I think I understood your problem fairly precisely. Your problem is to find suitable primary standards for the humic components. Note Humic acid is not the only component of Humic components. I suggest that you read the literature and try to find out if  not all, atleast the major components and try to locate the Rt values on GC. Also see the columns used and the detectors along with other parameters like oven temperature, injection port temperature etc. the correlate your observations with the published data. It should give you a fairly precise idea of your analysis.

You might want to review Patrick Hatcher's research (http://sci.odu.edu/hatchergroup/).  He has been working with humic acids for a long time using GC-MS.  Before moving to Old Dominion, he was at Ohio State.

Thank you both for replies. To clarify i should say that i need to measure the concentration of humic compounds not the building blocks or the the functional groups. so i dont want to decompose the humics, prior to measure them in GC. 

so the question is that if i can use them (in solution) as they are in the GC. As you probabely know, they are not volatile at all, so probabely i need to use an organic volatile solvent to extract them. i used Hexane, but couldnt get much of the humics soluble in hexane. Any idea what organic solvent i can use?

BTW, Thanks Sara for the link. I'm reviewing their work now. :)

Hello Mostafa,

Humic substances are not organo-soluble, but acido- (fulvic acids), alcalino- (humic acids) or insoluble (humin). So, it is not possible to analyse directly using GC.  Moreover their high Mw makes it impossible to volatilise in a GC. That's why we use pyrolysis. 

An easy way to measure their concentration is to determine the DOC.

Dear Laurent, Thanks for your reply. You are absolutely right. Since i have them in a microbial culture (with other organic carbons), DOC is not representative of Humic concentration only. The best way is Liquid chromatography, but since we didnt have it in the lab, i decided to try my chance with GC. I tried Hexane as the solvent prior to do the GC, didnt work. I guess the bottom line is what you said, with GC, its simply not possible! 

Dear Mostafa,

With hexane you will never solubilise humic substances.

The fractionation protocol is as follows : 

-extract organo-soluble compounds (lipids) using dichloromethane/methanol 2/1

- the residue is humic substances

-solubilise humic and fulvic acids using NaOH 1M

- the remaining fraction is humin

-separate fulvic acids from humic acids with HCl (humic acids precipitate)

Then you have the weight of each fraction and you can obtain the Organic matter content of each fraction using an oven (500°C)

Dear Laurent,

I have an article (see the link attached below) which used the same fractionation procedure (more or less) based on the solubility of different humic compounds on different pH (alkali, acid). the results suggest that its not accurate enough. even based on the source of the humic compounds the accuracy changes. Do you have any experience using this method personally? whats your view on the accuracy of it?

http://pubs.acs.org/doi/abs/10.1021/es0709223

BTW, there is a LC method for measuring humic substances called LC-OCD (liquid chromatography organic carbon detection) which separates the fractions of humics (HA and FA) based on their solubility in Alkali/Acid and then do couple of measurements like UV254 and DOC on the collected fractions. It seems accurate, i saw the results. Although its very expensive. The funny thing is that the principle is what you just mentioned, but when its done manually, its not accurate enough. 

The analysis of humic substances via direct GC is not possible due to the high molecular weight nature and functional groups content of humic substances. Even fulvic acids, the low Mw portion of humics, is not analyzable via GC. You could try GPC even though you won't be able to detect the chemical nature of your substances; anyway due to the fact the denomination is only operational you could quantifiy what you consider humics by calculating GPC areas.

Mostafa, I have been using the IHSS protocol of fractionation of humic substances for something like 20 years. You can see my publications for more information. For me it's accurate, since it is reproductible and there is no other way to separate the different pools of organic matter. You must know that the fractionation takes one week per sample.

DEar Riccardo, Thanks for the answer. i will look up GPC. 

Dear Laurent, THank you for the follow up. Its being nice. My setup is including 10-15 jars, and im working on the degradation of humics. At the end i will have at least 10 samples per week to measure, considering your time estimate, it is not really possible to use it, right? 1 week per sample, 10 week per 10 sample per week!! :) 

I think i should go with HPLC/SEC.

If you want to monitor the degradation, you should use pyrolysis after lipid extraction. The best way is PyGCMS but  if you don't have a pyrolyzer you can use a sealed tube  in an oven.

Dear Laurent,

Sealed tube would be great. Do you know an article using the sealed tube that i can read? or, maybe you can help me with the protocol! it would be great!