Particles in flow cell?

[shennong]

Hello,

I met some problems with our HPLC. Before I do more checks or cleaning, I'd like to hear some opinions. Thanks!

The HPLC is Shimadzu, SPD-M20A PDA detector. Binary gradient. Solvents are 0.1% formic acid and acetonitrile.

A few weeks ago I've seen spikes in the chromatogram across all wavelength (see picture.)
http://s42.photobucket.com/user/shennon ... e.png.html

According to the manual, the spikes are air bubbles flowing through flow cell. So I disassembled and cleaned the flow cell and replaced the lens. The spikes were gone.


Last week I ran some enzyme reaction products, and spikes were observed at the end when pressure dropped. I ran a lot of solvent washes because sample concentrations were too high in the reaction products. Then I started to see the abnormal spikes in my chromatograms when I ran blanks (50% methanol) (see picture).
http://s42.photobucket.com/user/shennon ... c.png.html

Later I started to see the wide absorption in the chromatogram (see picture) after I purged the autosampler (function of SIL-20AC).
http://s42.photobucket.com/user/shennon ... 5.png.html


I think spikes are bubbles in the flow cell. Wide absorption is caused by dirt in the flow cell. I'm thinking of trying the following things.
1. Remove column and wash the flow line with 95:5 acetonitrile/formic acid at 2 mL/min for hours. Hopefully, dirt and air will be washed out.
2. If the problem is not gone, take out the flow cell and flush with solvent.
Following is what I found on the forum. viewtopic.php?f=1&t=2394&start=0&hilit= ... +flow+cell

Flowcell cleaning procedure:
Flush with methanol followed by water to remove all of the methanol.
Flush with up to 30% nitric acid. Leave the acid in the flowcell for 5-10 minutes then continue flushing.
Flush with water until ALL of the acid is removed then flush again with methanol.
If this doesn't work, you will have to find the detector manual and take apart the flowcell to clean the windows. Be aware that you may need to buy new flowcell windows and gaskets.

In this procedure, is flush done using syringe? Any tips about avoiding air using syringe? And what does 30% nitric acid do?
3. If this does not help, I will have to disassemble the flow cell and sonicate the lens and cell window.


A final question is about the spikes at the end. I guess air bubbles come out of solvent when pressure drops, like bubbles come out when you open a coke. But we have never seen that before.
We have a 2-foot narrow tubing after the outlet. Is there general guideline about how much tubing it needs?
We use house vacuum and filter to degas 0.1% formic acid and never degas acetonitrile. The vacuum is very weak than it used to be, taking longer to degas same amount of solvent now. Is it possible that degassing is not sufficient?

Any suggestions will be appreciated. Thank you a lot in advance!

[shennong]

I tried posting the images but could not open them. Let me know if you have troubles in viewing the images.

[tom jupille]

Is it possible that degassing is not sufficient?

Not only possible but probable. Vacuum degassing is generally sufficient only for premixed isocratic mobile phases, after the water and organic have been combined. As soon as you stop the vacuum, the liquid begins to re-equilibtrate with the atmosphere. For on-line mixing you should really be using a membrane degasser.

As far as flushing the cell, you *pull* the liquid though the cell with a syringe. The nitric acid passivates the stainless steel (phosphoric acid is often recommended instead of nitric).

[shennong]

Thanks, tom jupille. That is very helpful.

Yes. The vacuum degassing is a big problem. Our building house vacuum is really weak now. I used a pump to filter degas 0.1% formic acid and ran blanks again. Most spikes are gone. Two spikes at the end of the run still remained there. I will try to get a membrane degasser, but not sure if I can get one...

We had been using two instruments for 5 years without any problems. Since we moved to another building, problems are more frequent. We have to change plunger seals more often. I have seen these spikes on both instruments. The second one was fixed with an attached tubing after flow cell outlet. The same bottle of solvent gives no problems on the 2nd one, but gives bubbles spikes on the 1st one. I think there is other problem with the 1st HPLC besides the degassing.

Last time, I tested where bubbles were introduced by disconnecting joints and sticking outlet into water and watching for bubbles. It is that after flow cell strings of bubbles were observed, so I decided to clean the flow cell. I will need to test this step by step if problem remains.

[Creal]

For me it looks like an air buble problem. When I aquire chromatogram similar to yours I disconnet column and run 100% IPA on whole system. IPA have higher viscosity compering to water based solvents, sometimes it helps.

Did you run any normal chromatography recently and didn't flush the whole solvent? Maybe there is some n-Hex somewhere in your system.