Chromatography Forum

First of all, I would like to thank everybody for the replies to my previous post "Advices about Internal STD method performing".
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 in advance
jurty

You need to be realistic. Ideally, it would be best to have the IS/analyte concentration in the reference mix be the same as what's found in the product sample. But in real life, like in your situation, not always practical. Sure, if I added IS to match analyte level expected early in the reaction, by the end the analyte would be at higher amount, so the IS/analyte concentration would be different. Run a linearity curve and calculate ratio of the peak areas to concentration of each.

Now, don't use a tiny IS or huge IS in reference to the analyte peak, that's for sure. I'dd add the IS as early as possible. Tips for ideal IS choice would be similar structure, similar properties, INERT to your conditions, available pure, commercially available, non-toxic, etc.