Chromatography Forum

I was trying to run under these conditions:

Agilent 1100 HPLC with MWD and RI
Phenomenex Rezex RHM-Monosaccharide
80 C Column Temperature
Water @ 0.6mL/min

I was getting these strange peaks:

I took the column out and flowed directly in to the UV and RI, but still have peaks, sometimes they are narrow. I have purged the reference cell repeatedly. My UV baselines @210nm and 190nm were fine, and the pressure was stable.

With no flow, the baseline is stable. I connected another pump to my system and saw the same thing, so I guess it has to be my RI detector causing this. It was working fine just a couple of days ago when I ran a simple caffeine reverse phase method with a mobile phase of 30%acetonitrile/0.1% acetic acid, so there shouldn't have been any solvent miscibility or buffer precipitation issues.

Could it be the lamp or flow cell? What kind of problem would cause this?

From the "peak" shapes, I would have guessed lamp problems, but your no-flow test eliminates that. A leak at the inlet of the flow cell sucking in air bubbles, maybe?

I think air bubbles could be the problem: at 80C water will readily form air bubbles in the detection cell. I suggest putting a constrictor after the cell to give some pressure in the cell and prevent outgassing. A few metres of fine teflon tubing may be enough ( can be knotted to create extra back pressure)

Have a look at the outlet from the detector and see if can observe any air bubbles.

Thanks for the replies. I've run this same method before with no problems, so a leaking flow cell seems to be the most likely cause at this point. I'll have to open up the box and troubleshoot it today, and if I manage to fix it I'll post what I did.

Well I observed a bubble passing from the flow cell to the waste line after each peak, and I did not have the column or column heater attached.
So, now I have to figure out why this is happening now as opposed to last week or the last time I ran this method.

A leak or outgassing are pretty much the only possibilities.

Thorough sparging of your mobile phase (continuous or a few minutes per hour via solenoid/timer) with Helium and/or some back-pressure regulation at the flow cell's exit (as prevoiusly mentioned) can help solve the problem if the problem is outgassing.

If it's an air-sucking leak, new fittings and/or tubing from the column to the detector are in order.

80°C? That's pretty warm. If it's at the upper limit of the temp. range for that detector, you might have to worry about any drafts from your lab's HVAC system, though baseline disturbances caused by drafts tend to be more sinisoidal than waht you posted...

Yep, 80 - 85 C is recommended for this method for carbohydrates to speed up the anomer rotation, else you may get broad peaks. I've run the same method before with no problem.
Also, the same thing was happening at ambient temperature with no column, so that is not the culprit.

I did notice that my degasser had a bad fuse and was not working, so I hope that when I replace it that will fix the problem. If not, I guess I"ll try the pressure restriction after the RI detector.
Would pressure restriction cause additional baseline noise?

The restrictor might cause one noise spike if it was too restrictive and cracked your cell.

I've encountered this problem before......check all fittings and make sure mobile phase is properly degassed. In my case, I had to open up the detector and finger-tigthen the PEEK fittings where the fluid path splits or thereabouts. A loose fitting can draw in air with great periodicity as the mobile phase moves pass it.

Hope you find the cause soon.

I suspect the problem will go away when you get the degasser working, but if not, there is an internal setting on the Agilent 1100 degasser module that allows the degasser pump to run continuously, which is how mine has been set for the last year or two.

If you don't want to go inside the degasser module, and want to be able to switch degasser operation modes, you can buy the cable for the Aux socket at the back, and join pins 1 and 3. Remove the plug, and the unit should return to normal mode. Details should be in the manual.

The vacuum pump life will be shortened, but it does solve some degassing and baseline noise problems. Mine hasn't required service yet, after two years at 24/7.

Please keep having fun

Bruce Hamilton