Chromatography Forum

I have been having problems with getting consistent chromotograms for analyzing Azithromycin. The retention time changes drastically with the ph of my sample solution and so do the peak shapes. Does anyone have a robust method for this compund?
Thanks
- Kiran

What buffer are your running in your mobile phase?

A 50 milli Molar Pot Phosphate Buffer with Acetonitrile.

I believe Tom was also looking for a pH for your mobile phase buffer, and this is even more important considering that you are using a phosphate buffer. It seems (at least, I've read many posts here recently) many folks inadvertently use phosphate buffers at pHs that are outside of the actual buffering capacity of the phosphate. Inadequate buffering can lead to reproducibility issues in chromatography.

You are using 50 mM phosphate buffer and may have a solubility problem in acetonitrile, although the water ratio probably is high.

I don't have a method for this compound, but I am ready to bet (a substancial amount of Swedish Krona) that it will have a quite nice retention on the ZIC®-HILIC column.

[quote="url_mutual"]I have been having problems with getting consistent chromotograms for analyzing Azithromycin. The retention time changes drastically with the ph of my sample solution and so do the peak shapes. Does anyone have a robust method for this compund?
Thanks
- Kiran[/quote]
We use for assay and dissolution test (drug analysis): TSK OD-2PW column, TosoHaas, 0.05 M K2HPO4 (pH 10) :AcN - 45-55, detection at 206 nm. Solvent for samples - mob. phase.

We use for assay and dissolution test (drug analysis): TSK OD-2PW column, TosoHaas, 0.05 M K2HPO4 (pH 10) :AcN - 45-55, detection at 206 nm. Solvent for samples - mob. phase.

Is it safe for column lifetime using phosphate buffer at pH 10? Have you try other type of buffer, i.e. TEA (pKa= 10.7), 1-methyl-piperidine (pKa = 10.3 ), glycine (pKa = 9., or ammonia (pKa 9.2)?

pKa's for phosphate are 7.8 and 12.5.

At pH 10.7, phosphate is not much of a buffer, which is probably why the pH of your sample is changing things. Suggestion would be to either change the pH or the buffer. Bill Tindall may have some suggestions here (Bill?).

With regard to column stability, my recollection is that "PW" columns from Tosoh are polymer-based, and so not subject to same pH limitations as silica-based columns.

There must be something missing here as I don´t see any mention of pH by Kiran (url_mutual.

Here is a procedure for the product in human serum. The same parameters will work for your analysis.

Found at https://www.macherey-nagel.ch/web/MN-WE ... =HPLC00361

Remember to change the language to English.

You're right (as usual! ), HW. The pH 10 value was suggested by OlaVes. I still don't know at what pH url_mutual is operating. However the symptoms (retention and peak shape changes depending on pH of sample) are certainly consistent with a 50mM buffer at an "unfavorable" pH.

Allright, this can be continued hypothetically as it is interesting (real life problem would still be better). The buffers in that range are not so appetizing, at least what I have seen. Ammonium carbonate (pH 8.25-10.25 and 9.25-11.25) is not clearly defined. Amines (see syx) oxidize easily. Glycyne (mentioned by syx, I have refs giving the pH range: 9.2-10.6) is not so hot at low UV wavelength. CAPS (3-[cyclohexylamino]-1-propanesulfonic acid has been used in chromatography (J Chrom, 514, 97 (1990), this is a Fluka ref who sell this stuff), purity and low UV compatibility? The last two are zwitterions, might complicate things.
Anybody have some better ones?
There is an interesting slide show type article I copied out of Waters´website on basic chrom on X-Terra, I don´t have the URL ready.

Here is a procedure for the product in human serum. The same parameters will work for your analysis.

Found at https://www.macherey-nagel.ch/web/MN-WE ... =HPLC00361

Remember to change the language to English.

Thanks, Steve. The problem is the detector: electrochemical, guard cell at +1V, dual electrode cell at +0.7 and +0.8V (as in USP). Could the method be run in most common detector (UV-Vis/PDA)?