HPLC RT's

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11 posts Page 1 of 1
Hi to all,
I am observing variations in RT's of my HPLC analysis. I am using Isocratic method. My method did not say the RT of my analyte. If I use the same column, sample and moble phase in different system I am getting diffent RT. Could any one tell me the allowable % of variations in RT's. Is it same variations for both Isocratic and gradient. If different how much will be allowable % variation. Please guide me. Thanks in advance

with best regards
Babu
Hi Babu,

you will get variation from system to system for your retention times, each system will have a different "dead volume" in the tubing if nothing else. I certainly wouldnt expect >minute however so it depends what you're seeing whether you should worry or not.

lynz x
what is your buffer?
do you have a set pH?
are you pre-mixing the mobile phase? are you using the pump to mix?
what systems are you using?
do you use a column oven?
do you have a lab with climatic temp control?
there are many
Thanks unmgvar and lynzjm for your valuable replays. Yes I am using phosphate buffer pH is 7.1. I am pre mixing the mobile phases. One is having 10 % organic solven and another is 30 % organic solvent. I am using simadzu systems. I know that RT's will change from system to system. I agree with you. For changing the RT's there will be some limitations I hope. How much will be the limit of allowable change like RT's in min or % of RT's. Is any guide line suggests this or any regulation would suggests this limit. If you have any information please let me know. Thank you once again.

with best regards
Babu
I'm not aware of any guideline stating "allowed" discrepancies of retention times. And, especially with gradient elution, I would'nt find it any wise. Depending on the "Hardware" (Dwell volume!) and the method itself, rather large differences might be found between different HPLCs. Think of a dwell volume difference of 1 mL - there might be even bigger differences when comparing different HPLCs - and a gradient method with a rather low flow rate, say 0.5 mL. This translates in a dwell time difference of 2 minutes. Assuming that the retention time of your analytes is only dependent on %B - might be oversimplified, but it's justified for this example, i suppose - you will get a retention time difference of 2 minutes, even when using the exact same column and eluents!

I would look at this problem from a practical, pragmatic point of view: Does the retention time difference have any negative effects on the separation? Or to be even more provocing :D: Who cares about retention times as long as all peaks are nicely separated :lol: . You're using standards to identify your analytes of interest?
(OK, you might have hard times arguing with an auditor about this... :? )

If you are developing methods that are to be run on different HPLCs, you should look at the differences of these machines at an early stage - might prevent you from trouble later.
Transferring methods between different HPLCs CAN be quite troublesome - and sometimes impossible without changes to the methods themselves, which can be really nice :cry: if you're in a regulated environment...
I also dont know of any guidelines into RT variation but this should be assessed in your method validation robustness testing. Either validate and run your method on the one HPLC system or do robustness testing over a variety of systems to establish the RT variation.

As above poster says if you're running standards they will have comparable RTs to your samples, perhaps the criteria in your method could instead focus on a RT shift from standard to sample allowable limit rather than a sample peak RT criteria. We used to give RT windows of around 2 minutes to expect a peak in on most methods as we had up to 4 systems we ran on and there was variation between the Agilent 1200s and the TSPs.

lynz x
Thank you HPLCaddict and lynzjm for your valuable replays. Why I post my question in this forum is, recently I have performed one method validation by using gradient method. In that precision day I got RT of my analyt peak is 7.0 min and Inter precision day I got 8.7 min. My manager questioned me how much will be the allowable RT variation in analysis. I said might be 2.0 min. He asked me which guidelines will say this. I am not aware of this. I am working in regulatory marketing company. To conform this I have shared with you people. Thank you for your quick replays. If you get further valuable information regarding this topic, please share with us. Thanks once again.


with best regards
Babu
It looks like others have given some useful advice. I would like to add that the flow rates should be checked by measuring the flow from a waste line into a volumetric flask or cylinder and using a stopwatch. While systems may have differences in dead volume, differences in the actual flow rate from the pumps can affect your R.T.'s just as much. Just because the system says it is pumping at 0.5 mL/min does not mean that it really is exactly 0.50 mL/min. There are differences in how various pumps handle liquids of varying viscosities. The choice of pump seals, their age and condition can also affect the flow rate. For example, I would start with the system that produced the longer RT. Are the pump seals in that system in good condition or has it been a while since they were replaced?
Mr nschwartz I used very good systems for entire validation process. The company in which I am working is manufacturing regulatory marketing products . The system are well calibrated ones. Like HPLCaddict and lynzjm said might be the dwell volume places role in RT shifting. I agree with this. I looking for the allowable RT. How much could be change in RT's. If you have any information regarding this please share with us.

with best regards
Babu
have you checked the dwell on the systems?
which instruments do you have in the lab?
i can try to trouble shoot you.

1 what is the current Rt
2 what is RSD of 5-8 replicate injections of well equilibrated system from 5-8 separate vials
"If your experiment needs statistics, you ought to have done a better experiment." Rutherford
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