Chromatography Forum

The column was over pressure during the run. So I back-flashed the column first.

Then I detached column and turn on the flow, let it drain from column inlet line (disconnected with the detector). The system pressure without the column showed very high (eg over 1500psi for 100% H2O at 1 mL/min, over 1000psi for 50% ACN at 1 mL/min). The sample was diluted in buffer (KH2PO4 and saline) for injection, I was thinking salt precipitate in the system resulted in hight pressure. I washed the system by using Water, then organic solvent, but didn't decrease pressure much.

What else I can do to lower the system pressure?

You probably have a clogged in-line filter.

Hi Flora,

I’m almost certain that the sample comprised one or more protein/s. So, you should analyse the situation as follows: Was there any reason for dissolving the sample in a buffer?
The answer would be yes. What I’m trying to say is; neither pure water nor ACN will dissolve the precipitated protein on the inline filter or whatever is clogged.
Wash the system with 0.1M NaOH mixed with some 20 – 30% isopropanol. Remember to disconnect the column and the detector.

If I was right about the sample composition you might like to revise the mobile phase so that no precipitations occur in the furure.

Best Regards

The column was over pressure during the run. So I back-flashed the column first.

Then I detached column and turn on the flow, let it drain from column inlet line (disconnected with the detector). The system pressure without the column showed very high (eg over 1500psi for 100% H2O at 1 mL/min, over 1000psi for 50% ACN at 1 mL/min). The sample was diluted in buffer (KH2PO4 and saline) for injection, I was thinking salt precipitate in the system resulted in hight pressure. I washed the system by using Water, then organic solvent, but didn't decrease pressure much.

What else I can do to lower the system pressure?

First find out where the problem is located:
If you can do a wetprime without the high pressure problem, then you tubing from the injector to the column is clogged.
If you still have the high pressure problem even with a wetprime, your inline filter is probably clogged (most common)
Try to untighten the filter and see if your pressure is normal. If so, replace your inline filter.

Ace

I called tech service. They said it might be pressure transducer's problem. Because it always shows negative system pressure reading (~-25psi) in the control pannel even the flow is zero. But I am also think the in-line filter is clogged. I will see how Tech service to deal with it.

Thanks for the suggestion.

The column was over pressure during the run. So I back-flashed the column first.

Then I detached column and turn on the flow, let it drain from column inlet line (disconnected with the detector). The system pressure without the column showed very high (eg over 1500psi for 100% H2O at 1 mL/min, over 1000psi for 50% ACN at 1 mL/min). The sample was diluted in buffer (KH2PO4 and saline) for injection, I was thinking salt precipitate in the system resulted in hight pressure. I washed the system by using Water, then organic solvent, but didn't decrease pressure much.

What else I can do to lower the system pressure?

First find out where the problem is located:
If you can do a wetprime without the high pressure problem, then you tubing from the injector to the column is clogged.
If you still have the high pressure problem even with a wetprime, your inline filter is probably clogged (most common)
Try to untighten the filter and see if your pressure is normal. If so, replace your inline filter.

Ace

there is no protein or peptide. I was testing a small molecular which is very soluable in the buffer.

Thanks for your suggestion, i will save it for later protein analysis.

Hi Flora,

I’m almost certain that the sample comprised one or more protein/s. So, you should analyse the situation as follows: Was there any reason for dissolving the sample in a buffer?
The answer would be yes. What I’m trying to say is; neither pure water nor ACN will dissolve the precipitated protein on the inline filter or whatever is clogged.
Wash the system with 0.1M NaOH mixed with some 20 – 30% isopropanol. Remember to disconnect the column and the detector.

If I was right about the sample composition you might like to revise the mobile phase so that no precipitations occur in the furure.

Best Regards

I was testing a small molecular which is very soluable in the buffer

The overall question is: Is it soluble in the liquids you were trying to wash the system with (i.e. pure water and ACN/water mixture)?
What is your mobile phase btw? And why is the sample (protein or not) dissolved in phosphate buffer?

Best Regards