Pressure stability issues - He sparging related?
Posted: Mon Feb 23, 2009 5:27 pm
Dear Chromatography Forum,
I am rethinking the He-sparging of the aqueous mobile phases in my experiments because I have been having some experimental problems in the last few weeks. I have a Waters Delta 600 pump and I sparge the aqueous mobile phase (ionic strength 0.01M typically) with He at 15 mL/min as recommended in the HPLC manual.
The problem is that the pressure becomes unstable during the middle of experiments. Our experiments proceed just fine for several hours and then the pressure just becomes very unstable and the flow drops off. At the start of experiments I try to make sure that the pressure is stable with all of the tricks that I have learned over the years. In particular, when putting a new mobile phase in the active reservoir (we pump 100% B) I by-pass the pump and draw out an excess of mobile phase to be sure that there are no bubbles in the lines from the reservoir to the pump. I also pump several mL of mobile phase through the pump (and out the column by-pass) to ensure there are no gas bubbles in the pump.
I pack my own columns with fairly large particle size (100-800 micron) quartz sand substrates (for my geochemistry research) and so the backpressures are not large. For example, when I use 20x4.6mm columns, the backpressure is about 150-300 psi.
I understand that sparging drives a large fraction of dissolved gases out of the mobile phase and I furthermore understand that when He is used as the sparging gas it has such a low solubility that it doesn’t stay around in any significant residual concentration. It is my understanding that dissolved gases in the mobile phase are undesirable because of the pressure drop after the column. In particular, I understand that when the pressure drops after the column bubbles can come out of the mobile phase at that point which can create problems for the detectors. I have optical detectors; one dual wavelength absorbance detector and one fluorescence detector. My first question for the HPLC community is what kind of post-column pressure drops, if gas-saturated, i.e. non-sparged mobile phase liquids were being used, would generate bubbles? I wonder if the relatively low backpressure in my experiments (in the range 150-300 psi) could even create post-column bubbles.
I do possess a 75 psi back-pressure regulator that I could install somewhere in the system, such as after the detectors.
I have performed some troubleshooting tests to look at the check valves and pump seals and I haven’t found any problems. The best guess that we have at the current time is that the He sparging gas is building up in the mobile phase reservoir and then bubbles are entering the solvent inlet line. This hypothesis is supported by the observation of gas bubbles in the line between the reservoir and the pump.
How tight does the cap of a He-sparged reservoir need to be? I used to make the cap fairly tight and even wrap the cap with parafilm, but now I leave it loosely fitting. However I am still concerned that there is a sufficiently tight seal that an overpressure builds up in the reservoir over the course of hours.
While I am at it I am going to make a long post even longer! Do you feel that He sparging would offer any pH protection to aqueous mobile phases? It occured to me that the He sparging would decrease the amount of dissolved CO2.
Thank you for your consideration, and thanks in advance for any useful input. I know there are several questions in here; I am open to your input on any or all of them.
David
I am rethinking the He-sparging of the aqueous mobile phases in my experiments because I have been having some experimental problems in the last few weeks. I have a Waters Delta 600 pump and I sparge the aqueous mobile phase (ionic strength 0.01M typically) with He at 15 mL/min as recommended in the HPLC manual.
The problem is that the pressure becomes unstable during the middle of experiments. Our experiments proceed just fine for several hours and then the pressure just becomes very unstable and the flow drops off. At the start of experiments I try to make sure that the pressure is stable with all of the tricks that I have learned over the years. In particular, when putting a new mobile phase in the active reservoir (we pump 100% B) I by-pass the pump and draw out an excess of mobile phase to be sure that there are no bubbles in the lines from the reservoir to the pump. I also pump several mL of mobile phase through the pump (and out the column by-pass) to ensure there are no gas bubbles in the pump.
I pack my own columns with fairly large particle size (100-800 micron) quartz sand substrates (for my geochemistry research) and so the backpressures are not large. For example, when I use 20x4.6mm columns, the backpressure is about 150-300 psi.
I understand that sparging drives a large fraction of dissolved gases out of the mobile phase and I furthermore understand that when He is used as the sparging gas it has such a low solubility that it doesn’t stay around in any significant residual concentration. It is my understanding that dissolved gases in the mobile phase are undesirable because of the pressure drop after the column. In particular, I understand that when the pressure drops after the column bubbles can come out of the mobile phase at that point which can create problems for the detectors. I have optical detectors; one dual wavelength absorbance detector and one fluorescence detector. My first question for the HPLC community is what kind of post-column pressure drops, if gas-saturated, i.e. non-sparged mobile phase liquids were being used, would generate bubbles? I wonder if the relatively low backpressure in my experiments (in the range 150-300 psi) could even create post-column bubbles.
I do possess a 75 psi back-pressure regulator that I could install somewhere in the system, such as after the detectors.
I have performed some troubleshooting tests to look at the check valves and pump seals and I haven’t found any problems. The best guess that we have at the current time is that the He sparging gas is building up in the mobile phase reservoir and then bubbles are entering the solvent inlet line. This hypothesis is supported by the observation of gas bubbles in the line between the reservoir and the pump.
How tight does the cap of a He-sparged reservoir need to be? I used to make the cap fairly tight and even wrap the cap with parafilm, but now I leave it loosely fitting. However I am still concerned that there is a sufficiently tight seal that an overpressure builds up in the reservoir over the course of hours.
While I am at it I am going to make a long post even longer! Do you feel that He sparging would offer any pH protection to aqueous mobile phases? It occured to me that the He sparging would decrease the amount of dissolved CO2.
Thank you for your consideration, and thanks in advance for any useful input. I know there are several questions in here; I am open to your input on any or all of them.
David