Chromatography Forum

i am doing prednisolone assay for tabs. isocratic 68%H2O, 27%ACN, 5%THF +0.5gr TBAHS/lt. The method says that i have to mix them in this particular order. Why is that? Second question: TBAHS suggests that i am doing ion pairing? Thus the mobile phase looks very "sensitive". Every time i do it(2-3 per month) i get different retention times(one day 25min the other 45min). It is not the column nor the LC system or the temperature of the lab(and column. I accuse the mobile phase rather. Any ideas?

Differences may come from different equilibration times and different pH. Did you check pH?

The concentration of TBAHS might not have been optimized under the method develpment. Try adding more of it (f. eks some 25%, 50% and 75% more) and see if it isn't eliminate the variation to some significant extend.

Best Regards

PH is about 2.2-2.3. Is there a possibility that due to the poor accuracy of our cylinders(plastic) the amount of THF is a little different evey time.? And why the method says to mix them in that order?(H20,ACN,THF,TBAHS)

Poor volume accuracy could be a valid thesis. It deserves an investigation by weighing the volumes before mixing them and compering the masses, next time you prepare the mobile phase again. If urgent, then just weigh out the volumes you're dispensing several times and check for significant deviations.
OK to one of you're original questions: The required order of mixing could be due to the procedure for measuring all the volumes. If you measure all the volumes separately and then mix them, I don't see any significant reason for comply with the described order. If you – on the other hand - measure the first couple of components and then fill up to volume with the last one then the order of mixing will be important – naturally.

Best Regards

what i mean is, naturally i would solv first the TBAHS in water and then proceed with ACN and THF. Why they say the TBAHS last?

It beats me

Anyway, I have this hunch that you need to investigate whether or not there's enough of it.

Best Regards