GC-FID for free Volatile Fatty Acid quantification - issues

Discussions about GC and other "gas phase" separation techniques.

25 posts Page 2 of 2
Thank you for the update

"The only follow up I have for this is... The Restek tech person said this column is designed for pH 5 - 9. Would it be a problem to run my samples at pH 2 -3? "

Stabilwax-DA is a cross bonded phase, however, I would go with the experience and advice of the Restek person.

http://www.restek.com/chromatogram/view/GC_CH00284

Rather than getting tied down to only thinking about, and continuing to try to overcome, problems created by this analysis in terms of aqueous injections and pH -

If it continues to be a problem then perhaps you should consider an alternative approach of using DCM or MTBE liquid/liquid extraction of your aqueous samples (after pH adjustment) to overcome possible pH and aqueous injection problems ?

e.g.

Determination of volatile fatty acids in wastewater by ... - ResearchGate
https://www.researchgate.net/...acids/. ... +volatil...
30 Oct 2013 - fatty acids collected at the elutriation units of Unit 3, 4 and 5 at Johannesburg Water-Northern Works ... the ratio of propionic to acetic acid was good since the ratio o did not exceed 1.4with the exception of the samples collected on .... able to separate using the Stabilwax-DA column and then detected by the ...


Looking forward to your updates
Regards

Ralph
With a 50:1 split I do not anticipate there being enough water on the column to cause problems.
Peter Apps
to anahita.b:

Some comments:

1. VFA peak areas increased with increasing concentration FA via desorption process (pKa FA < pKa VFA). VFA a very "stiky".

2. Add Internal Standard (isobutyric or isopentanoic acid etc.)

3. Vinj (for H2O samples) = 0.2 - 0.5 ul (must be optimized)

4. Split = 1:30 - 1:60 (must be optimized)

5. Liner and wool (little) must be deactivated; better - Liner with Jennings Cup

6. Tinj = 250 - 280 C (must be optimized)

7. Restek StabilWax-DA have normal resistance to water injection (from my experience)

8. Take careful attention to cross-contamination (inject blank after STD injection to check). Syringe washing liquid must contain FA (2%v etc) or other acid with pKa < pKa VFA. If water solution don't help, try to use MeOH or IPA instead of H2O and use two step washing cycle (2%v FA in MeOH/IPA then 2%v FA in H2O etc.)

Best regards, Alex
Hi Everyone,
Quick update... I have optimized the conditions and found that 0.5 uL with 50:1 split injection with 2% formic acid was giving a very low signal, especially for 0.5mM concentration. So, I have decided to use 1 uL injection, 50:1 injection in 2% formic acid. Thanks for your feedback.

This information is really helpful, Alex. I mostly use the same conditions as you had described. I have been having issues with cross-contamination due to carry over of sample. I guess the wash isn't enough. I do two washes each with 85% methanol, 50% methanol and DI water. But I have not used 2% formic acid. I will try that next time. I would appreciate other suggestions as well.

Would you be able to suggest any other internal standard as my VFA mix contains formic, acetic, propionic, isobutyric, butyric, isovaleric, valeric, isocaproic, caproic and heptanoic acids. It is a commercially available mix.

I have tried (1) crotonic acid (2) 4-methylvaleric acid (3) 2-ethylbutyric acid. The first two were coleluting and cannot be used. The 2-ethylbutyric acid is separated "better" but still bit of overlap with valeric acid.

I use a deactivated liner. Can you explain what is "Liner with Jennings Cup"?

Thanks a lot again. This is helping me so much!!
anahita.b wrote:
So, I have decided to use 1 uL injection, 50:1 injection in 2% formic acid. Thanks for your feedback.
If you'll have bad RSD of peak area go to 0.4-0.5ul and split 1:30.

anahita.b wrote:
do two washes each with 85% methanol, 50% methanol and DI water. But I have not used 2% formic acid. I will try that next time. I would appreciate other suggestions as well.
2%v FA in MeOH (IPA) - 3-5 times then 2%v FA in H2O - 2-3 times.

anahita.b wrote:
The 2-ethylbutyric acid is separated "better" but still bit of overlap with valeric acid.
Try to optimize GC oven programm (decrease gradient rate or add/prolongate isotherm part etc.).

anahita.b wrote:
I use a deactivated liner. Can you explain what is "Liner with Jennings Cup"?
http://www.gcferrules.com/ecommerce/product/344/
Thank you for the info!

Update:
I tried 0.5 uL with 30:1 split but the signal is still not great for low concentration such as 0.5 uL. So, I may have to stick with 1 uL with 50:1 split.

I have also decided to use isocaproic acid as my internal standard since it is not found in my samples. However, it is present in my standards and so, I cannot have it there. I can still quantify the accuracy of my standards in other ways (through duplicate runs or check standards).

Question:
The fresh problem I am having is with the needle wash. I tried MeOH, 85% MeOH, 50% MEOH, Acetone, 50% Acetone, DI water, EtOH. All of them still leave residue after 5 - 10 washes with these solvents. Among these, EtOH wash works best. Would you guys have any other suggestions? I read somewhere that a gas-tight syringe (even though these are liquid samples), can help with this. Is that true?
It was also suggested to me that wax columns in general cannot take aqueous samples very well. That's certainly been our experience. Then we came across the note:

https://www.agilent.com/cs/library/eseminars/Public/water%20injections.pdf

We use DB-Wax, and we didn't believe the results presented (Chromatography looks fine after 2000 injections of water). So we tested with one of our older DB-Wax columns. Sure enough, after a week of continuous 100% water injections, we ran one of our FAME test mixtures, and the chromatrography was as good as it was prior to the water injections.

So it's not the water alone (at least with DB-Wax), but the nature of the aqueous sample. If we do a split injection of our lower layer from our FAME heptane/aqueous work-up, it results in immediate column failure.

Just thought this was worth sharing.
anahita.b wrote:
The fresh problem I am having is with the needle wash. I tried MeOH, 85% MeOH, 50% MEOH, Acetone, 50% Acetone, DI water, EtOH. All of them still leave residue after 5 - 10 washes with these solvents.
Are you added formic acid (or other acid) in wash liquid?

anahita.b wrote:
I read somewhere that a gas-tight syringe (even though these are liquid samples), can help with this. Is that true?
Yes, it can help a little.
Hi everyone,

UPDATE:
(1) As suggested, I retried 0.5uL with different splits and 10:1 seems to work well. I continue to have a lot of issues with carryover. I use a glass wool liner. I switched that out to a new one and did a run. That really helped reduce the carryover issue (although it is still there). So, I conclude it is the liner and not the syringe. But it seems tedious to have to replace the liner so often (maybe everyday!). Do you guys have any thoughts or experiences with liners for aqueous samples?

(2) On the pH front, I guess the bottom line is that pH effects the area. So, I am going to try to get all standards and samples within the same pH range for homogeneity.

Jake, thank you for the interesting ideas. For my clarification, do you mean that using a pure aqueous sample worked better than a mix of water/solvent?

Sav, for the wash liquid, I add use 2% formic acid in DI water which is my last wash. The other solvents do not have any acids.

Looking forward to more advice! As always, thanks everyone!
Hi again everyone,

I am very close to completing my method development for VFAs on GC-FID. I have the following parameters:
(1) Inj temp: 250 deg C
(2) Column temp: 70 - 200 deg C
(3) Time of run: 15 min
(4) Column: StabilWax DA
(5) FID temp: 275 deg C
(6) Inj volume: 0.5 uL with 10:1 split

I maintain the pH of standards and samples between pH 1.6 - 1.8 using 2% formic acid.

The last (and big) issue I am having is with carry over. This is particularly bad with higher concentrations and is prevalent across all VFAs (acetate to heptanoate).
The solutions I have tried is:
(1) Increasing the injection temperature to 250 deg C... This made a big impact. The carry over issue is better but not completely gone.
(2) Running the column "empty" with no sample... this showed there was no residue in the column
(3) Cleaning the injection port, septum and liner... helped a little but not too much
(4) Tried a variety of wash liquids... It is definitely not the needle.

I currently put 2 -3 blanks in between each run which makes it a little better.
This is increasing the run time to 1 hr! I would really appreciate any other advice. Thank you, as always!
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