Pay now or Pay Later!6 Jul 2022
All too often in tech service, we field complaints about column-to-column HPLC reproducibility. Even though at Restek all lots of bonded silica are tested to consistent specification requirements and all HPLC columns are individually tested to assure consistent packing, we cannot possibly account for all the myriad uses and different methods, often presenting their own unique set of challenges and nuances.
Proper robustness testing involves using at least three lots of silica. Sometimes folks neglect this all together, and the method is built upon a single column, which can in a worst-case scenario become the "outlier", as future columns are tested. Also, if the single column has been used for other purposes it could be changed by the use, care/handling, and matrix components injected. The customer's expectation becomes centered around the resulting chromatography from this singular column, and all future columns (even if in agreement with one another) are deemed "bad" by the customer.
NOTE: Three different serial numbers do not necessarily signify three different lots of silica. Each column from Restek comes with a QA report which lists the lot of silica used. Other manufacturers may provide this information in a different manner.
Both the ICH (International Conference on Harmonization) and the USP guidelines define the robustness of an analytical procedure as a measure of its capacity to remain unaffected by small but deliberate variations in procedural parameters, providing an indication of the method's suitability and reliability during normal and expected use. (2,3)
In addition to evaluating fresh columns from different lots of packing, other variables should be investigated during the robustness study to ensure long term success of the method. These would include mobile phase pH, composition, buffers, temperature, flow rate, gradient slope, length, and hold time variations.
Evaluating robustness early in the method validation process can often save a lot of time, energy, and expense (i.e. FRUSTRATION!) later, perhaps preventing the necessity to revalidate.
For more detailed information on validation parameters-
Method Validation and Robustness (chromatographyonline.com)
(2) Int. Conf. Harmonization, Harmonized Tripartite Guideline, Validation of Analytical Procedures, Text and Methodology, Q2(R1), November 2005.
(3) Pharmacopeial Forum 31(2) Mar.-Apr., 549 (2005).