Chromatography Forum

Hello Colleagues,
I have a problem: high pressure in the flow cell of diode array detector.
This reaction was formed after the analysis of insulin in drug discovery.
As the mobile phase was used sulfate buffer (90%) and 10% acetonitrile.

Can anyone encountered this problem? I can not in any way to lower pressure.

Done: Rinse cell with water, isopropanol, nitric acid solution. It does not help.

Thank you!
With best regards!
Dmitry

Of course sulphate. It's classic!
People working with insulin are quite conservative. Have used high sulphate concentrations in many decades. OK it's another discussion. What you're dealing with is protein/insulin precipitations.

What you need to do is: Rinse the whole flow path and at least the flow cell with 0.1 M NaOH. Start with low flow (f. ex. 0.2 ml/min) and after half an hour or so go to 1 ml/min for half an hour. Then rinse whit water and the system is ready for new challenges You have removed the/any column prior to all that naturally.

Best Regards

Of course sulphate. It's classic!
People working with insulin are quite conservative. Have used high sulphate concentrations in many decades. OK it's another discussion. What you're dealing with is protein/insulin precipitations.

What you need to do is: Rinse the whole flow path and at least the flow cell with 0.1 M NaOH. Start with low flow (f. ex. 0.2 ml/min) and after half an hour or so go to 1 ml/min for half an hour. Then rinse whit water and the system is ready for new challenges You have removed the/any column prior to all that naturally.

Best Regards

Thank Danko!
Excellent Recommendation!

And if it is no secret what other mobile phase can be used by me, except for sulfate?

With best regards!
Dmitry

I might think about flushing the cell in reverse, start with low flow.

I might think about flushing the cell in reverse, start with low flow.

Thank you for your answer!
I tried to do this procedure, but the result is the same: high pressure.

will try to work with flow cell tomorrow, as advised by Danko.

if it is no secret what other mobile phase can be used by me, except for sulfate?

There are many possibilities, depending on different factors, such as the type of insulin (is it conventional Human Insulin or one of many modified variants?) desired separation mode or degree of separation (is it an assay or related substances analysis?) etc.
If you're mimicking a method from some pharmacopoeia, then you're stuck with the current mobile phase - good or bad.
Let's solve you immediate problem first and then the need for new ways will be more (or less) obvious.

You might like to get back here with an update.

Best Regards

Once I got blockage in the tiny tube leading to the detector flow cell from its SS union, ended up replacing that tubing.

Danko, in the beginning I want to thank you for your help in solving the current problem. I have prepared a 0.1 N solution of NaOH, and further along your recommendation. Within two hours the pressure was different from 180 to 130 bar, then increased to 220 bar and after sharp downturn to the normal operating pressure. (30 bar).

Danko, you're right, this is the method method from some pharmacopeia. And most likely to retreat from it, the user can not. I just tip, this action flushing the daily maintenance of the device, after the analysis of insulin in the lab, yesterday.

With best regards
Dmitry

Hi Dimitry (bratushka)

You were welcome.
Good luck with future challenges/assignments.

Best Regards

I have just seen your posts... But one should pay attention to the flow cell of the DAD Detector it might not support all the pressure... check on the manual for the max pressure that can be supported. High pressure might break your flow cell.
We also are experiencing such troubles after analyzing human insulin. We have to clean the whole system.

I have just seen your posts... But one should pay attention to the flow cell of the DAD Detector it might not support all the pressure... check on the manual for the max pressure that can be supported. High pressure might break your flow cell.
We also are experiencing such troubles after analyzing human insulin. We have to clean the whole system.

Absolutely, Nour! We must pay attention to the maximum pressure in the cell. It is written on the body of the flow cell. For this case, the limit set in the program. Only after that should be cleaned the whole system of prepared solution.

With best regards
Dmitry