The Multipurpose Technique for Testing Reversed-Phase HPLC Packings in the Method Validation Study

Konstantin Sychev, R&D, Elsico-Eesti OU, Tallinn, Estonia
Evgenia Okunskaya, R&D, Elsico Eesti OU, Tallinn, Estonia

To develop a new, or to reproduce an existing HPLC method successfully, one has to possess comprehensive and reliable information about HPLC stationary phases that he owns.

From a viewpoint of method developer, such information is crucial to study the robustness of the developed method during the method validation study. A chemist who reproduces that method needs the open information about different HPLC packings to find the column of choice for the given separation.

Theoretically, a perfect HPLC packing test should provide the most vital information about chemistry and quality of an examined stationary phase, such as:

• a stationary phase type: the prevailing HPLC mode and HPLC modes with minor contribution to retention and selectivity;
• the average ability to retain analytes in the prevailing mode (for example, the hydrophobicity for RP packings of any type);
• chemical inertness of a stationary phase (i.e. its ability to elute chelating compounds as symmetrical peaks).
  

Besides, an informative HPLC test should be also applicable to track batch-to-batch reproducibility and quality of purchased HPLC columns, both new and used. Therefore, such test should provide the information about:

• packing efficiency of a column;
• batch-to-batch reproducibility of a column packing.

In other words, by analogy with qualification of HPLC equipment, column testing should present the complete procedure of HPLC column qualification.

The goal of our presentation is to introduce the original method for testing the selectivity and quality of commercial reversed-phase HPLC packings, and to show how this information can used in the method validation study.

Our test is applicable to any hydrophobic packing with the prevailing RP separation mode. A packing may possess some minor contribution of HILIC and CT (charge-transfer) separation modes, which are responsible for altering the conventional RP selectivity.