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Topic: What should be done to decrease the pressure of the HPLC column?  (Read 1073 times)

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Offline User47

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What should be done to decrease the pressure of the HPLC column?
« on: February 16, 2022, 03:47:53 AM »
After using an HPLC (C18) Column for several times, now it doesn't do what it has to do, when ever we want to do a research by a substance, the machine shows a every high pressure on the column and the process would be stopped on its own and won't work, we have washed is with methanol, water acetonitrile but non of them have been effective till now, what are we ignoring about this? What should we do to decrease the pressure of the column?

    Available pump heads Max. flow rate in ml/min is 10

    Max back pressure in bar 400

    Recommended particle size is 5 μm
« Last Edit: February 16, 2022, 05:03:01 AM by User47 »

Offline kriggy

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Re: What should be done to decrease the pressure of the HPLC column?
« Reply #1 on: February 16, 2022, 05:46:34 AM »
It seems that there is a some sort of precipitate inside your system. You might want to wash with solvents for longer time than you did now.

Also, depend on size of your column, smaller diameter requires smaller solvent flow. Just yesterday my column wash stopped because I did put 1ml/min on 3 mm column and the pressure increased over the specs which stopped the flow.

It also depends on solvent, IIRC organics increase the pressure therefore if you have high% of organic solvents it also might be the cause

Offline rjb

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Re: What should be done to decrease the pressure of the HPLC column?
« Reply #2 on: February 16, 2022, 07:49:35 AM »
You have a couple of options if your column is clagged up with precipitate which is most likely to be your problem:

Begin by back-flushing your column. This involves taking off the column and replacing it so that the flow is in the reverse direction i.e. flow enters the column outlet. The input (which is confusingly now the output) must go straight to waste to avoid contaminating the detector. Backflush with water followed by ACN and if necessary MeOH/H2O. A flow rate of something like 0.5ml/min for maybe 1hr (or longer if it's really clagged up) might do the trick.   

If this doesn't work, you could potentially consider replacing the column inlet frit. This is something that I've never done before myself, so I can't give you any advice other than to be careful; you can easily degrade the column.

Please note that I am assuming that you don't have a guard column installed. If that is the case consider installing one as it will make life a lot easier if (when) this happens again!



Offline User47

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Re: What should be done to decrease the pressure of the HPLC column?
« Reply #3 on: February 16, 2022, 10:29:18 AM »
Thank you so much, but I tried these ways and they weren't effective, BTW our column has a guard column, could the problem be about the machine itself not just the column?

Offline User47

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Re: What should be done to decrease the pressure of the HPLC column?
« Reply #4 on: February 16, 2022, 10:31:32 AM »
Thank you, @kriggy and @rjb

Offline User47

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What should be done to decrease the pressure of the HPLC column?
« Reply #5 on: February 16, 2022, 10:34:00 AM »
mod edit
I merged this entry with the rest of your posts on the topic
we do not cross post here
see forum rules




After using an HPLC (C18) Column for several times, now it doesn't do what it has to do, when ever we want to do a research by a substance, the machine shows a every high pressure on the column and the process would be stopped on its own and won't work, we have washed is with methanol, water acetonitrile but non of them have been effective till now, what are we ignoring about this? What should we do to decrease the pressure of the column?

    Available pump heads Max. flow rate in ml/min is 10

    Max back pressure in bar 400

    Recommended particle size is 5 μm

I have done what my friend recommended here, but none beard fruit, could the problem be something other than the column?x
« Last Edit: February 16, 2022, 12:19:12 PM by billnotgatez »

Offline rjb

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Re: What should be done to decrease the pressure of the HPLC column?
« Reply #6 on: February 16, 2022, 11:19:59 AM »
Thank you so much, but I tried these ways and they weren't effective, BTW our column has a guard column, could the problem be about the machine itself not just the column?

It's certainly possible. I guess the first thing is to isolate the problem if you can. I suspect that you have done most of this, but just in case not...

I would begin with guard column which would obviously take more of a battering in normal use than the column itself. Does replacing the filter or guard column itself (depending upon design) help to lower pressure to nominal values? If so this is (at least part of) the problem.

I would then move on to the column and after backflush (which you have done) I would look at whether a another column (even one from another HPLC that you know works OK) alleviates the problem. If so, you've isolated the problem to the column. At this stage you can change the frit (which you've done) and if this doesn't work buy a new column.   

If changing the column doesn't fix the problem, then the last thing that I can think of is the pump frit which should be user replaceable. I'm led to believe that these are supposed to be replaced fairly frequently (not that ours are!) and that if dirty, they can increase pressure fairly significantly.

Beyond that, I suspect that it's a job for an engineer  :(
 

Offline User47

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Re: What should be done to decrease the pressure of the HPLC column?
« Reply #7 on: February 16, 2022, 11:29:25 AM »
Yeah, you're right, I got to start with guard column.
any way, Thank you so much for the tips you just mentioned, they were being so useful.

Offline kriggy

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Re: What should be done to decrease the pressure of the HPLC column?
« Reply #8 on: February 17, 2022, 02:12:46 AM »
Thank you so much, but I tried these ways and they weren't effective, BTW our column has a guard column, could the problem be about the machine itself not just the column?

Of course it could :D :D Try different column if you have one to see if there are same problems. If not, problem is in your column, if yes its likely in the machine.

Offline User47

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Re: What should be done to decrease the pressure of the HPLC column?
« Reply #9 on: February 17, 2022, 03:03:23 AM »
Thank you my friend.

Offline Babcock_Hall

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Re: What should be done to decrease the pressure of the HPLC column?
« Reply #10 on: February 17, 2022, 05:05:21 PM »
Occasionally the matrix decomposes or collapses (I am not sure of the correct terminology).  The column has to be replaced at this point.

Offline User47

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Re: What should be done to decrease the pressure of the HPLC column?
« Reply #11 on: February 17, 2022, 08:20:34 PM »
Right! Thanks

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