There may be column washing problem because some impurities may stick on the column. That can obstruct the flow and increase in the pressure.

You should wash the column with hot water and then wash with methanol.

Gudluck

:)

Dear Huq, check out the whole tubing starting from Pump to the Column inlet for any air trapped, purge the pump for few minutes. If still you have the same problem, checkout the rest of tubing following the column outlet, and as Robin Joshi suggested it could be also due to blockage in column, so better remove the column and use "tubing joint" and then purge, I am sure the problem will be resolved iA.

Hi

It also depends upon what analysis you have done previously. What were the samples run before your present experiment. Sometimes previously run samples are trapped in the column because of inadequate washing. Did you use any herbal extract samples or plasma samples before. Anyways, Just do one thing. In the morning when you start the work, wash the sample with methanol for 1-2 hours, after adequate purging initially. Then run the column with water for 4-5 hours. After this again purge and start washing with methanol before shutting down for 1-2 hours. Do this process continuously for at least a week. You will notice a change in pressure and it will stabilise as well as an increased pressure will come down to normal.

Just do this and inform abt the status. Feel free to ask. I m sure this will help

Regards

Thanks to all for your suggestions. Actually the pressure goes up to high and then just after that directly drop down to zero psi. Again it rise up gradually and falls down to same zero level. Now i will try with your advises.

Are you working with buffers in mobile phase? This situation can result in

formation of precipitates at the pump… try remove and clear pulse damper imput. anyway check the attached file, may be helpful.

This probem usually is due to some impurities and particles which are stuck inside the columns. I would suggest to wash your column with gradient H2O/ACN overnight., this all hydrophilic particles will be washed out by H2O and all hipophilic particle will be washed out by ACN...Hopefully this will work.

Thank u brother.

Thanks to Tiago for the attached file. this looks very useful.

Pressure changes mainly because of the bubbles. First you should sonicate your mobile phase and if you have mixtures between acetonitrile or methanol and aquous buffer it is preferred to mix and sonicate then use from one inlet, try to avoid the mixing in the pump if possible. Blockage of the tubes normally cause high pressure not flacculations. You may be able to open the pump head and see what is going on, sometimes you can see an air bubble moving between the pistones then you need to purge, you can use syring manually to do it.

@Moyeenul Huq, I would like to know the nature of sample previously used in this system and the sample you are working with now. Once we know that what is the nature of the sample, it would be easier to solve the problem because then we can suggest the ways to remove the traces of that sample if it is sticking to the column. Whatever it may be, impurities, serum, herbal extracts or buffers etc., you need to find out the nature of the problem causing agent.

Yes I agree with the suggestions regarding the mechanical work out of your system. You can just work out the pipings, tubings and have a look at the pump also. Hope you get the favorable result and your problem is resolved by this effort. But what if it doesn't works. So the knowledge about the impurity is important.

Hope you got my point. It is just like the hardware and software problems of the computer, haha. If it is hardware (here your HPLC system parts), then it is fine, but if it is software (eg. traces of any leftover samples), then you need to remove the bug out of your system.

Anyways keep washing the column continuously with the solvents suggested previously. You can do the plannings side by side. But purging and washing should be continued regularly.

Gud Luck

Regards

Dr Alok@ thank you very much for your knowledgeable discussion. I am running MeOH crude extracts of 3 plants just for analysis them. I ran 2 species successfully but the problem appeared during the 3rd species.

Instrument was working well till you ran the first two plants. So there might be some handling problems and problem of air being retained can not be ruled out also. So do the mechanical workout firstly so it is confirmed that your system is perfectly all right. Then see, if the pressure is ok, it is fine. It should be :)

Now initial problem is the herbal extract only and more importantly it is sticking to the column. It was a methanolic extract so need to wash continuously with methanol. Be patient and see the result after washing for a week. It will stabilize once the extract is washed off the column.

Lets see. Inform accordingly.

Gud Luck

Regards

Thank you again for your advise. I shall let you know the result.

Hi, 

I have read your discussion and I have a similar problem : I am running samples of collagen cross links and my pressure is not too high or too low but it is oscillating between 1000 psi or so and zero. I am using a binary gradient of solvent B and the more the more concentrated in B is the solution, the littler is the period of the oscillation... And at the moment I put my 100%B solvent for the washing, the pression plunges do to zero.

Any ideas ?

Thanks

Once I faced the pressure fluctuations in our waters-HPLC system then I washed the system with HPLC-Grade water at a flow rate of 0.2ml/min overnight and after that I didn't find this problem with the buffers.

Hello, I am facing the same problem with the pressure. it is jumping from 30 bar to 39 and comes back to 25 bar. I need to address this problem.  what do you think if we change the column?