Chromatography Forum

We have GC Agilent7890A with ZB-WAX column (30m x0.32mm x0.25um) and FID detector. I want to determine VFA in water media. I have tried some methods but not working. Any expert suggestions will be appreciated.
Thank you for cooperation.
Saiful, PhD student

Hi Saiful

Welcome to the forum.

The column can separate the acids you are interested in. That is all anyone can tell you unless you give a lot more detail about what you are trying to do, and what methods you have tried already. What concentrations are the acids in the water, what else is in there ? Have you run standards ? What instrument conditions are you using ?

The more you tell us the more we can help. The more you don't tell us the more we can't help.

It is also useful to know your level of experience with GC.

Peter

Thank you Peter
Now I am using standards of above mentioned acids in water (0.01 mM, 0.1 mM, 1 mM, 10 mM and 50 mM). One of the methods I have tried is injector temp. 220 C, Column temp. 80 C for 2 min then increased to 130 C (10 C/min) stay at 130 C for 2 min and then increased to 220 C (10 C/min) stay at 220 C for 5 min. Total run time was 22 min and FID detector temp. was 250 C. Our GC model is Agilent 7890A.

If any other information please ask me.
Regards.
Saiful

Hi Saiful

Please post all the details of the method: injection volume, split ratio, flow rates etc etc.

What exactly is the problem?, no peaks, poor separation, too many peaks, poor linearity, noise and baseline drift, repeatabilty ?. "Not working" can mean almost anything.

Peter

Sum of my methods:
GC Agilent7890A with ZB-WAX column (30m x0.32mm x0.25um) and FID.
Injector temp. 220 C, Column temp. 80 C for 2 min then increased to 130 C (10 C/min) stay at 130 C for 2 min and then increased to 220 C (10 C/min) stay at 220 C for 5 min. Total run time was 22 min and detector temp. was 250 C.
Injection volume: 1uL, Carrier Gas (N2): 1 mL/min.
Problems:
1. Many peaks observed but at 3, 9.5, 10.9, and 11.6 min I have found 4 big peaks almost all of the samples even when injected only solvent (water).
2. When concentration of an acid sample (Acetic Acid) increased the peak area increased but not the expected fold.
3. Confusion increased with no of sample increased.
4. Other things like baseline, separation, noise was OK.

Are you injecting split or splitless ? If split, what is the split ratio, if splitless what is the splitless time ?

Peter

My sample injection was splitless.

My sample injection was splitless.

Myself - I wouldn't even try splitless for stuff so volatile, no way will those VFA "condense" on the column head.

My sample injection was splitless.

Try with a split ratio of 20:1.

Peter

VFA from a direct injection is not easy. We run them headspace; you can do this manually using septa vials. Run the headspace at around 70-80C, gas phase split injection.

I have tried by 20:1 and 50:1 split ratio, but now two peak. One of them area increased with concentration but no relation with the increase of concentration. If any other possible way, I could try.
Thank you everybody for your cooperation.

Are you doing manual injections, or using and autosampler ?

Do you now see only two peaks instead of the "confusion" that you used to see ?

What is the source of the water that you use ?

What kind of inlet liner do you have in the inlet, and does it have any packing in it, glass wool for instance ?

Try with acids dissolved in a polar volatile solvent like dichloromethane, what do you see ?

Peter

Hi Peter
Thank you for your responses.
Autosampler I am using and this time I am using standard and diluting with DD water.
One good thing is now I have seen three peaks of Acetic, Propionic and Butyric acid and one solvent peak I think. But the peak area were not increased 5 fold when I have injected 5 fold concentrated standard. Inlet liner is glass wool.
I tried 20:1, 50:1, 100:1, 150:1 split ratio. With the split ratio increased the acetic acid in lower concentration disappeared, like 2 mM peak disappeared in 150:1 split.
Any suggestion. Thank you again for your cooperation.
Saiful

Hi Saiful

What solvent are you using ? - you mention only water, which the FID will not detect.

If the 150:1 split makes the peaks too small then use a smaller split ratio.

Peaks in the blank mean that you water is not pure.

It is difficult to get repeatable results when injecting water to a hot inlet - you will probably need to adjust the pre- and post-injection delays. Try reducing the injection volume also.

Peter

I make isobutyl esters using isobutyl chloroformate. That way I can use a db-5 or db-1 column. I am looking to see if I can replace the pyridine with triethylamine instead as the pyridine peak is close enough to the isobutyl acetate peak (acetic acid) to sometimes cause peak broadening.


Here is my FFA method it works on either db-5 or db-1

Samples can be dissolved in isobutanol if it is fat based or 0.1 N NaHCO3 if it is aqueous/spray dry.
Dilute down to the range of 500 to 25 ppm of each fatty acid.

I make a 1000 ppm 4-methyl-valeric acid in isobutanol ITSD solution
I make a fatty acids std at 1000 ppm also in isobutanol.

The reaction I do in an autosampler tube and cap. Cap when vortexing but loosen it up after to allow gas generated to escape.
25ul pyridine
75ul isobutanol (25ul ITSD 50ul of sample or isobutanol)
200 ul 0.1 N NaHCO3
Syringe 40 ul of isobutyl chloroformate
Immediately vortex vigorously for at least 10 sec (critical)
Wait a few minutes
Add 33ul of 7N NaOH
Syringe in 40 ul of isobutyl chloroformate
Vortex immediately
Add 400ul of hexanes and 200ul of saturated NaHCO3 and NaCl to neutralize the derivatizing reagent (It makes a big peak of carbonic acid diisobutyl ester)
Transfer the organic layer to a new tube.

I inject 2ul 10:1 split at 280 deg inlet with a typical split liner with wool.
Ramp
Start 40 deg for 5 minutes (enough time to get isobutyl acetate ahead and separated from the big pyridine peak only bit before.
16 to 325 5 minutes

I’ve gone all the way up to stearic. It also does linoleic and oleic. It doesn’t do the TCA cycle acids to many side products. I do Lactic, Fumaric, Succinic, Malic, and Citric using ethyl chloroformate along with the amino acids. I have a few experiments I am working on that method to reconcile that carnosic acid like the 0.1 N NaHCO3 method yet glutamic acid like having at least 0.1 N NaOH in the reaction mix or the yield goes down about 1/3-1/2 yet carnosic acid has almost no yield in NaOH above 0.05N.