In my reverse-phase LC method, why do only one or two peaks shift in retention time, while other peaks in the same method do not shift? - WKB229859

ENVIRONMENT

• Reverse-phase LC
• Troubleshooting
• Peak shift
• Retention time

ANSWER

Potential causes:

• pH of mobile phase is drifting, which can alter retention time of ionizable compounds.
• Too high of a percentage of strong organic solvent in sample diluent, which can cause variability in the earliest eluting peaks.
• Selectivity differences for the specific analyte under the specific method conditions, from batch to batch.

ADDITIONAL INFORMATION

If the mobile phase pH is less than 2 pH units above or below the pKa of the analytes that are shifting, try adjusting the pH to at least 2 pH units above or below the pKa for all analytes to have a more robust method.

Waters highly recommends screening three columns from three different batches (gel lots) of columns when validating a method. This is a critical point.  Sometimes only one batch is used, although there were many "lots" screened.  For most Waters columns, the first four digits of a lot number (or serial number) indicates which batch (gel lot) was used to pack the column. Waters sells many of its columns in Method Validation Kits (MVKs) that include three columns, each from a different batch of packing. Any variability among the three batches used for validation indicates that the method is not robust and is subject to batch variation during the lifetime of the method.

See:

How do I translate the 14-digit serial number for Waters columns?

Method Validation Wall Chart