I have a following problem:
I have to analyze impurities in our final product (ointment). The product has two active ingredients and total of 6 impurities (3 from each API). The problem is the huge difference in amount of API’s (3,0% and 0,05%). The concentration of a sample for determining imp’s from second API causes overloading of the first one and therefore huge tailing. The impurities show up on tail. What are my options? Can I quantify those impurities?

I am looking forward to your suggestions.

Best regards

Depending on your method, there should be more or less changes to enhance the seperation.
- Changing the dimension and the particle size is one possibility (e.g. from 250 x 4.6 mm and 5 µm to 150 x 6 mm and 3 µm). Even the volume of the two dimensions are nearly the same, 3 µm particles could enhance seperation and peak shapes.
- If you're running right now an isocratic method, switch to gradient.
- Use temperature as an tool to enhance seperation and/or reduce peak tailing.
- Use another column for better selectivity and/or peak shapes.
- If possible change wavelenght to archive more signal intensivity and reduce injection volume. This should reduce peak tailing.

To discuss usefull alternations to your method you should give more information to the method in use.

Dear Anonymous;

Probably you need two methods.

Benjamin

Two methods will work.
I assume that the imputities showing up on tail belong to the first API. Some things would be intersting to know: what are retention times, separation factors and, most interesting, spectroscopic properties of your APIs and impurities.
If you can analyse the second (0.05%) analyte more specifically that would help. Is it active in fluorescence? Does it absorp at higher wavelengths?
Andreas: I see the point in using 3 µm material, but what is the use of a 6 mm diameter column? I would expect the analytes coming out of the column more diluted: that will decrease tailing of API1 but also decrease sensitivity on impurities.
From my experience switch from 250x4, 5µm column to 150x4.6 (or better 125x4mm), 3µm column will result in similar resolutions. retention times shorten, sometimes gradient hve to be adjusted.