Chromatography Forum

Hi folks

There is aquestion that puzzles me at the moment. I want to add an internal standard prior to sample prep to compensate possible loss of volume etc. What happens, when after sample prep the analyte concentration is above the calibrated range? Without ISTD I would dilute the sample and walk off happy, but if I dilute by factor 10, the ISTD concentration will be far away from its normal response - is that still reliable?
The best option would be to dilute the sample prior to sample prep, but I would have to know the result before doing this. Trial and error is no option because sample amount is very limited.
Any idea how to handle this?

Kind regards
Jörg

ps.: I thought about diluting the samples with ISTD solution at the target concentration. It should be possible to compensate this, but will work only if the ISTD extraction efficiency is 100% ?

Presuming that you are calibrating by taking the ratio of analyte area to IS area, then diluting the sample with clean solvent is not going to help, because the analyte: IS ratio will stay the same, and so will still be out of range. An exception to this might be if the out of range response is caused by column overload of the analyte.

Diluting with IS solution defeats the object of adding the IS in the first place.

So you are probably stuck with re-doing the prep with a smaller quantity of sample.

Peter

Thanks Peter

This is what I also ended up while web-searching. I have found a new approach: I will go for two calibrations with different ISTD concentrations (say 100 mg/L and 10 mg/L), but using the same analyte concentration range. This will allow me to do the sample prep with constant amount of ISTD, but I will be able to dilute the sample and use the second calibration curve for evaluation. This will be less work than doing the sample preps twice (if possible at all, depending on availability of sufficient sample)

Regards
Jörg

Hi Jorg

That might work, let us know how it goes.

Peter

Would using 2 different compounds as ISTD's work? The first compound would be used to compensate for sample prep variation. The second compound for GC variation. If you needed to dilute your sample, then the conc. of second compound could be held constant. I'm sure I don't understand completely what you are doing, but it's just a thought.

That might work, let us know how it goes.

This was a good approach, even though the internal standard prove unsuitable (but this is an other story).
Calibration with two different concentrations of ISTD was good, and repeated measurements of standards before and after dilution were in good agreement.

Regards
Jörg

Thanks for the update, Jorg

Are you going to publish anything on it ?

Peter

Would using 2 different compounds as ISTD's work? The first compound would be used to compensate for sample prep variation. The second compound for GC variation. If you needed to dilute your sample, then the conc. of second compound could be held constant. I'm sure I don't understand completely what you are doing, but it's just a thought.

This is commonly done, one standard for extraction/sample prep recovery/efficiency, one standard for injection/detector variation. I learned them as "surrogate" and "injection internal standard" (or IIS), respectively.

Thanks for the update, Jorg

Are you going to publish anything on it ?

Peter

No, sorry this was during a non-disclosure study. But it can be found here as a good starting point if anyone else runs into this kind of question.

Jörg

I agree with MMJ88; this is precisely why you use both a "surrogate" standard and an "internal" standard. The internal standard is used to compensate for injection variability as well as being used as a reference for quantification. If you had done the analysis this way you would have merely diluted the sample, added back the appropriate amount of internal standard and then reinjected the diluted sample. The recovery (surrogate) standard would have been at low concentration, but it would be moot since your analyte is clearly present at quite high concentration.