Chromatography Forum

Hello everyone!

I am currently validating a method for the determination of metformin in human plasma by HPLC-UV. I'm having problem with inconsistent retention time the moment I make a new mobile phase. But all throughout the run, the retention time is constant. A change of about 5-7% in the retention time for both active and IS is evident everytime a new MP is prepared.

Info about the method:

Mobile Phase: ACN/ 0.02 M KH2PO4 (pH 6.2) , 65:35 (Isocratic)
Column: Phenomenex Cyano Column, 150 x 4.6
Flow: 1.5 mL/ min
Equilibration time: 30 minutes
Extraction Method: Direct precipitation with ACN and back-extraction with DCM, aqueous layer is injected into HPLC system

What could be the problem?

Thanks

Ghie

Hi,

some questions:

do you prepare your mobile phase exactly in the same way ?
how do you mix the buffer and the ACN:
65 volume ACN, of 35 volume buffer an than mixng in one bottle ?
or adding the buffer to 65 volumes of ACN till 100 ?

phosphate buffer ( different lots?)

pH adjustment ?

regards

philippe

Hello!

I prepare stock solution of 1M Phosphate buffer. From there, I make 0.02M then adjust the pH to 6.2. I measure 35 of the buffer and add it to the pre-measured 65 of ACN and then mix. The final pH is then checked and found to be 7.28

Thanks
Ghie

Hello,
Are you using the same cylinder/flask for measuring? Perhaps you may have to switch to Class A glassware if you method is that sensitive to slight variations of mobile phase. If you are using 2L cylinders they can have a +/- 10mL bias. I've got a method that is very sensitive to mix which causes a 2-3 min retention shift by adding 10mL extra reagent to a 2L prep.

Another work around to try is to let your pump mix for you. Assuming you have at least a dual channel pump. Prepare each solution in separate containers. Set Channel A 65% (ACN) and Channel B 35% (buffer). If you need to perform a check on the pH after the solutions are mixed collect some of the mixed solution from your pumps waste line and measure it.

I also thought that mixing is our problem. I tried using volumetric glassware Class A to measure everything accurately. But still, I cannot obtain the original RT. Could it be possible that the column is the problem? I checked the column again based on CA, but the RT of the analyte used in the CA did not change. So i assume that the column is ok.

1, column stabilization;
2, mp temperature stabiization;
3, buffer pH stabilization
4, column temperature stabilization

I'm with ym3142. Can you regulate regulate temperature?

Perhaps it is in the sample prep technique (non-sufficient aqueous layer separation prep to prep).

I would bet the price of a pizza that the problem is pH.

Thow away your pH meter and prepare your buffers by weight (good gravy!, I'm starting to sound like Bill Tindall ).

ghie,
please let us know how you solve the problem so one of us can win pizza from Tom.

I'm starting to doubt that it is because of the pH meter. The final pH measurement is changing after mixing the contents of the MP. However, I am also looking at the possibility that the CN in the column is changing in form at high pH. could that be the reason?

Help!

Ghie

I am not a fan of CN columns. They are much, much, much... less stable than C18 columns.

I assume that you are not vacuum filtering your mobile phase?. If you are, make sure that you don't leave it under vacuum for too long.
It would have to be a looong time to lose significant amounts of CH3CN and affect retention, and the mobiel phase would probably be colder than ambient, but....

One test would be to make a couple of batches of mobile phase, run some samples, swap mobile phase batch, run the samples again, and revert to the orginal mobile phase and retest, to see if the initial RT returns. If it does, the solution is in your mobile phase preparation.

Please keep having fun,

Bruce Hamilton