the sample diluent is underivatized (to see if anything seen in this blank is from underivatized contaminants).
   • Perform a blank injection where the sample diluent is derivatized (to see if anything seen in this blank is from derivatized analyte and/or contaminants).

When a potential source has been identified, take steps to correct the issue.

ADDITIONAL INFORMATION

*In the double gradient test:

1. Create a gradient table where you start with the initial method gradient up to the point you get to the re-equilibration point.
2. At that point, add in a 20-minute hold during re-equilibration. 
3. Then, add in a duplicate of the initial gradient. 
4. Make sure to run one blank injection using your regular gradient and then immediately run a blank with the double gradient.

If there is a peak seen in the first half of the double gradient but not in the second half, this means that the source of the peak is likely not in the mobile phase but in the sample, vial, septa, or injector.

If there is a peak in the first half of the double gradient and the second half of the double gradient, look at the peak area counts. If the peak area is higher for the peak in the second half of the double gradient, this means the source of the issue is likely the mobile phase, perhaps the water source.

If there is a peak in the first half of the double gradient and the second half of the double gradient, look at the peak area counts.  If the peak area is similar for both peaks, this means the source of the issue is likely the column (something has built up on the column and a similar amount elutes with every gradient).

See: Carryover_Ghost Peak Troubleshooting.pdf for a little more information on this type of troubleshooting.

Also see:  Blog - Carryover troubleshooting - includes double gradient - for Waters Knowledge Base.pdf