Chromatography Forum

Hi everyone,

I am a university student looking at the nitrogen fixation rates of soil bacteria, using the acetylene-ethylene reduction assay. I inject acetylene gas into the headspace of gas vials containing bacterial culture. I take samples once a day and measure them in a GC.

The issue is I am not seeing any change in ethylene levels which are very minimal. The acetylene levels seem to be decreasing significantly but this may just be because I am removing some acetylene by taking samples. I am certain that the bacteria are growing and fixing nitrogen as I am growing them in nitrogen free media and have measured growth rate in separate tubes at the same time. It takes about 48 hours for the bacteria to grow exponentially, and reach an absorbance of about 0.100. I have tested for ethylene detection in the GC using a sample of ethylene gas. I do not understand why there seems to be no change in ethylene levels.

I am inexperienced in using GCs and would appreciate any help or suggestions. Thank you!

You know where ethylene elutes on your column by shooting various dilutions of ethylene (in air, say) and seeing the peak you think is ethylene respond accordingly? Also, you are using an FID which has enough sensitivity for the levels of ethylene you are trying to see? You are shooting a large enough volume for the detector to be able to see the concentration you are trying to see?

Tell us more about your GC and conditions in order to get more useful advice.

Best regards,

AICMM

You know where ethylene elutes on your column by shooting various dilutions of ethylene (in air, say) and seeing the peak you think is ethylene respond accordingly? Also, you are using an FID which has enough sensitivity for the levels of ethylene you are trying to see? You are shooting a large enough volume for the detector to be able to see the concentration you are trying to see?

Tell us more about your GC and conditions in order to get more useful advice.

Best regards,

AICMM

Yes, I have injected pure ethylene so I know where the peak appears.
I have tried varying the volume from 1uL to 100uL and no change occured.

I use a gastight syringe to take 10uL aliquots from a 125mL containing 25mL of inoculated media. The vial contains 50% acetylene, although at first I tried 10-20%. I have also tried changing the aliquot volume from 1uL to 100uL.

My chromatographic conditions are:
Temp = 250C
Pressure = 25.63 psi
Total flow rate = 36mL/min
H2 flow = 40
Air flow = 450
Make up flow (N2) = 50
Back signal (FID) = 50Hz/.004 min

Let's stay with this one. In the future, there's no need to post the same thing in 2 different boards. Lots of us participate in multiple discussions.

What about the location (retention time) of the ethylene compared to the acetylene?

Two critical things that you did not share are 1) what is the carrier gas? and 2) what is your chromatographic column and what are its dimensions (length, diameter, and film thickness)? Perhaps your column is a stainless-steel tube? This is called a packed column and they generally require much larger flows.

26 psi is a very large inlet pressure (very high flow) unless you're using a packed column. Even for a 60 m x 0.53 mm i.d. column (considered quite long and wide by most standards), the head pressure should only be 5-8 psi (gets you a linear velocity of 25-40 cm/s - optimum flow for helium carrier gas). You can go faster if hydrogen is your carrier. If you're using a packed column, then none of that matters. Acetylene and ethylene are only different by 2 hydrogen atoms and a little bit of pi-electron density. If you have the wrong stationary phase, you're likely going to have a hard time separating them from each other.

I found this on the restek.com website. It's the only app note I could find that was able to show a separation of ethylene from acetylene using a thin-filmed column. But, you can see that the column is extremely long. You might have to paste the link into your browser. I'm not sure it will hypertext when I post this:

http://www.restek.com/chromatogram/view ... %7C74-85-1

All of the other app notes on the restek site that involve separation of acetylene and ethylene are porous polymer columns. It's not a trivial separation.

I wish I had some acetylene and ethylene, I'd fool around with it a little. I looked up this "Acetylene Reduction Assay" but in all of the procedures I found, I didn't see where anyone called out the column used to perform the separation.

A while back, someone else was having trouble with this analysis. I'm not sure if they ever resolved the problem but a good thing to do when you have questions like this is to search keywords in the search box for this forum. Chances are, there's been a long discussion on the problem already. I just searched "ethylene acetylene" and Sinkdo was having the same problem as you:

viewtopic.php?f=2&t=25621&p=121906&hili ... ne#p121906

butters,

I agree with rb6banjo about difficulty in separation. I am also disturbed by your comment about no change when running 1 uL to 100 uL. An FID should pick up the difference in these two concentrations (is one peak much wider than the other?) If it were me, I would mix about 10 uL of each (acetylene and ethylene) in a blank vial and see if you can discriminate.

You don't tell us if you are running packed (assume no) and you don't tell us if you are running splitless or split. Might be pertinent since you are saturated with low and high volume injections.

Essentially, forget assay's for a moment and just work with standards put in clean vials to see if you can separate and detect.

Best regards,

AICMM

Without knowing what column is used, the temp mentioned by the OP of 250C seems rather high for ethylene retention. My minimal experience is with a packed column; we use a column temp of 80C to separate these gases for our acetylene reduction work.