Chromatography Forum

Hii,
In order to evaluate the efficiency of some catalysts I used to performed almost daily GC-FID analysis. Normally I used to add a solution of an internal standard to the sample I withdrew from the reaction vessel with (making so a new solution made of equal volumes of internal standard solution and the sample solution collected from the reaction vessel). Basically I diluited the real sample to 1:2. Obviously I made the previous calibration curves in the same way, that is preparing different solutions of the analyte ( A + B + C ) and adding to each, before the injection, the same quantity of the standard solution (D); practically injecting so new solutions ( A’ + B’ + C’).
In other research groups I saw a different procedure:
They just add a known amount of internal standard at the very beginning directly in the reaction vessel along with the reaction mixture. So they avoid adding the STD solution to the just collected sample ( because now the collected sample solution has the already the standard in it) Injecting directly inside the GC. Obviously they prepare calibration curves according to this procedure that is the standard solution and the analyte solution in the same flask...
I would like to know your opinion about these two methods of quantifying… which one you think is the more correct?

Many thanks
jurty

I would not say one is better than the other, it depends on what you are doing.

Only thing is if the reaction could affect the internal standard in some way, I would stay away from adding the internal standard to the reaction vessel.

If you are using expensive compounds (e.g. isotope labelled) as standards then the smaller quantity used in spiking only the sample is clearly going to save money.

Peter

But avoid situations where when combining two solvents you don't get the full additive volume, as this can change your concentration slightly (several percent is possible) when adding different amounts of solvent together. Sometimes 50 plus 50 equals 95.

best wishes,

Rod

And as long as the internal standard does not react or partition out of solution onto the catalist support or the reaction apparatus...

First of all, I would like to thank everybody for the replies. Personally I consider that adding the standard directly into the reaction could lead to a “fake” evaluation of quantification values.. I better explain myself with an example:
We have to quantify a certain product (our analyte) from a reaction vessel.
We put our solvent, reactants, catalyst and Internal STD. Internal STD directly in the reaction vessel. We let the process start and collect reaction samples at regular intervals. At the beginning our analyte/STD ratio assumes a certain value. This value tends obviously to increase because product amount increases by time. Nonetheless when we will consider our calibration curves equations to calculate the amount of analyte we will assume, as theory teaches us, that Internal STD concentration is constant, but actually the more samples we collect from the reaction vessel, the more in proportion, the STD concentration originally presents will diminish, yielding so through calculation, an higher-than-real ratio analyte/Internal STD and so an bigger-than-real amount of analyte/product. That’s why, according to me, Internal STD shouldn’t be added since the beginning inside the reaction vessel along with solvent, and reactants…What do you think about this?
Thanks
jurty

Unless there is a change in liquid composition - the internal standard remains at the same conentration and the total volume of the entire reaction mixture decreases by what volume is removed. And unreacted reagents and final product are removed in similar proportion -- no?

Don

I don't think we know enough about the reaction and the reagents to make that claim.

I can only assume the concentration in the final liquor is the measurement desired, which would require the std addition analyte to be added after the reaction was complete.

Rod

If an internal standard is to be added to the reaction vessel, then it should be added, with good mixing, just before the sample aliquot is withdrawn. Adding it before the fermentation is jsut asking for the microbes to convert some of it into substances unknown.

Peter

I left the question mark intentionally - so we might get the answer that begins "Well, actually..."