Hello fellow researchers,
I recently have a problem with an HPLC coupled to an Orbitrap mass analyzer.
I usually use some protein mix to make sure the equipment is ready to separate and read real samples. The thing is, after checking this acquisition was right, I ran those samples with no valuable chromatogram achieved (like I injected water or something). Wanting to know if it was a sample problem or equipment problem I ran like 4 times in a row the protein mix I was referring to before.
The first two acquisitions were ok, but the ones after were like I was using a blank. No useful chromatogram at all. Seems like sometimes work and sometimes don't.
What could have been happening?
More data:
- I work on nanoLC flows (0.2 ul/min)
- Buffer A is Water 0.1% FA and Buffer B is Acetonitrile 0.1% FA.
- The pressure is ok.
- I checked the autosampler in case it doesn't take the samples correctly, but the volumes taken were accurate.
- I tried to work on microL mode (skipping the valve), connecting the autosampler with a C18 column and it worked very well, so an issue on the ms analyzer is discarded.
Have you checked for a leak? These can be difficult to spot at low flows.
John S Wishnok Thanks for your answer. I have checked every fitting and no leaks apparently. Also, if there was any leak, pressures would have a noticeable dropdown, but they are ok.
Hi Víctor,
Make sure hat the MS is working proparly and the next- LC. I think you are working with trap column. If so, make sure that the your analytes are not washing out (change trap column, program, etc)
Start with the basics. Running at such low flow rates makes it difficult to spot leaks, flow disturbances or even changes to the mobile phase. Make sure the HPLC system is running the method 100% first. Check the settings on the MS to make sure you are still collecting data in the same manner as before. Check the MS settings, gas, temp etc too. Re-check your sample vials. Do you have enough sample in each one? Is the injector working correctly? Check, then verify the operation. Step-by-step is the way to proceed.
John S Wishnok So I have an autosampler with a loop injection (I used ulPickUp). Then goes the column oven with a 10 port valve. A C18 guard/trap column followed by a C18 Column. That goes right to a NanoSpray Flex and then the Orbitrap system. I do not know if that answers your question or not.
Tigran Margaryan The thing is, this methodology has been working for a long time. It is now that we have been having problems with it. We use a trap column as I said before, but it hasn't been the problem as far as I'm concern because we changed it a week ago trying to solve this problem we have, but no improvements.
William Letter OK. I'll do that (the flow change). The settings are OK, HPLC injection goes perfectly. To make sure it works well, I fill 3 different vials with 10 ul. On the first one I programmed to take 3 ul, the second one 6 ul, and on the third one 10 ul. Once it finished, the vials were 7, 4 and 0 ul.
As I said in the post, microL flows (skipping the 10 port valve) worked perfectly just a week ago, after the problem on nanoLC. So I think no injection, no syringe, no MS problem. It is either the nanoESI or the valve. We disassembled the stator of the valve and observed the interior was rusty so I washed it with methanol following the instructions of a technician I asked.
I hope those answers were explanatory. It is the first time facing a problem like that and I am trying to be as clear as possible with the answers. As I said, everything worked with this HPLC-MS methodology before (gradient, MS values, etc) so a problem must be mechanical and not in the method itself.
Thanks again for the answers!
This makes no sense to me?: " To make sure it works well, I fill 3 different vials with 10 ul. On the first one I programmed to take 3 ul, the second one 6 ul, and on the third one 10 ul. Once it finished, the vials were 7, 4 and 0 ul. ".
*Under no circumstance would we fill an empty vial with 10 ul, then draw 10 ul from it. The vial should always have some liquid left inside so as to not draw up any air. Depending on the vial dimensions and injector configuration (depth), this might be 3 ul in your case (an estimate only). **This may be unrelated to your original question, but following good practices is always important.
Whenever there is a change ("worked before, but does not now"), something obviously has been overlooked. Check the system step-by-step. A clogged calibration port, valve not triggering, bad connection, fouled line or column and the list goes on and on..... Too many things to solve over a web forum. If you can, get another set of local eyes on the system to look it over.
Víctor Caballero if it was working before it does not mean that it is not the cause of the problem. Since you checked the analytical column with other flow and MS you need to understand if the sample reaches to MS or not. Inject without trap column or modify the lc method to have a very short washing step. Then you can understand where the problem is.
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