Sinusoidal pattern and no peaks after HPLC maintenance

[Arne]

Hi all,

a graduate student accidentaly ran our HPLC out of solvent and since then there have been problems with it. We did a lot of maintenance on it (replaced pump seals, valves, cleaned inside of pump).

Now all I see when I do test injections is s sinusoidal pattern of peaks spaced 0.25 min apart (see picture) I do not see analyte peaks at all in standard samples.

Is this something you have observed before? What do you suggest to fix it?

The HPLC runs at the expected pressure and delivers the correct amount of solvent at all mobile phase compositions.

Best and thank you,

Arne

Image: http://imgur.com/J74TgFh

[juddc]

Perhaps there's an air bubble in your detector. Have you tried flushing with neat methanol?

[Gerhard Kratz]

Please disconnect your column from the System and flush with high flow rate with Isopropanol, Methanol and Acetonitrile. Check flow of each magnetic valve. It Looks like that it is the pump. But an air bubble in the detector flow cell can effect this phenomenon also. Good luck.

[aceto_81]

Do you see a flow running out of your column/detector outlet?

Ace

[James_Ball]

Hi all,

a graduate student accidentaly ran our HPLC out of solvent and since then there have been problems with it. We did a lot of maintenance on it (replaced pump seals, valves, cleaned inside of pump).

Now all I see when I do test injections is s sinusoidal pattern of peaks spaced 0.25 min apart (see picture) I do not see analyte peaks at all in standard samples.

Is this something you have observed before? What do you suggest to fix it?

The HPLC runs at the expected pressure and delivers the correct amount of solvent at all mobile phase compositions.

Best and thank you,

Arne

Image: http://imgur.com/J74TgFh

Is this measured by collecting the effluent from the detector into a 10ml graduated cylinder and measuring the time to collect 10mls?

It can sometimes be caused by an air bubble in the detector as one explained. If the pressure is steady and does not fluctuate with the signal, then it is probably a bubble in the detector or maybe column. If the pressure fluctuates with the signal then maybe it is in the pulse dampener or pump head. You can try running the flow up to something like 4ml/min with bypass valve open, then quickly close then open the bypass valve, this can cause any air bubbles to compress then expand which can knock them loose to flush out of the system. Just keep an eye on pressure and make sure not to over pressure the pump.

[Arne]

Thank you so much for all the replies!!

I do see flow (at the right rate out of the detector end).

I solved the problem. It appears that there was air trapped somewhere in the pump. I did some isocratic runs with just solvent A or B and then isocratic runs of a mixture of A and B, and finally gradient elution to zero in on where the problem lies.

I did monitor the pressure and found no fluctuations after flushing the system (again, I had done it before posting).

Arne

[Arne]

Oh, and I measure the flow-rate by hooking up a 500 uL empty syringe without the plunger to the column effluent. We do small-bore chromatography at flow-rates of about 200 uL/min.

[James_Ball]

Oh, and I measure the flow-rate by hooking up a 500 uL empty syringe without the plunger to the column effluent. We do small-bore chromatography at flow-rates of about 200 uL/min.

We have used a 5ml disposable syringe the same way for measuring larger flow also. It is a good way to do the flow measurement without much mess.