Dear V mani Rathnam

(1) You need to read a simple text on the fundamentals of quantitation in gas chromatography.

(2)Your faculty advisor should have given you this fundamental guidance before allowing you to use the instrument.

(3)None of these instruments is an 'answer machine'. The best technique for quantitation depends on the conditions, sample and hardware.

**** I personally do not trust any commercial software to do the job right, so I export all raw signal data into Excel worksheets and do my own analysis of the data. I strongly recommend that students and professionals alike do this until there is proof that the instrument and software are truthful.

Dear V . Mani Rathnam,  Since you have done the calibration curve of the two compound  and followed the procedure to calculate the concentration of your compounds in the liquid mixture, if the concentration ratio of the two compounds of interest is same in both 1000 micro litre and 20 micro litre liquid mixture , you should get same area ratio for your different sample. 

one need to be calculate the concentration of the individual compounds is the liquid mixture in mmol per ml  in the liquid mixture. If the concentration ratio is same for both the compounds in the different liquid mixture, one should get the same area ratio for the same sets of the liquid mixture

If you still get the different results then you have to be sure that the mixture is a homogeneous one, if it is not homogeneous and you may be injecting different concentration of the compounds in to the GC

Dear Bikash Sharma,

Firstly, thanks for the reply. 

I think you didn't get my question correctly. I have a liquid mixture consisting of two compounds of unknown concentration. I have to find the individual concentration in the liquid mixture. For that, I have prepared two GC samples of 1000 micro litre solution. First 1000 micro litre solution consists of 10 micro litre of liquid mixture and the rest 990 micro litre is internal standard plus solvent. Second 1000 micro litre solution consists of 20 micro litre of liquid mixture and the rest 980 micro litre is internal standard plus solvent.

After injecting these two samples into the GC, I noticed that the 20 micro litre liquid mixture sample has more concentration than the 10 micro litre liquid mixture sample. Here, my question is why the concentrations are different for 10 micro litre and 20 micro litre samples that are prepared from the same liquid mixture (unknown concentration). And, is this possible to get the same concentration of the liquid mixture compounds by taking whatever amount of the sample from the given liquid mixture?

Dear V. mani Rathnam,

Obviously the sample solution with 20 micro litres of liquid mixture will have more concentration and hence more area of the GC peak since it is two times more concentrated than the sample with 10 microlitres of liquid mixture  as both are finally diluted to 1 mL. 

Dear V mani Rathnam

(1) You need to read a simple text on the fundamentals of quantitation in gas chromatography.

(2)Your faculty advisor should have given you this fundamental guidance before allowing you to use the instrument.

(3)None of these instruments is an 'answer machine'. The best technique for quantitation depends on the conditions, sample and hardware.

**** I personally do not trust any commercial software to do the job right, so I export all raw signal data into Excel worksheets and do my own analysis of the data. I strongly recommend that students and professionals alike do this until there is proof that the instrument and software are truthful.

I share Bikash's bewilderment and agree with Scott.  Instruments/computers/software will always give you an "answer".  The question you need to ask yourself is "Does the answer make sense?",  "Is it reasonable?" and then " If not why not?".  Your answer to these questions can only be arrived at from a basis of knowledge, logic and experience.

Go back over your description of your sample preparation.  One of your dilutions to 1mL contains twice as much of the original sample as does the second dilution.  It must ipso facto contain twice as much of the two analytes whose concentration you are interested in measuring.  Perhaps you need to include this factor in your calculations.

When you are doing an Internal Standard quantification, please note that the internal standard concentration should remain same in all the calibration standards. 

Thank you Bikash Sharma, Scot Abbott, Charles H Hocart, and R. Girijan Menon for your valuable suggestions. 

How did you derive a calibration curve if the liquid mixture is of "unknown concentration"?

10 uL and 20 uL are VERY large injection volumes for GC. It takes skill, experience and knowledge to adequately perform large volume injections, which, from your post, are 3 assets which you are lacking. 

As per Scott, and completely in accord with Scott, you really need a basic GC training course before you proceed.

Dear Mark Krause, I derived a calibration curve by taking the pure compounds at different concentrations. I am taking the training now. Thank you for your valuable suggestion.

What Mr. menon told was correct. The internal standard area should  be consistent for all the calibration Standard levels.