If one lab has a holdup of 1000ul and my lab has a holdup of only 650ul from the pump to the column, how would the Rt change if the all else remains the same?
Presently the other lab gets a 5 min Rt and I get 3 min Rt using a RP column.

Gradient program:
ml/min Water Acn
Start 2 65% 35%
1 min 2 65% 35%
11 min 2 35% 65%

The difference should be at most .35/2 or .175 minutes. This doesn't explain the 2 minute discrepancy. How did you determine the holdup volume?

Are the columns the same packing from the same supplier? Is one of the columns older or used more than the other? As the column ages, I would expect retention to decrease, though probably not to the extent you are seeing. Have you checked the flow rates through the systems? If one system is delivering a greater flow rate than the other, the retention time would be lower. Have you checked the gradient performance of the systems? If the gradient is slower on one of the systems, the retention would be greater. What are you using as water? Are you both using HPLC grade water? Is there a buffer or other additive used by one lab and not the other? Just a few thoughts.

Reply to Chris and Russ
Chris: that is what I figured, about 0.2 min which does not explain the 2 min diff. I got the holdup vol from waters, I'm using a 2650 module which has 650ul holdup and they are using a separate 600E pump which is 1000ul as least.
Russ: Yes, their column is older and more used, mine is brand new. Both are identical manufacturers and the identical gel lot packing. The other chemistry stuff is the same.
Thanks for your replies.
I was thinking if drylab would have some isocratic/gradient conditions to bring the elutions closer.

Hi Rick,

Changes in dwell volume can affect the selectivity of gradient methods, especially at the begining and the end of the gradients.

Sub 1ml dwell volumes are small, I would verify the actual system dwell volumes using an acetone tracer at 270 nm.

You can check if the different dwell voulmes are affecting your selectivity by making a compensating change in the flow rate. The 0.175 min calculation would be right if it was an isocratic method.

Depending on the autosampler used on that 600 system, the volume could be approaching 3 mL. You should measure it. The 2690 is usually around 650. Drylab is a good idea, but you need to measure both system volumes experimentally (its not hard to do).

Gradient accuracy could also be playing a role. There will be system to system differences and larger difference if one system uses high pressure mixing and another low pressure mixing. Also there could be lab to lab differences. One lab could prepare a 65% ACN solution volumetrically and another v/v and another with online mixing. Good luck.

You need to measure the dwell volumes yourself. The plumbing of your system may be different than what the manufacturer thinks it is. For example, the loop in the injector could make all the difference. I guarantee that after you have measured it, everything will be as clear as chicken broth.