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Matching HPLC Methods to Different Columns
Thomas Jupille and Lloyd Snyder
Adjusting mobile phase composition in response to differences in stationary phase chemistry is a common problem in HPLC:
• to compensate for column aging
• to compensate for lot-to-lot variation in column packings
• to adapt a method for a new column when the original column type is not available
• to implement a compendial method for which the stationary phase is defined "generically".
Assuming that the columns under discussion show similar efficiency, the problem reduces to one of adjusting retention and selectivity as necessary to meet system suitability requirements for the method being used. If we limit the discussion to reversed-phase separations, there are only a limited number of ways to adjust retention and selectivity by changing the mobile phase:
• changing mobile phase strength (isocratic percent organic solvent or gradient steepness)
• changing mobile phase type (the familiar "solvent triangle" using acetonitrile, methanol, and tetrahydrofuran)
• changing temperature
• changing pH (for ionizable analytes)
• changing additive type or concentration.
There are two questions to be answered:
• is there any combination of mobile phase changes which can compensate for a given stationary phase change?
• what specific combination of changes will provide the closest match in resolution and retention to the "reference" ("defined", "system suitability") values?
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Rheodyne LLC
IDEX Health & Science Group
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