I'm performing a RP-HPLC method for a bio-molecule and am having a new problem with retention time variation. The method is as follows:
A = 90% Water / 10% ACN 0.2% TFA
B = 90% ACN / 10% Water 0.2 % TFA
0 min 37.5% B
1 min 37.5% B
11 min 57.5% B
12 min 100% B
14.5 min 100% B
15.5 min 37.5% B
18 min 37.5% B
Reading at 280 nm
Column : 4.6 X 50 mm Vydac C4 (241TP5405)

Historically, the RT of the main peak within a run has been tight (within 0.02 min). Within a receint run the RT variaiton was 0.02 min with each injection of material (test and reference)for a total variaiton of over 0.2 min (well outside of our identity specificaiton).

What factors should be investigated in this case?

Your column could be getting dirty, especially if you are running a biological matrix. A good back flushing of at least 10 column volumes each with strong solvents such as TFA and ACN could help. I would also flush with methanol. If you do this, be sure not to send the flow to the detector. Your column could just be getting old, too.

What type of pumping system do you have? RT shifts can be a change in %s being pumped. In my case a bad proportioning channel has been known to create rt shifts.

I think you have inadequate re-equilibration time. Best practice is to use 10-15 column volumes at original MP composition for re-equilibration. Your column has a volume of around 0.5 ml. At flows of 1ml/min, you would need 7.5 min to generate 15 column volumes.

Thanks everyone for the quick responses. To Michele's response, the product being tested is actually a highly purified biological molecule intended for injection so there are no blood/serum products present. We're not seeing a change in backpressure.

John B, we use HP1100 systems with binary pumps. We ran an additional experiment on the same instrument the next day and saw no difference in RT (same mobile phase)

jclark, that is a good point and will be looked at. One odd thing is that we've run this method for many years (using different column lots) and this is the first time we've seen this occurance.

Keep them coming

why don't you run the same reference with previous column if you still keep them? then compare with current column? i beleieve more due to column variations if other accidental factors like MP freshness is excluded. usually 5 -8 times flushing volume is enough to re-equilbrate the column.

On the 1100 binary pump, there is a small "screen" frit on the outlet check valve(the one on top) Dollars to donuts the one running the water is somewhat plugged. One thorough test is the Agilent leak test,built into the software on the Diagnosis screen. All you need is IPA and a plugged fitting. Give it a try...