HPLC high back pressure problem from newbie

[car66]

Hi Everyone...

I am quite inexperienced with the HPLC and hope to get some suggestions/advice.

We have a Waters 2659 system and it is giving me very high back pressure. I unhooked the column, then guard column and the pressure is still very high. I then changed out the inline filter, and still very high. 0.5ml/min was just water was around 800-900 psi.

The hplc was mainly used to separate proteins using size exlusion column. Isocratic flow with 50% acetronitrile and 50% TFA(0.1%). UV detector is used.

I am not sure if I need to or is able to provide anymore details. I hope that someone can provide some advice on where to start looking for the problem.

Thank you very much!!

[ym3142]

work backward from detector to pump, remove one components at a time and then check the pressure after each removal, until you find one of the component cause the pressure,

enjoy

[markreyes]

if you have high back pressure, especially if you know that the system was used to run for proteins. I would checked the inline filters and the check valves.

you can see if the check valves are clogged by just shaking them next to your ear.

inline filter is bad when you see residual salts on it.

[AdrianF]

Also try changing the tubing fine HPLC tubing can become crimped (PEEK) or patially blocked and create tremendous back pressures.

[car66]

thank you so much for the replies!

The inline filters look ok, it was recently replaced. I tried the check valves and shaked it, it does sounded like there is something in there. ^_^

I will change that out first and hopefully that is my problem.

[HUH?]

The easy test for tubing and check valve problems is removal of columns, guards, and pre-column filters and plumb straight through (with a union). If you still experience high back pressure, then tubing or check valves may be a problem.
Add each inline component and flow. You should find the source of the high back pressure.

Good luck!

[Hugh]

High back pressure will never be caused by check valves. Transducers are typically plumbed after the pump and will measure the pressure created by everything downstream of them. Since the check valves are upstream, they won't be part of the problem. Clogged check valves typically cause lower pressure because of restricted flow, not higher pressure.