Chromatography Forum

Hi

Has somebody experienced problems with the L60 (USP) phase columns. We are using the columns for analysis of Nevirapine and by the time 3-4 sets of analysis is complete, the column performance goes down and we have to replace the column. We have tried two brands, both from Sigma-Aldrich. Supelco ABZ+ and RP Amide columns. The mobile phase is Ammonium acetate buffer: Acetonitrile = 80:20. We are only injecting clean samples of Nevirapine dissolved in mobile phase. Mobile phase, injection solutions are all filtered through 0.45u membrane filters.

1) What's the pH? If it's above 8.5, it would compromise column life

2) Are you using a guard column? If not, I'd strongly recommend one. Amido columns are quite susceptible to extreme pH, so a guard column would be a good idea, if a high pH is unavoidable.

Hi Srinivas,

Thanks for your reply.

The pH of mobile phase will be around 5.0. Details of mobile phase preparation is as below.

0.025M Ammonium phosphate buffer : 2.88g Ammonium phosphate monobasic (NH4H2PO4- Ammonium dihydrogen phosphate) in 1000ml flask, dissolve in 800ml water, adjust pH to 5.0 with 1N NaOH, make up with water. Mobile Phase : Amm Phos. Buffer+Acetonitrile:4:1.

(in my first posting I had wrongly mentioned the buffer as
ammonium acetate)

Regs

Maybe there is some problem with your method rather than with the column. Ammonium phosphate (if that is what you are using) is not a buffer at pH 5. I imagine it is quite hard to adjust the pH to 5! If you want to use pH 5.0 then acetate would be a better choice. This really is a buffer at pH 5.0

Hi Victor

Thanks for your reply. But my mobile phase is as per the USP 28 monograph for Nevirapine. I dont have much choice here!!!

Regs

Venkitesh

What is the mode of failure? High pressure, retention shift, efficiency loss, selectivity change, symmetry loss, etc?

Hi

With one column the pressure went up drastically and now we are not able to use. The moment we put the mobile phase into the column, pressure goes up and system stops.

With a second colunm, pressure is still normal, but the peak shape has deteriorated and resolution lost with normal calibration mixtures. Looks like the column surface is no more active as it was for the peaks to separate !!!

Regs

Venkitesh

Actually, both cases sound like severe contamination. High pressure is most often caused by particulate contamination. This could come from unfiltered buffers, bacterial growth in the mobile phase, unfiltered samples. Your mobile phase is not harsh enough to strip the bonded phase off the silica in such a short time. Contamination by hydrophobic substances, particles or surfactants could cause loss of peak shape and resolution.