Chromatography Forum

hello,

i am trying to detect volatile fatty acids (c2 - c7) from an aequous solution (buffered around pH7) via GC with an zebron zb-wax column and a FID as detector, but the results are not great - i have the following problem and and would like to have your help:

1. at the time, i am using c6 as an intern standard - but that is not that great because i believe, c6 is in my solution as well - what other standard may i use to get better results?

2. i lower the pH from 7 to pH3 to get the organic acids volatile, but even the calibration does not function right, if i apply a 1g/l solution, the result of the acetic acid is only about 0.5 g/l

3. i also don´t get clear peaks, sometimes 2 oder very large peaks

does anybody now an online tutorial, paper or can share his/her knowledge?

thanks in advance!

Especially with acetic acid, one has to have the solution at a very low pH to get good results for organic acids from water.

This is an old problem which has been beaten to death over the years.

The best solution is to use cation exchange chromatography column. See any good HPLC catalog.

You could try putting a good amount of formic acid into your solution if that is not possible. Formic does not detect well with a FID. It also elutes AFTER acetic acid on your column. It may interfer with quanitation of propanoic acid. HCl and H2SO4 tend to be troublesome.

best wishes,

Rod

What is your sample preparation ?, are you extracting the acids into a solvent, or injecting the aqueous solution directly ?

At what stage do you add the internal standard, and what is it coreccting for ?

Do you have an equilibriumm headspacer that you could use ?

What concentration are the acids ?

Peter

the first step for the sample preparation is to centrifuge and then to filter the supernatant with an 0,4µ sterile filter.

then 90µl are brought together with 10µl of the intern standard with h3po4 to buffer the solution from 2-3 -

1µl of this is injected into the gc.

the acids are around 0-2 g/l.

any help?

You are not likely to achieve full recovery of acetic acid under these conditions.

best wishes,

Rod

well - i don´t have a hplc-system here - just a gc. so how can i make it better?

You could try using SPE cation exchange and then desorb the organic acids for injection from the elution solution. Your problem lies in the solution contains a base (counter ion of the acids). Even though you acidify the solution to a low pH, when you evaporate the acidic solution your organic acids will (to a degree) not all convert to the free acid in vapor phase, some will remain as salt in the injector (reduction of recovery).

You could try H2SO4 instead of phosphoric acid (use a very large excess) and increase your injector temperature as high as possible (this will help some).

Sometimes no matter how big your screwdriver is, the nail just doesn't want to be driven straight in.

Using an aromatic hydrocarbon might be a better choice than hexane, naphthalene comes to mind or ethyl benzene.

Replacing your injection liner frequently will also help.

Good luck. I hope you can improve your recovery.

Rod

hi, Dear

this is dhruvil
i guess u r using Head space method to quantify yr acetic acid, isn't it ?
well u can never get acetic acid by head space.
i think u should go for a liquid injection in GC. i have several times done acetic acid quantification on GC only.

i made use of DB-WAX column (30 * 0.53 * 1 µm ) column. and do bring down the pH of your sample solution to around 3 by Trifluoro acetic acid.

i think this may help u solving your problem.
do inform me or do tell me the porblems you faced in this. ok so that i can guide you further. ok


ok then all the best .
cu soon
bye

regards,
dhruvil

Hi roador

When you say that your internal standard is "c6" do you mean hexane or hexanoic acid ? I hope the latter because hexane is not soluble in water.

Assuming that you have C6 acid as the internal standard - if you add 10 ul to 90 ul of sample you have 10 %, which is 100 times as much as the analytes. Ideally the concentration of standard and analyte need to be about the same.

Microlite volumes are not easy to dispense repeatedly - is there a reason why you use such small quantities ?. What is the concentration of the phosphoric acid and what volume of it do you use ?

You could consider coverting all the acids to esters - whaich are easy to analyse by GC.

Peter

hello dhruvil,

no i don´t use headspace - i already use liquid injection, with glasswool at well. as i said i have a wax column, - but to bring down my ph to 3 i use H3PO4 - why should trifluor acetic acid be better?

@peter: my internal standard is iso-caprone acid, not hexan.
and yes, you are alright, i did write it wrong - the concentration of my standard is about the same of the analytes.

convertion to esters is not possible.

the real problem now is, that c2 (acetic acid) just dissappears. i do not get comparable or reproductive results.

Dear Roador,

Your results have been seen before, if that is any comfort.

If you do seem to find a way of performing an analysis don't think your work is done.

Use another GC and another column to demonstrate ruggedness of the method. I think you will eventually come to the conclusion that GC is not the technique for this analytical problem.

The water plug will most likely diffuse any acetic acid plug you may be able to inject onto the capillary column, that is if you can get a good recovery from the salt to the free acid. A packed column would be a better choice, specifically, a 1% SP-1000 on Carbopack B in a fused silica coated metal column with on column injection.

I wish you success and hope you don't spend too much of your valuable time in finding a solution.

Rod

Dear Rod,

i really do get the point - as it seems, i don´t really have the right equipment for my analytical problem - but what else can i do? i tried to get hands on an hplc - no luck- and the wax column was just bought -

just trying to make the best out of it.

sorry - i don´t really understand what you mean by "the water plug will most likely diffuse any acetid acid plug?"

thnx

The large water plug will be on the surface with the acetic acid plug. But the broad water plug will inhibit the focusing of the acetic acid so whatever does enter the column will be diffused (flattened out) by the water on the column surface. If you injected acetic acid in hexane then this would not happen. If you injected acetic acid in acetone it would not be a problem to the same degree.

But in addition with water, it acts as a base compared to acetic acid and besides not getting a focused plug, you have the problems with the two states of ionization of the acetic acid (free acid and cation).

Can you see why the acetic acid 'disappears' ?

Remember: if it were easy it would have been done a long time ago.
I tried doing such things by all kinds of manipulations almost thirty years ago. Esters are better, but not always possible with water present.

Better also is ion chromatography. The method is developed and documented. Just buy the parts and do the analysis with an accurate answer. It is so much easier to do it the right way. Doing it the wrong way means you get the wrong answer at a cost of wasted time and effort. But I understand that bosses don't always understand that point.

I know you are trying to do the best with what you have. Been there and done that. I would wish to save you frustration and wasted time.

wishing you every success,

Rod

Hi Roador

Why do you say that conversion to esters is not possible ? Given that you work with small quantities of sample (90 ul) you could add that and the internal standard to say 1 ml of acid alcohol (use propanol to get longer retnetions), heat for a while, add some saturated salt solution and extract the esters into 1 ml of hexane, and inject.

Rod is right - you are never going to be able to do free acids reliably by injecting them in aqueous solution, although dhruvil's method might be worth a shot. Use the minimum amount of glass wool in the inlet.

Good luck Peter

hello everybody,

i know use glass wool and glass beads in the liner of the GC, and, as i have to say it is much better - i get nearly symmetrical peaks, not tailing or fronting - i know, the concentration is not the acutal one - but i hope i can live with it.


thanx for your help