I am working in an assay of flubendazole in a suspension form along with methyl and propyl paraben.every thing is going well except that the retention time of flubendazole peak is varied from run to run (e.g. 12 then 14 then 15 then 12 and so on..). The retention time of methyl and propyl parabens did not affected and the operating pump pressure remained constant all over the working time.
the conditions was C18 column 250x4.6mm 5um , flow rate 1.5 ml/min.
Mobile phase: methanol water 55:45
I have tried to control temperature at 30 C but retention time is still varied.
Can anyone explain to me what is happening??

These are interesting problems, one compound misbehaving. It could be that flubendazole is not fond of your mobile phase (labile instead of robust conditions). For instance, some people use ACN as the organic, example: http://www.metachem.com/Applications/ODS3_C20.htm.

A buffer was also not used there, but might be a possibility if you donīt get it under control.

How about telling us about your results?

It sounds like you have an air bubble forming that is affecting the retention time. Is this pre-mixed moblie phase or pump mixed? How did you degass your mobile phase?

Mel,
is bubble formation more prevalent in pre mixed or pump mixed? just curious about this. I've always used pump mixed and never really had a problem. why would one be more problematic over the other.
Jim

Theoretically, the pre-mixed would cause less problems because the outgassing has already occurred and you can degass it further by sonication or sparging. I always use the LC to mix because I feel it is more reproducible. I sonicate my solvents before use and have an in line degasser. As long as my system is primed, I have no problems.

In some systems, when the outgassing occurs with the Methanol and water mixing it can cause bubbles and the bubbles can cause the check valves to fail temporarily.

How could an air bubble effect only flubendazole?

Propylparaben is a neutral cpd but flubendazole is not. So it is quite obvious that it was a buffer issue.

Mel,
Concerning Mobile phase , I tried to use both methods: the premixed mobile phase and the pump mixing . I degas the mobile phase by sonication and I have also in-line degasser. For both methods the phenomena still existing.
when I began my work, I used a C18 250x4.6 column with particle diameter of 10 um and I used buffers for this column. I found that the most critical factor in controlling retention time of flubendazole is the temperature rather than the pH. every thing was O.K. but I had to change the column as it shows forking in all peaks(it was an old column).I switched to a new column(Hypersil c18 250x4.6mm with particle diameter of 5 um.) the column was not used before that .the problem of changing retention time appeared after using that column.

Try to add some acid to you samples (for example 0.1 N HCl in Methanol or something).

0.1 N HCl will affect most HPLC systems which is made of stainless steel. Try something more gentle, let's say 25 mM phosphate.

I guess there is an mistake, I dont mean pump 0.1N HCl trough your system, I mean something like add 20 ml 0.1N HCl in methanol to your sample and dilute to 200.

It's a version of pH control, which should be done to control retention.

1st Anon. Oct. 22 (...is neutral):
If my info is correct, the parabenes are phenol esters, the phenol should be able to dissociate (acidic). The flubendazole is probably basic, having basic and acidic substances in one run might cause difficulty in optimization.
HM, any success yet?

the only success I had was on the old column but when I used the new column every thing has gone bad.
even in the old column the same conditions( methanol : water 55:45 , different temperatures) gave me reroducible retention times.Could it be due to the change of column.?

Changing the column definitely sounds like it is the source of your problems. You said that you changed from a 10 micron to 5 micron column. The 5 micron column is where you see retention time shifts. Did the retention time shift from 12 minutes (on the 10 m) to 15 minutes on the 5 micron? A 5 micron column will typically have a higher carbon load and therfore lengthen the time it takes some compounds to elute.

Just my 2 cents.

Melissa

HM,
If you have a basic compound in your assay, you should use pH control, i.e. a buffered mobile phase. If you don't do that, you get results like the ones that you are reporting - shifting retention times. My recommendation is to use a pH 7 buffer, phosphate, and see if the retention does not become stable. Temperature control is not likely to be the primary issue, but it does help if you control the temperature.
to Melissa: there is no reason why a manufacturer should change the carbon load going from a 10 micron particle to a 5 micron particle. I have my doubts that the creators of Hypersil would have done something that irrational.