How to Troubleshoot a Double-Peak HPLC

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High performance liquid chromatography, or HPLC, is a technique used to separate compounds from a mixed solution. A metal tube filled with silica beads, called a HPLC column, is used to separate the solution before it is detected by the HPLC detector. A double peak from the detector reading usually implies that something else is contaminating the system. Most of the time, the problem lies in the HPLC column.

  • Determine what type of solvent is being injected into the HPLC system. If the solvent is stronger than the liquid mobile phase that passes though, peak splitting may occur. An quick solution is to use the same solvent in the mobile phase as the solvent in the sample. If the problem persists, the HPLC column may be contaminated.

  • Wash out the column by pumping 100 percent methanol or acetonitrile though the HPLC system for at least 15 minutes. This will remove strongly attached materials bound to the column. Reverse the column and perform the same procedure with the same solvent. This process will remove any particulate matter that may be clogging up the frit—a filter located at the front of the column. If the chromatography continues to display double peaks, the column has been fouled with strong contaminants.

  • With the column in the reverse direction, pump 25 mL of water at 1 mL per minute. Then flush the column with 25 mL of isopropanol, methylene chloride and hexane successively. Reconnect the column in the proper direction and flush the column with 25 mL of mobile phase solvent. If the double peak continues to persist, replace the column.

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