Peak areas decrease with each injection using an isocratic method - WKB220111

SYMPTOMS

• Peak areas decrease with every injection when working with an isocratic, reverse-phase method
• After flushing the column with organic solvent, peak areas increase to initial values but then again gradually decrease with every injection

ENVIRONMENT

• ACQUITY UPLC columns
• BEH
• XBridge

CAUSE

Sample impurities gradually accumulate on the reverse-phase column, leaving less and less free binding sites for the analyte. This impurity buildup on the column leads to decreasing peak areas because the analyte cannot bind to the column.

FIX or WORKAROUND

The isocratic method requires an organic solvent wash step at the end of each chromatographic run. The wash step removes the impurity buildup on the reverse-phase column. This is especially important if the chromatographic method is run with a highly aqueous elution.

ADDITIONAL INFORMATION