Troubleshooting the Source of Contamination on Columns

Answer

This Information Applies To: Agilent HPLC systems

Issue

The presence of ghost peaks can be a symptom of a system contamination issue, caused by accumulation of sample and/or foreign substances in the flow path. One common source of contamination and carry-over of peaks from previous sample runs is the LC column. 

In addition to the presence of ghost peaks, some increase in pressure may have been noticed. This article focuses on troubleshooting a contamination issue that is suspected to originate from the column.

Items required

The following parts may be required during the troubleshooting process: 

• A column 
• A guard/precolumn and in-line filter, if applicable 
• A rotor seal if a column selection valve is installed

Steps to Follow

1. Bypass the column and column compartment using a restriction capillary or a zero dead volume union, then perform a series of blank runs on your LC system to check if ghost peaks or contamination is still present.
   
   

   Confirmation: A blank run is a run with only injecting the solvent that samples are dissolved in.

   A comparison of column vs. no-column blank runs should identify whether the noted contamination is due to carry over from the column or column selection valves (if present) in the flow path. If the ghost peaks still appear on the blank run with the column bypassed, then the contamination is located else where in the flow path and not the column.
2. If this test indicates that the contamination is localized to the column, column switching valve or capillaries that connect these items, then proceed to step 3. 
3. If a column selection valve is installed, bypass the valve but still keep the column connected to the flow path, and run a blank. If the contamination is not longer present with the valve bypassed, then contamination of the rotor seal is suspected. Change the rotor seal of the selection valve and then retest to confirm that the contamination issue has been solved.
4. If contamination is still present, then the column is suspected as the source of contamination, go to step 5.
5. If the method is using a gradient method, clean the column using a stronger mobile phase. Chose a solvent that should elute all the compounds from your samples, and is compatible with your column. See your column manufacturer's information for further advice on solvent selection for this task. If contamination remains go to step 6.
6. Clean the column by black-flushing. If contamination persists, go to step 7.
   
   

   Caution: Always check your column suppliers recommendations for a safe back-flushing procedure, and check what are the safe solvents to use for your specific column and application. Use of incompatible solvents or excess pressure can cause column damage

7. Change to a new in-line filter or guard/pre column (or both), if applicable. This will ensure that the LC system is free from any potential contaminants deposited on the in-line filter or HPLC guard column.
8. Check the tubing and fittings, especially the injector outlet to column inlet. If there is dead volume, contamination can accumulate in those spaces. Replace the tubing and fittings. Make another injection and check for contamination. 

If you cannot locate the source of ghost peaks after working through these steps, contact your local Agilent Support representative for further technical support.

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