We were trying to develop an HPLC method as an alternative to the titration method for active quantification for one of our products that contains two actives. We managed to develop the method using ODS Hypersil column. We had another equivalent column;ODS-2 Hypersil and we performed the test using this column but unfortunately, the peaks of both the actives did not separate.
When I compared the specification of both the columns(refer attached), the only main difference is the pore size in which Hypersil ODS 1 has greater pore size(120 Angstrom) with smaller surface area(170 metre square/gram) compared to ODS-2 with smaller pore size(80 Angstrom) with greater surface area(220 metre square/gram).
I need expert's opinion on this to help with this. Which parameter shall I focus during method optimisation in order to equate the method?
(1) Unfortunately, you are focused on the wrong area in understanding the problem (Pore size is not the variable that matters here). The support "pore" size relates mainly to the exclusion size of the molecules that are suitable (and the surface area available for interaction of the unmodified support). Provided that your samples are less then ~10K daltons (approx, as it depends on hydrodynamic volume in solution where we use 300A supports for larger Mw), then this is one parameter that you do not need to focus on. For most samples which are lower in Mw, pores sizes of 120A to 80A will be fine. The surface areas are slightly different, but the two columns you compared have different chemistries and are not alike.
(2) There are thousands of "different" HPLC column types/supports available for use in HPLC analysis. The suppliers/manufacturers do provide very basic information about the types of supports, but DO NOT provide details regarding the chemistries used to create them. As such, comparisons which may appear to show a difference in one specification, may not have anything to do with your observation (e.g. Pore size). So called, "equivalent columns" often are NOT equivalent. ODS and ODS-2 use different surface chemistries so they may or may not provide similar results (you will always have to try them to find out. As the sample changes, so will the results).
(3) To find a suitable HPLC method of analysis for YOUR sample(s) will require identifying a suitable column AND suitable mobile phase for analysis (possibly evaluating many columns and many mobile phases). Do not simply substitute a column in the method. Often, the method itself needs to be re-developed and optimized to see if it will work. This is best done by someone with many years of professional, hands-on experience. *As you do not yet have the basic training in HPLC needed, please consult with an experienced, professional chromatographer for local assistance.
- "Modern HPLC Method Development Tips (PART II)"; https://hplctips.blogspot.com/2016/08/modern-hplc-method-development-tips.html
- "HPLC Column PORE SIZE (or Pore Diameter) and Retention Time "; https://hplctips.blogspot.com/2014/12/hplc-column-pore-volume-or-pore.html
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