Chromatography Forum

Dear folks,

we have some trouble with our new HPLC system (Agilent G1312B 1260 Bin Pump). We run an LC method with a start gradient of 60% A (1.5% acetic acid, 1 mM NH4OAc, water) and 40% B (1.5% acetic acid, 1 mM NH4OAc, methanol) at 0.3 mL/min. The pressure at 60% A and 40% B is usually around 470 bar. The sample batch always works fine with similar pressures before the injections. After every batch, the LC is automatically turned off. Here is the problem: When starting the LC again the next morning, the pressure goes immediately high up to 600 bar. After rinsing for 1h, pressure is still the same, it doesn't normalize. To solve the problem, we turn off the pump for a while and after turning on again, the pressure is usually back to normal. Sometimes we have to turn off the pump again to solve the problem.
We tried to run the LC after the sample batch has finished at a low flow rate (0.05 mL/min) instead of 0 mL/min, and the first time it worked fine, normal pressure when adjusting to 0.3 mL/min. But the next morning, it didn't work, we had 600 bar again. Any comments and recommendations?

Hello

Few things:

1.For 0.3 ml/min pressure of 400bar is quite high (what column have you got and what temperature?)
2.Are you sure that when you switch on LC next day the column compartment is on? If it is off it is normal that pressure is high (if temperature is not reached)
3.I'd recommend to create standby method: flow 0.05 (you won't use a lot of solvents) and detectors lamp off

But first of all check LC flowpath : pump and sampler for blockages - needle seat is possible blockage place (if you have rich sample matrix and uplc capillaries), also rotor seal in sampler and mixer in binary pump (if you use buffers)

Regards

Tomasz Kubowicz

Do you have a notable pressure when purging?

If you have a blockage somewhere and can't find it, you can manually disconnect the flow path at certain points, put on the flow, and check the pressure. In this way you can narrow down where the blocking takes place.

As I understand it, the pressure comes from the column.
If no pressure is registered without the column, the answer is clear:
Some of your sample is precipitating upon mobile phase contact. That something (sample matrix) is not in your standard solutions.

You need to optimize your mobile phase or your sample preparation protocol.

Best Regards

As I understand it, the pressure comes from the column.
If no pressure is registered without the column, the answer is clear:
Some of your sample is precipitating upon mobile phase contact. That something (sample matrix) is not in your standard solutions.

You need to optimize your mobile phase or your sample preparation protocol.

Best Regards

I would recommend flushing the column with a water/ACN mix (no buffer) following each batch so that there is no buffer in the system when starting a new batch.