Chromatography Forum

Hello,

Recently, I've tried to quantitate ethanol as well as some organic acids (mainly acetic acid and lactic acid) in aqueous fermentation medium. In order to do so, I've run some standard solutions. Here are method details:
FID conditions: hydrogen flow rate: 30 ml/min; air flow rate: 400 ml/min; makeup flow rate: 25 ml/min; temperature: 250 ⁰C
Column: Agilent DB-FFAP; 15mx530µmx1µm; carrier gas is helium at flow rate: 3 ml/min.
The oven temperature was programmed at 40 ⁰C for 5 minutes, raised to 230 ⁰C at 10 ⁰C/min and held at 230 ⁰C for 7 minutes.
Injection volume: 1 µl; injector temperature: 250 ⁰C; split ratio: 20:1

I've prepared aqueous solutions of ethanol and acetic acid. In all my chromatograms I can see a water peak at RT about 5.9 min (which is weird, I didn't know FID can detect water..).
Another strange thing is ethanol 50 ppm chromatogram:

And ethanol 100 ppm chromatogram:

So in the first one I can see a little peak of ethanol while in the second one there isn't one. Also I can see about 20 times bigger "water" peak in the second chromatogram. I've run the experiment several times always to this effect.
I'm a beginner with gas chromatography with a little experience, so I would appreciate your help/tips very much.

The flame does not respond to water. With aqueous injections, you worry about the water extinguishing the flame when it comes out.

Where does ethanol elute when you inject it neat? Just pump your syringe full of ethanol but only inject the needle (nothing in the barrel). Or, you can just use a gas-tight syringe and inject vapor out of the reagent bottle. Have you done that for acetic acid as well. Carboxylic acids are retained on this phase unlike anything you'd expect (based on molecular weight).

Hello

1ul of water has vapour volume much higher than liner volume. So you're overloading inlet (you will have poor RSD).
Also with water injections I'd use retention gap between inlet and analytical column.

Please paste chromatograms so we can see profile.

Regards

Tomasz Kubowicz

Thank you for the replies.
Here's a chromatogram of ethanol's standard solution with concentration 50 ppm. I guess it's ethanol around 4 min and water around 6 min

Here's a chromatogram of ethanol's standard solution with concentration 100 ppm - only "water peak.


And also a chromatogram of acetic acid's standard solution with concentration 100 ppm. Acid's peak aroun 14 min.

Welcome to the forum.

I cannot see the images - possibly an internet problem at my end.

Under the conditions that you are using you will definitely not see any signal for water - why do you think that the peak at 5.9 minutes is water and not ethanol ?

At 50 "ppm", (presuming that you mean ug/g per gram or ug/ml and not mole fraction) and a 20:1 split you are putting about 2.5 ng of ethanol onto the column. That will not give you a very big peak (not a really small one either, but ethanol has a quite low FID response). While you are getting things working try increasing the concentration by ten times.

You first need to find out where the ethanol peak elutes. Inject from the headpsace of pure ethanol in a septum-topped vial. You should see only one peak - that will be the ethanol.

Peter

If you have the option static headspace would work best for you.
Other wise As Mr Kubowicz said you are over loading your inlet. Inject no more than 0.5ul and cool the inlet to 100-105C.

Sorry to disagree with other posters - but with a 20:1 split the vapour volume of the water is a minor issue compared to not having a peak that you can assign to your analytes.

You must determine the retention times using pure headspace.

Peter

Thank you for your help. I'll try with headspace and will post my results soon.

ethanol and acetic acid work well by headspace. I do total evaportive with acetonitrile as an ITSD. Lactic I'm not so sure. I usually esterfy it. In a normal wax column that I use it elutes at temperature max and is hard to make out in the column bleed.

That must be ethanol, water does not show up. I've been running this test for awhile at 1ul injection as low as 5:1 split.
What sort of liner are you using? Need to have glass wool for evaporation and catch fines from your medium. I try to stick to water only, but have had to inject brine water occasionally.