Chromatography Forum

I have problems with colistin sulphate determination. I have method from European Pharmacopoeia but my chromatogram is far from good separation. Do you have some experience in this topic? I will be grateful for some "working" procedures. I found some publications but I would like to know something from practitioner.

If it's in the EP, it probably works. What you need to do is tell us what you are doing different to what's in the method, and what you get for all the resolution requirements etc. Including a chromatogram would be nice.

Column = 150 x 4.6 x 3.5 um end-capped C18
Column temerature = 30C
Mobile phase buffer = 4.46g sodium sulphate dissolved in 900ml water., then ~2.5 ml phosphoric acid to give pH=2.3-2.5. Make up to 1 litre.
Mobile Phase = Take 780ml of the above buffer, add 220 ml acetonitrile. Mix well, filter.
Flow rate = 1.0 ml/min
Injection Volume = 20ul
Detector = 215 nm.
Sample = 25mg in 4:1 water:acetonitrile

Most likely issues are:-
Grades of acetonitrile and sodium sulphate. Need to ensure they don't absorb strongly at 215 nm
Buffer not prepared properly.
Column is not equivalent, or dead.

Bruce Hamilton.

Thank you Bruce for your voice. Yes, it should work. I applied all conditions from EP but column is the problem, I think. As you know there are few on the market "Column = 150 x 4.6 x 3.5 um end-capped C18". I used Zorbax SB-C18 4.6x150 3.5 um and... bed results. I tested column according manufacturer test and some other test and it works well.
Does somebody some suggestions?


Thank you

I applied all conditions from EP but column is the problem, I think. As you know there are few on the market "Column = 150 x 4.6 x 3.5 um end-capped C18". I used Zorbax SB-C18 4.6x150 3.5 um and... bed results. I tested column according manufacturer test and some other test and it works well.

As far as I'm aware the Zorbax Stablebond C18 columns are not end-capped as specified in the EP, and the -OH is still on the silica. I just checked the Phenomenex catalog, which also said they were not end-capped, but you need to check Agilent's data. I think the appropriate column would be the Zorbax Eclipse XDB-C18.

You haven't mentioned whether your solvent grades are suitable, but I'd fix the column issue first.

Bruce Hamilton

Went to EP site, searched, and found they list:-

Waters Symmetry 150 x 4.6 mm 3.5 µm;
Nomura Develosil ODS-UG-3 150 x 4.6 mm 3µm

for the assay and related substances tests on colistin. Maybe you should purchase one of those for your work.

Bruce Hamilton