Chromatography Forum

Dear all,

I have a special problem regarding GC method which focuses on separation rather than detection.

Our lab purchased a solution of 14C-labelled TCE dissolved in methanol. My microcosm experiments require addition of pure TCE without methanol. I am trying to separate the TCE from the methanol by injection onto a packed column (1% SP 1000 on 60/80 Carbopack B). The GC is set up so that the column effluent exists the GC and terminates in a needle. 1 uL of the TCE/methanol mixture was injected into the GC. Methanol will come out of the column first, followed by TCE. During the time when TCE elutes, I insert the needle into the serum bottle to collect the outcome gas.

This method was selected because some one in my lab has done a successful separation of Vinyl Chloride in Toluene solution with the same column. However, in my case the methanol peak is so large (and thus has a long tail) that even after the elution time of TCE there is still methanol coming out of the column. The current column temperature program I tried is staying at 140C for 8min (when TCE comes out), then raising the temperature rapidly to 230C and stay there for several minutes. I am ready to try lower temperatures, but the observation that methanol would come out of the column even after the elution time of TCE really upsets me.

I have never done such separation work with packed column before and I am not quite familiar with the specification of different GC columns. So any suggestion about temperature program, column selection, or even physical separation method of TCE-methanol would be highly welcome.

PS: I heard that capillary column may achieve better separation. After reading some references about capillary column, I found two major concerns:
1) A feature of capillary column is its split injection. However, we cannot afford lose a large percentage of the 14C-labelled TCE (it's quite expensive), so the split ratio cannot be too big.
2) If we go with low split ratio, or even splitless inlets, the 1 uL methanol might overload the column. Does anyone here knows the highest amount of methanol that can go into a capillary column at one time?

Thanks in advance!
Rong

The simplest solution is simply to inject a smaller sample into your present column at the present flow rate.

You are flooding the column, either through backflashing the sample in the injector, or just overwhelming the packing of the column.

A reduction in size of the sample, or running the column at a cooler injector and oven temp is advisable.

For a HUGE separation try using a porous polymer column to separate the two analytes. Almost any will do, N Q, P, S, or A polymer should be fine.

An increase in flow rate should also reduce the tailing as long as you are not backflashing the sample in the injector.

best wishes,

Rod

Hi Rod,

Thank you so much for you suggestion!

I didn't know that there is a potential risk of backflashing the sample into injector. And after some reading on this issue, I think it's highly possible the cause of my problem.

My inlet temperature (200 C) is much higher than the boiling point of methanol (65 C), and the volume of vapor produced by 1 ul methanol would be up to 400 uL, almost comparable with my flow rate (~450uL/s).

Another observation which might serve as an evidence was a methanol peak in air injection. Following the addition of neat methanol, I ran a no-injection blank and got a flat baseline, but an air injection after that would show a peak at methanol retention time. I think it indicates that the column is clean but the injector has residual methanol (but I am not sure).

Your suggestion of reducing the inlet temperature sounds a good idea, and I will start from that. Based on my current flow rate, do I need to go down to a really low level (e.g. less than 100C), or a medium temperature (between 100-150C) may be enough?

Also, I am really thankful to know that there are many other types of columns which can do a better job at such separation. It's great to have some alternatives! Will definitely look into them.

With my best regards!
Rong

Methanol will tail quite a bit on that column, if I recall the early days with that stuff. I think Rod may have invented that stuff , so he is the man here.

I think I still have some of that packing under my fingernails.

That material can be packed poorly which causes tailing, and it can be coated poorly with the same result.

Tailing can happen if the column is depressurized quickly with the packing being disturbed, or if the column is dropped.

Still, if the injection volume is reduced to a small volume (less than 0.25 microliters for example) and there is still excessive tailing, then the column is installed poorly, or the column is damaged internally.

The 1 microliter sample should be injected SLOWLY over 5 seconds, not in an instant or 'burst' manner.

I would use a P polymer or Chromosorb 101 column and start at 80C and program at 10C/min to 200C with a 40mL/min flow rate. I would also recommend an inlet packing drop of 3-6 inches from the end.

Use a final hold as needed.

Good luck,

Rod