GC-MS Volatile Fatty acids

[Peter Apps]

Hi,

Now i am really confuse. Yes i am using a Hamilton gastight syringe. About the sample prepration:
I have made a standard solution using the above mentioned acids. Then i put 5 ml of the solution in 10ml vials.what kind of closures (caps) do you have on the vials ? Then i put all these vials in a waterbath with a temperature of 85°C (This is the temperature in the headspace of the vial). when the required temperature is achieved i wait for 15 minutes and after that take out the sample using syringe. With no autosampler this is the simplest way in my opinion. The sample equilibration is OK, but let me repeat - you will not get good results trying to do headspace with a syringe that is cooler than the sample. There is no way around this problem with the setup that you have.
I have normally heared/read a sample volume of 0.5-10 uL is sufficient for GC-MS analysis.This is for liquid samples, you are injecting headspace which is a gas I have tried using higher volumes but the chromatogram becomes worse In what way does it become worse ?, wider peaks, more variable peak sizes ? What higher volumes did you use, and how quickly did you inject them ?.

Zia

[Peter Apps]

Hi Zia

I am still not clear on why you want to use headspace analysis. Have you tried liquid injections ?

Peter

[ziarehmann]

So so so,

1. Why Headspace? I want to measure the concentration of fatty acids in the biogas digester using maize, swine manure and different organics. The sample ist a semi solid with lots of fibers present in it. Taking a liquid sample from that would be difficult. Thats why Headspace. Now as it seems that headspace would be best suited for automated analysis. I have one more basic question. As i say i have very fibrous sample, lets say i centrifuge my sample and seperate liquid phase and inject that liquid (0.1-2uL) in GC. Will the concentration of acids be same as in case of my original sample??

2. Caps: Crimm caps with 3mm septa

3. Yes with the needle temperature i cant do a lot. But how big would be the influence of that?

4. About the volume today i am using a volume of 100uL. The peaks are fine. But they are like the pic what i have posted yesterday. So the is that really possible?? Than i should use 2uL instead of 100uL. The time between sampling and injecting needle in the inlet is max 20 sec.

[MSCHemist]

If you are going to centrifuge you should spike first with an internal standard. Some not naturally occuring carboxylic acid. That way the internal standard should correct for the fact you are probably not taking a representative sample for GC.

Another reason for headspace is your matrix is probably water based. Injecting water is bad for the GC. It has a huge expansion coefficient when injected so don't inject more than 0.5ul and it carries all sorts of nonvolatiles like salts, fibers/carbohydrates, proteins etc that just sit there and mess up your inlet and could wreck your column.

Some suggestions. I beleive a sample prep for ffa would be to acidify the sample, saturate with salt (salt out) and extract with ethyl acetate or diethyl ether.

Headspace SPME is also a possibility. It is a head space technique but becuase you are binding the analytes to a fiber you don't need to worry about it condensing in a cold needle. You will need a good internal standard to help account for variations in SPME. When I do SPME I typically add 1g of material, 1g NaCl, 3ml of water and a stir bar in a 22ml headspace vial. I then warm it to the optimal temp for FFA probably 85deg C or watever as FFA aren't as prone to degradation as some other analytes [for general analysis I do 50 deg C] and wait 15 minutes. Then I expose the Fiber to the headspace for 30 min then put the fiber in the GC inlet [Restek 2093 liner 260 deg 2:1 split] for 1minute to desorb analytes into the GC.

[MSCHemist]

I've been considering getting into FFA as well as they are relevant flavors. I currently have sort of a flavoromics technique where I do the TCA acids and amino acids together on a 1701 column by derivatizing with ethyl chloroformate.

I do lactic, citric, fumaric, succinic, and malic acids as well as every amino acid except arginine. It also does the free fatty acids though some of the smaller ones are covered up by the solvent peak and I can't tell acetic acid [which is turned into ethyl acetate] from natural ethyl acetate].

[James_Ball]

Have you tried using a 1ml injection with a 100:1 split ratio?

This would give you as much on column as a 10ul splitless injection but should also give much sharper peaks. Keeping the injection port temperature near 200C would also allow quick evaporation and removal of any condensed water in the syringe.

[Peter Apps]

So so so,

1. Why Headspace? I want to measure the concentration of fatty acids in the biogas digester using maize, swine manure and different organics. even if the analysis of the acids was working well this would be a very challenging project because there are a lot of other volatile organics that come off such fermentations, and you must separate your acids from those organics as well as from one another. Also, with a lot of supsended and dissolved solids you must do your headspace calibration in a matrix that matches the samples, or use method of known additionsThe sample ist a semi solid with lots of fibers present in it. Taking a liquid sample from that would be difficult. Thats why Headspace. Now as it seems that headspace would be best suited for automated analysis. I have one more basic question. As i say i have very fibrous sample, lets say i centrifuge my sample and seperate liquid phase and inject that liquid (0.1-2uL) in GC. Will the concentration of acids be same as in case of my original sample?? Probably not, but the errors are very likely to be smaller than trying to do manual headspace

2. Caps: Crimm caps with 3mm septa

3. Yes with the needle temperature i cant do a lot. But how big would be the influence of that? At a guess your repeatability will be worse than 30%, and your LOD will be at least ten times higher than if you had a heated syringe or an automated system

4. About the volume today i am using a volume of 100uL. The peaks are fine so what IS the problem ?. But they are like the pic what i have posted yesterday so in other words increasing the injection volume does not give poorer performance as you previously stated. Are the 100 ul peaks bigger than the 2 ul peaks ?, how much bigger ?. So the is that really possible?? Than i should use 2uL instead of 100uL NO - why do you insist on using a volume that is so small ?. The time between sampling and injecting needle in the inlet is max 20 sec.but the syringe cannot be at 85 C, or you would not be able to hold it

[ziarehmann]

Hi,

I have the possibility of centrifuging the sample and then using that for the analysis. But as MSCHemist said it could cause problems or damage the capilary.
You are right peter yesterday i did analysis by taking the same sample amount from 6 parallel vials and the difference in peak areas was around 40%. Frankly saying i dont have lots of options. I must optimize the whole thing with the means i have. If i can reduce the repeatability to 10% thats also for the moment ok with me. Liquid injection is also an option but if it damages my column then thats also not a good one.

Any ideas where i can improvise?

[Peter Apps]

Reviewing so far from memory, the suggestions that have been made are:

acidify and do headspace

acidify and extract into ether, then inject the ether

SPME

increase the headspace injection volume

decrease the sample temperature so that the syringe is not colder than the sample (combined with acidify and increase volume, this might still give the peak sizes you need)

derivatize the acids

Before we come up with more ideas, have you tried any of these besides increasing the injection volume ? (which you variously say does not work, or works as well as a small volume) ?.

Peter

[MSCHemist]

I am working on a chloroformate method. the main issue are acetic and propionic as they are very volatile, tough to get out of water, and with ECF ethyl acetate and propyl acetate will probably get covered up by reaction mix componenets (pyridine) and solvent. I be sure to share if I am successful.

[ziarehmann]

Hi,

Increasing the volume i have checked, in that case for the first three minutes i get lots of trash and then all my peaks. I simply subtract the first three minutes and the results are looking good apart from reproducability.
Today i am goona check the effect of acid addition. I will be acidifying using 34% Phosphoric acid (As suggested by some literature).
Tomorrow addition of salt and phosphoric acid.

I will share my results when i have them.

Regards!

[Peter Apps]

Hi,

Increasing the volume i have checked, in that case for the first three minutes i get lots of trash and then all my peaks. I simply subtract the first three minutes and the results are looking good apart from reproducability.
Today i am goona check the effect of acid addition. I will be acidifying using 34% Phosphoric acid (As suggested by some literature).
Tomorrow addition of salt and phosphoric acid.

I will share my results when i have them.

Regards!

Hi Zia

Good plan. You will almost certainly find that the peaks get bigger with the addition of acid, and they may get bigger with the salt. You may then be able to reduce the sample temperature, which will help with the repeatability by reducing the temperature difference between syringe and sample.

Peter

[ziarehmann]

Hi Again,

Now i have a basic question. Should i add the acid with open vial i.e. first i add acid and the clamp the cap or should i add acid with closed vial?
In an article i have read the addition of acid is performed with open vial so that CO2 can escape in atmosphere where as the addition of salt with closed vial.
What's your opinion?

[Peter Apps]

Hi Again,

Now i have a basic question. Should i add the acid with open vial i.e. first i add acid and the clamp the cap or should i add acid with closed vial?
In an article i have read the addition of acid is performed with open vial so that CO2 can escape in atmosphere where as the addition of salt with closed vial.
What's your opinion?

Two things to bear in mind. Unless there is a lot of carbonate or similar in your sample, adding acid should not cause gas production. If you crimp the cap and then add the acid, the only way you can do it is to stab a needle through the septum. That will cause a leak when the vials heat up and you will lose a variable amount of the headpspace. So put the sample into the vial, add the acid, then put the cap on.

Peter

[ziarehmann]

Hi Again,

Now i have a basic question. Should i add the acid with open vial i.e. first i add acid and the clamp the cap or should i add acid with closed vial?
In an article i have read the addition of acid is performed with open vial so that CO2 can escape in atmosphere where as the addition of salt with closed vial.
What's your opinion?

Two things to bear in mind. Unless there is a lot of carbonatein my standard samples i dont have such thing but later on in the original fermenter i will have carbonate present. So at the moment i am doing it acidifying and then closing. Hope for the best results or similar in your sample, adding acid should not cause gas production. If you crimp the cap and then add the acid, the only way you can do it is to stab a needle through the septum. That will cause a leak when the vials heat up and you will lose a variable amount of the headpspace. So put the sample into the vial, add the acid, then put the cap on.

Peter