Sample peak is moving 1-2 minutes in either direction

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

8 posts Page 1 of 1
Hello,

I have been using 2 mL sample loop on Waters 2695 separation unit to inject 200uL injection volume. I have noticed that the main sample peak keep moving in both direction. For example RT is 40 minutes, then you can also get RT 38 minutes or 42.5 minutes etc.,
I tried some of the things like cleaning/replace solvent bottle filters, changing primary check valves, cleaning head assembly etc., but still no not resolved. When I ran the same method on other HPLC which has 200uL sample loop, it rans perfectly fine. RT is always the same.
Will anyone help me to understand and resolve the peak shifting issue ? Is it because of large 2 mL sample loop?

Thanks in advance.
Hi Amitlad,

I have a few ideas for what may be the culprit, but I would like to know more about your chromatography before ushering a guess. Could you give us some more details of your method, column, oven, etc? How long has this problem persisted as well? The entire time that you have run this method, or some issue that just recently popped up.

Thank you,
Tyler Smith
Hello Tyler,

Thank you for your reply. The method column is C18, 2.7um particle size, 4.6x150mm, column temperature is 50'c. This problem is there from the beginning. Most of the time peak shift is small but this type it is larger and also the peak area/counts are very different. I hope this will help.
Hi Amitlad,

Interesting, when you said you were injecting volumes up to 200ul, I immediately assumed that this was preparative chromatography due to those volumes but it seems like this is an analytical type column (please correct me if I'm wrong). If that is the case, then something that you definitely want to change is that sample loop and injection volume size. I'm not too familiar with Waters columns, however, Restek (after a quick google search to here: https://www.restek.com/en/frequently-as ... and-UHPLC/) recommends injections (on that sized column) to be between 8 and 40 ul. Then again, I'm not exactly sure what you mean by "separation unit" and this could be a normal part of your HPLC. However, I would attempt to change your sample loop size first. Another thought I had (because I assumed it was prep LC, which is what I mostly focus on) is that the temperature of your column is not as exact as you would believe. This could be due to things around the atmosphere of the environment as well, like other instruments or AC. The temperature should not be too much of an issue, but I know some columns (with particular acids) actually degrade much faster below their temperature cut-off limits due to those mobile phase additives. It seems like, from your trouble shooting, that the sample loop should be the first thing to try. Now on the other HPLC, was the exact same column used or was it the same type but different physically (aka just another copy)?

I hope you get everything working quickly! I'll be here to help,
Tyler Smith
If this is a manual injection into such a large loop, could it be possible you have some air getting into the loop?

Normally when you do a manual injection into the loop the sample pushes out the exit end the same amount that you push in with the syringe, in your case 200ul. If the waste line drops to a container below the bench, it is possible that gravity is causing it to pull on the sample loop and pull in air as you remove the syringe if the septa is not sealing well, before you can rotate the valve to inject position. If this is the setup you have start by putting the waste container at the level of the injection valve to prevent a siphon from forming.

This can also happen with automatic samplers but is not as common.
The past is there to guide us into the future, not to dwell in.
That's a truly enormous loop. For a column like that, you're probably running at about 1mL/min, which means it's a 2 minute loop. It'd make 2 minutes' difference depending on which end of the loop the sample started at (but an autosampler ought to load loops in a repeatable fashion, albeit with the valid concerns in the last reply).

Are all your samples in the same matrix? The reason I ask is that 200uL is a big injection on a column that size. You'll probably get away with it if the sample is in a solvent that is at least as weak as the running solvent at the point you make the injection, but if some samples for some reason are in a stronger solvent, they will probably elute early and with bad peak-shape. The other thing to bear in mind is what's in the rest of the 2mL of loop. If you want consistent retention times, you're going to need a loop that is in a consistent state at the start of each injection: sample plug in the same place, remainder of the loop containing the same solvent, and it should be a solvent that isn't strong enough to endanger your chromatography (i.e. if you're using a system where the loop is filled with needle-wash, be careful what your needle wash actually is).
Good luck!
Hello All,

Thank you for your suggestions. I tried the column and 2mL sample loop on the other HPLC unit. The retention time of the main peak do not shift and remains consistent. I tried both 200uL and 2 mL loop and it RT is consistent on HPLC unit. So, I think there is something wrong with second HPLC unit. I tried to clean and replace solvent line filters, primary check valve, flow rate etc., What else I should try ?

Amit.
amitlad wrote:
Hello All,

Thank you for your suggestions. I tried the column and 2mL sample loop on the other HPLC unit. The retention time of the main peak do not shift and remains consistent. I tried both 200uL and 2 mL loop and it RT is consistent on HPLC unit. So, I think there is something wrong with second HPLC unit. I tried to clean and replace solvent line filters, primary check valve, flow rate etc., What else I should try ?

Amit.


On an autosampler it can be affected by the rotation of the valve. If it is sticking and not rotating fully each time then the restriction when it does not completely move will cause lower flow, then when it does completely move you have higher flow. That is one possibility that I have seen happen before.
The past is there to guide us into the future, not to dwell in.
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