Gas bubbles out detector when inject real sample only??

[seravitae]

Hi guys,

I inherited a Varian ProStar binary setup (PDA, Prep heads, vacuum degasser) and I seem to be having some trouble with analytical injections.

Currently running a waters C18 4.3mm analytical column with a SecurityGuard cartridge, running MeOH/H2O mixes.

The system will run fine if left to pump by itself, blank MeOH injections into either pure MeOH or anywhere down to 10:90 MeOH:H2O is fine, a very small peak due to solvent composition change or small impurity, but this is okay for our application.

The problem is that when I take my analyte, dissove it in either 100% meoh or 50:50 MeOH:H2O, and degass the solvent (sonicator), injections into the system cause lots of gas bubbles to come out of the detector.

I thought maybe the rheodyne was leaking but i would have seen this if my blank injections were gassing out. I carefully prepare my syringes (gas-tight, fill past injection volume, vertically purge out headspace) but still I only get gassing out when I inject a real sample.

Chromatogram is a single signal at about 5 minutes, a very broad one, with high frequency noise about 50% of the vertical intensity of the main peak height. For those of you familiar with NMR it looks like a septet essentially. Corresponding to the high frequency transients are loads of gas bubbles out of the PDA line, much much more than I could have possibly injected into the rheodyne. Tried purging/washing out the injector loop, didn't help.

Not sure where to go from here - anyone have any idea why my sample is gassing out?

edit: If it helps, C18 TLC shows three resolved spots, of which the sample is a "click" product - aliphatic alkyne and azide --> 1,2,3-triazole. I suspect 2 spots are starting material and 3rd one is product.

Regards,
Seb

[HW Mueller]

You don´t tell us what volume you are injecting, but you tell us that you can´t possibly inject enough to explain the amount of emerging bubbles. After that you talk about your sample gassing out. Idon´t see how that goes together. Did you ever inject air at your usual volume?

[seravitae]

Hi HW,

Injection volume is 20uL of 1mg/1mL solution.

I dont understand what you mean did I inject air at my usual volume, unless you are being condescending.

Regards,
Seb

[HW Mueller]

If one has an air problem of your type it seems obvious, not condescending, that one would inject air (in your case 20µL) to check what the maximum in injected air would do.

[seravitae]

Ok sorry english not fantastic here, no hard feelings. I will do an air injection. However I changed from MeOH to IPA on suggestion of someone here and there is massive signal being produced even after 1 hour (obviously IPA will elute analyte quicker), I think the column is extremely dirty, possibly even totally covered with analyte so I am going to flush it until i get a baseline and then start trying again. Possibly even step up to more nonpolar solvent.

Also I am suspecting the rheodyne may be faulty or need rebuilt because I just got sprayed with solvent out of the needle port when switching from inject (loop inline to column) back to load (loop to needle port and waste). It does not happen when i switch from load to inject...

Dirty columns and unhealthy rheodyne, really thrown in the deep end with this gear for a newbie!

Cheers,
Seb

[HW Mueller]

OK, I suspect that you do something slightly different when you inject sample as compared to blank. One can imagine that if something is wrong with the Rheodyne that it could easily lead to drawing in air in some situations.
A spitting valve may not be due to a damaged rotor, it could just be a wrong tension on the rotor/stator. Drawing a lot of air is sometimes caused by syphoning out of the loop via wrong waste lines. It is best to have just short waste tubes attached to the valve, going straight out, with the exit holes being at about the same level.