Pressure & retention fluctation Agilent 1100 (with gasse

[abbidmajid]

Hello all, I have a issue with pressure & retention variation. Following conitions usd: Channel A - ACN (40%), Channel B - Phosphate buffer pH 6 (60%), Flow rate 1ml/min, 15cm column. Pump Quanternary.

Pressure initially stablises at approx 50Bar, than fluctuate between 15 - 55Bar. This is with/without injections. I have tried used a premixed phase of 40/60, but problem remains. Any suggestons??. I have changed the seals, plungers, gold seal and swaped the MGCV (for one that i know is ok)

thanks :

[Consumer Products Guy]

Have you purged ALL four channels of your quat pump (even though you are only using two or one) ? That's important, purge each for 5 minutes at 5 ml/min.

Are you degassing, like with Agilent Vacuum degasser? Important. Remember, a vacuum degasser has a hold-up volume of the previous solvent, another reason to fully purge.

Do you have a small leak somewhere?

If that doesn't take care of it, I'd use the premixed mobile phase (I'm assuming nothing precipitated out, and that you filtered it), put ALL four leads into that, purge ALL the channels, and try. That way the multi-channel mixing valve is essentially out of action, as it wouldn't matter how it mixes. Or you could just use one channel and use the adapter fitting to bypass the multi-channel valve and go directly into the active inlet valve.

If that's still fluctuates, I'd go back to IPA and run the Agilent pump flow test protocol to see if my pump was OK.

[Bruce Hamilton]

Have you cleaned/swapped the solvent reservoir filters?.

Also, if I recall correctly, the aqueous phase is preferred to be in channel A ( lower channel of gradient mixing valve ), and the solvent phase is preferred to be in the channel above it ( in this case, B ). If you have them reversed, that might cause problems.

As well as the options suggest by CPG, I would also fill both A and B channels with 1:1 H2O:CH3CN and throughly flush each channel and the injector ( without a column ) at 5 ml/min for 10 minutes, then insert the column, flush it, and try a gradient using the same 1:1 H2O:CH3CN solutions.

If that gives stable pressure, then I'd try your mobile phases again.

Bruce Hamilton

[abbidmajid]

Thanks chaps - i will try these suggestions tomorrow.

[Anthony_Ng]

Will it be the problem of Active Inlet Valve? Or the ruby ball inside the AIV cartridge cannot seat properly?

I am not sure if too concentrated phosphate buffer will deposit somewhere?

[Consumer Products Guy]

Will it be the problem of Active Inlet Valve? Or the ruby ball inside the AIV cartridge cannot seat properly??

Possibly the AIV or its cartridge; but first he should use what Bruce H. and I suggested previously, to see if the issue lies with the pump or elsewhere. Substituting parts randomly is not a good strategy.

"Change one thing at a time"

[abbidmajid]

Hello all, I have primed/purged all the channels. Set channel A as phosphate buffer and Channel B with ACN. Pressure fluctuation still present.

Primed all lines with IPA, performed a leak & pressure check using the key-pad then checked the flow-rate. ALL PASSED.

Then primed channel A with premixed mobile phase of 40/60 ACN/phosphate buffer,pH6 - again problem not resolved

One thing which i was not able to do, is the "Seal Wear-in Procedure". for standard seals (5063-6589).

It requires the following: adapter (0100-1847), restriction capillary (5022-2159). I have ordered these for future use.

I have the engineer in tomorrow for another problem, will ask him to do this.

....hope this resolves the issue. It is rather strange, that i never have come across. then i never changed the pump-seal before, as performed as part of PM.

[Consumer Products Guy]

Sounds like you didn't try the premixed mobile phase as I suggested earlier: put ALL four leads into that, purge ALL the channels, and try. That way the multi-channel mixing valve is essentially out of action, as it wouldn't matter how it mixes.

And since you have ordered adapter fitting 0100-1847 already, when it arrives you could just use one channel and use the adapter fitting 0100-1847 to bypass the multi-channel valve and go directly into the active inlet valve.

FYI, we (or our Agilent engineers) haven't used any seal break in procedures.

[abbidmajid]

Hello Consumer Products Guy, thanks for replying. I will try premixed phase in channel A (& purge all) in morning.

thanks

AM

[Bruce Hamilton]

I suspect that you may have just identified the problem - assuming you didn't have temporary brain fade and install the seals incorrectly - you haven't performed the seal wear-in procedure, or equivalent.

As you are using non-volatile buffer, and if you do not have one of the seal wash options, it's quite likely the plungers have some surface roughness. When I perform wear-in, I usually just install an old 250mm column onto the pump outlet, and I don't worry about the adaptor.

I just crank the IPA flow up to pressure about 320 bar for 20 min or so and then slowly switch down to zero flow. Then I usually go through a gradual transition of IPA to water keeping the pressure at normal.

Also, its' not clear to me that you tried the 1:1 Water:CH3CN that I suggested, which could have helped dissolve any residual buffer deposits. You should be regularly flushing your system with high water mobile phases to remove buffer deposits within the solvent system and pump, especially if you don't have a seal wash option.

Please keep having fun,

Bruce Hamilton

[Consumer Products Guy]

Yeah - what Bruce said - if your plungers are scored then you'll get all kinds of crazy pumping and pressure issues.

[abbidmajid]

Thanks for the tip Bruce, will rinse system with ACN/water and also perfrom the wear-in using a old column.

The plungere were replaced same time as the seals.

Will keep you posted

Fingers crossed for tomorrow!!!

thank you