Back Pressure Regulator Problem

[steve2]

I am trying to run an assay that has been established for about 10 to 15 years. Unfortunately, I'm running this assay on a different system. The assay was developed using Shimadzu equipment, and I'm using Waters. Normally, that wouldn't be much of a problem, but I've had nothing but problems trying to get this assay to work on the Waters system.

Last week I informed the group who developed this assay of the recent problems I've been having, namely that even without degassing the mobile phase, I get air bubbles. The mobile phase has dioctyl sulfosuccinate (DOSS) as an ion pairing reagent. Why this was chosen I'm not sure, but because DOSS is in the mobile phase I can not degass it. Anyway, I told the group that I was having baseline problems, and I was told to use a backpressure regulator because the DOSS will still form air bubbles in the system without it.

Today I put on the backpressure regulator and the detector's flow cell blows. The backpressure regulator (Upchurch #U-610) is rated at 40psi and the flow cell can take 145 psi. There shouldn't have been any problems.

The entire system normally runs at about 150 psi at 0.1 mL/min while running the mobile phase. However, once the backpressure regulator in put on the psi jumps to over 900. I've contacted both Upchurch and Waters. Upchurch tells me that the psi shouldn't be that high and Waters tells me not to use the backpressure regulator.

How can I correct this problem? I can't run the assay without the backpressure regulator, and I don't have time to develop a new one.

The system I'm running is as follows:

Alliance 2695 with a 2487 UV/Vis detector and a 2475 Fluorescence detector. Everything is from Waters. The 2487 is connected to the column and the 2475 is connected to the 2487, then out to waste. The backpressure regulator is connected to the waste of the 2475, and it's the 2475 that I'm using for the detection, which has the flow cell that was destroyed as well.

Who would've thought quantitating Topotecan would be so difficult.

[Uwe Neue]

First, let me ask you why you can't degas the mobile phase. I understand that you are using a surfactant, and maybe that it will foam. If it is foam that is the concern for off-line degassing, it could go away after a while, and you would be back in business. Alternatively, you could use on-line degassing.

If this is working, I am sure that this is better than making another attempt at blowing up the detector cell. However, I am not sure how the backpressure regulator works, and what can go wrong. Whenever I needed to get a bit of backpressure on a detector cell, I used a short piece (2 inches) of 9/1000 tubing connected to the detector outlet with a piece of teflon tubing. This provided enough backpressure with barely a chance of blowing a detector cell. At the low flow rates that you are working at, you may need a bit longer length.

[HW Mueller]

Maybe you ran this device backwards? Anyway, it´s a good idea to test run these things without anything on line before installing them behind a detector. Normally these are quite reliable, if clean. (Incidentally, I usually check all lines behind detectors, with solvent in a syringe, before starting runs).

[Rob Burgess]

Are your baseline problems prevalent on both detection systems i.e. UV and FLD?
I wonder if your baseline problems are actually due to air bubbles at all. Any chance of providing an image to this forum board? It may be that your poor baselines are due to something else (purity of mobile phase components).

Try removing the cell and looking at the window with mobile phase flowing through it... you can usaully see if bubbles are forming and thus causing you problems.

[folive]

I have had the same problems with bubbles forming in the flow cell of my fluorescence detector. I would suggest investing in an adjustable back pressure regulator (0-100 psi or so) as well as a in-line pressure gauge, so your back pressure near the detector can be monitored and adjusted accordingly. I recently bought both of these from Alltech and they seem to work well.

F. Olive

[steve2]

Thank you, everyone, for your responses.

Rob, I will work on getting some scans of the baselines. I checked the flow cell without the BPR and there didn't seem to be any air bubbles in the flow cell.

HW Mueller, I checked before starting anything that it was on correctly, twice. My mistake is that I didn't test it before installing it. Being relatively new to HPLC, I'm learning as I go. Unfortunately, these kinds of mistakes cost time and money.

Uwe, the DOSS in the mobile phase is fairly concentrated, so any attempt at degassing offline or on-line will generates bubbles or foaming. I tried using SS tubing with smaller diameters, bending them into up and down U shapes, but that didn't seem to help with the baselines.

folive, I may need something like you have, if not now more than likely later. Do you have a part #?

[folive]

0-100 psi backpressure regulator, Alltech #39021, $200
0-100 psi liquid pressure gauge, Alltech #19255, $140

Larger ranges are available, but if your max psi is 145, then these should do the trick.

You may also want to consider a pressure relief valve, which will divert the flow and release the pressure if a max pressure (0-200 psi) is achieved, to avoid blowing the flow cell. Have not tried this but may be worth looking into.....0-200 psi, Alltech #39025, $140.

Hope this helps....maybe Alltech will send me some goodies for the free advertising

F Olive

[steve2]

Retracted.

[steve2]

The following scans are of test shots with the mobile phase with topotecan standards done at different times and different days:

[Mark Tracy]

You can use an inline vacuum degasser without fear of foaming. Rheodyne makes a good one of modern design. (Lots of people OEM it too.)

[Uwe Neue]

I am with Mark. The on-line or inline degasser is your best bet. Your system should have one, and you should not need to buy one. Look at the manual.

[steve2]

I used the in-line degasser before, but was told to turn it off. However, I had the assay working just fine with it on until last Monday when the current problem with the baseline occurred. I'm not sure what the problem is, but it's causing a great many headaches.

[Uwe Neue]

Well, turn it back on...

[HW Mueller]

steve2, you got me a bit confused now. The problem occurred right after you cut out the degasser? If that´s the case you know the source, as Uwe suggested. If it happened with the degasser still on line than you got gobs of gunk into your system, or the degasser quit (possible?).
If air is the problem than it looks more like dissolved air or air foam (probably need not be visible) than bubbles which cause either spikes or sawteeth. In case of dissolved air a restrictor doesn´t help much. (Now we discussed before whether dissolved air absorbs light. It shouldn´t, but I did experiments showing that N2 and He strongly raise baselines and noise, probably via scattering)

[HW Mueller]

I just read Mark´s comment in "Mysteries of ion chromatography" on "stuck bubbles", that reminded me that especially when testing mobile phases (UV detector) I regularily open the plumbing (quickly to produce a jolt) between the UV and a restrictor (or sometimes a restriction). Very often that lowers the baseline, so Steve2´s baseline could be due to a stuck bubble also.
(The trick to close off, temporarily, a capillary with a finger has also been used successfully here. I have never been able to increase the pressure with this method to endanger the 100bar rated cells).