Chromatography Forum

Hi,

I am using LC Packings Ultimate-Switchos-Famos coupled to Applied Biosystems QStar Mass Spec. I believe it is a typical set-up for most people doing shotgun proteomics.

There is a PepMap 100 C18 trap column prior to the capillary column with the same material as the trap. The trap column has just recently replaced (3 days ago) together with the new capillary column. So as the rotor inside the Valco valve.

But I had encountered this problem, when the Switchos valve switched from 1-2 to 10-1, the capillary column's pressure dropped from 180 bar to 55 bar. A typical run should see the pressure drop of not more than 10 bar and recover within minutes. But the pressure actually never recovers unless you swing the valve back to 1-2 position.

We tried to change the valve, the trap, the connection lines, but it never solved the problem. The best explanation is a leak, but we couldn't see any sign of liquid around the valve. Could it be air bubble trapped inside the valve? We purged the system thoroughly and increased the flow for overnight, but still haven't solved this problem.

Wondering anyone come across this problem?

Thanks for viewing,

Gan

Hi Gan

This is just a guess because I do not know the system that you are using - you probably have a leak between two ports of the valve, the liquid from your flow stream is getting into another fluid path without leaking out of the valve.

Unfortunately the most common cause for port-port leaks is a scractched sealing surface, which can be expensive to fix.

Peter

Thanks Peter,

I changed the rotor and the valve, inspected for scrathes, but still couldn't fix it.

Gan

Hi Gan,

I am a nanoLC specialist for Dionex in the UK, the company LC Packings is owned by.

I have seen this problem previously, and is almost exclusively caused by the installation of the trap column. You state that you changed the precolumn 3 days ago, did you see the problem occur when you replaced the precolumn? Or did you replace the trap because you saw the problem? I am guessing it is the first.

It could be the tubing, or the fittings involved. Are you using the newer design tubing with PEEK fittings (as opposed to the older ones with pre-installed SS fittings)? It may be that the PEEK fittings are just not quite tight enough against the precolumn. When you switch the valve to 10_1, it is very difficult sometimes to see a leak of a a few 10's of nanolitres a minute, which would give you pressure drops as you've seen.

If you have the new type precolumn tubing, check the installation of the precolumn. To confirm correct procedure: The 15cm 30um tubing has PEEK fittings at each end, one is a single-piece fingertight, the other a 2-piece nut and ferrule. The single-piece fitting should go into the valve, the 2-piece into the precolumn holder. If you are using the older 10cm x 30um precolumn tubing with the pre-installed SS fittings, I strongly recommend getting the new type. It offers a much better, more accurate fitting.

Slide the ferrule from the 2-piece up the tubing a couple of millimetres, this ensures that it is completely butted up against the precolumn when you tighten it.

Then install the end of the first tubing assy into the valve, next to the port from the loading pump. Run the pump at 25-30ul/min, check the pressure is OK. (Should be approx 25-30 bar). Stop the pump, if it is running when you connect the precoluyn cartridge, it could introduce a dead volume. Connect the other end of that tubing into the precolumn holder, insert the precolumn, and then the second piece of tubing. When this tubing is new, it needs to be quite tight, to ensure that the ferrules seal properly against the precolumn. Turn the pump on again, check pressure again, to ensure you have no blockage or restriction in the tubing. If OK, connect the other end to the valve and try it.

Dead volume is critical here, even if the tubing is only a fraction of a mm out it can equate to minutes of delay at 300nl a minute! So also re-check your analytical column installation. You don't mention that it takes longer to get to operating pressure when you start the pump, if it does then the column installation could also be a problem. With the symptom you describe, I don't think this is the problem though.

Even if you have used new precolumn tubing, it could be worth trying another set, maybe there is a bad fitting or something? (Although I haven't had any problems like that before). Also try another precolumn cartridge for the same reason.

Feel free to get in touch if you continue to have problems.

Thanks Vanquish,

Yes, problem occured when we replaced the trap column. We had always suspected the dead volume in the trap, but no matter how we assemblied the trap column connection, they just refused to behave.

I am not sure the tubing that we are using is with newer design. The catalog no. for the tubing is P/N 160182. I will order another set of tubing. For the time being, we managed to control the pressure drop by changing the trap column configuration to an up-right position rather than the horizontal position.

Thanks again for your valuable comment.

Gan