Chromatography Forum

Hello all,

I'm trying to use USP method for API verification. It's a gradient one and takes 72.1 minutes. Analytical column is 4.6-mm 25-cm; 3-mm packing L1, MP-A is 10 mM ammonium acetate and 5 mM sodium 1-heptane sulfonate, and MP-B is Acetonitrile and methanol mixture. The column and the system were equilibrated for 3 hours. At the beginning, we've got perfect peaks -Tailing factor: NMT 1.5 for both peaks-. However, after 10-15 injections, we lost system suitability; unacceptable peak shape. The column is new and used only for this analyze. Since it's USP method, we have to use it. Is there any suggestion? Regards.

More information would help:
- did the peak shape deteriorate gradually or abruptly?
- "peak shape" is a bit vague; is it simply tailing or something else (e.g., a shoulder0
- are all the peaks in the separation showing the same problem (or are some peaks still well-formed)?
- was the problem accompanied by changes in retention time?

The reason for asking all of this: if it's a "physical" problem (column headspace or partially-blocked frit) is should affect all the peaks in the separation in a similar manner, and the retention times should be unchanged. If it's a headspace, it will usually come on gradually; a partially-plugged frit will usually come on abruptly.

If it's a "chemical" problem (e.g., deterioration of the bonded phase), it is likely to affect some peaks more than others, and the deterioration is usually gradual.

Dear Tom,

Thank you for reply.

- The peak shape deteriorated gradually
- mostly shoulder
- There're five peaks in the chromatogram ( main peak -R+S-, İmp A,B,C,D and E. We observed all the same problem for all peaks
- There were no change in retention times

As you explained bellow, it should be physical problem. Using pre-column filter could resolve problem. We'll try and let you know result. Thanks

Also, we observed that there was precipitate in MP-A (ammonium acetate probably). Is there any tips to prepare/store MP contain ammonium acetate? We prepare MP daily and filter from 0.45 micron filter.

More information would help:
- did the peak shape deteriorate gradually or abruptly?
- "peak shape" is a bit vague; is it simply tailing or something else (e.g., a shoulder0
- are all the peaks in the separation showing the same problem (or are some peaks still well-formed)?
- was the problem accompanied by changes in retention time?

The reason for asking all of this: if it's a "physical" problem (column headspace or partially-blocked frit) is should affect all the peaks in the separation in a similar manner, and the retention times should be unchanged. If it's a headspace, it will usually come on gradually; a partially-plugged frit will usually come on abruptly.

If it's a "chemical" problem (e.g., deterioration of the bonded phase), it is likely to affect some peaks more than others, and the deterioration is usually gradual.