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!

Please share with us in detail, your chromatographic (column, flow conditions, temperatures, detectors, etc.) AND sampling conditions (static headspace, gas-tight-syringe injection, etc.).

Are you able to separate ethylene from acetylene in a gas standard at comparable concentrations to what you expect in your bacterial broths?

Please share with us in detail, your chromatographic (column, flow conditions, temperatures, detectors, etc.) AND sampling conditions (static headspace, gas-tight-syringe injection, etc.).

Are you able to separate ethylene from acetylene in a gas standard at comparable concentrations to what you expect in your bacterial broths?

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

I have not tried injecting a mixture of acetylene and ethylene, but I can seperate acetylene from the gas vial samples and I have injected pure ethylene and got a clear peak.

butters,

What column are you using and at what oven profile? What type of injector are you using and under what conditions? How much ethylene are you expecting to see? At 10 uL if you are using split injection you may simply not be putting enough material on column to detect it.

BTW, 50 hz is awfully fast, do you really need that fast?

Best regards,

AICMM