GC and Internal Standards

[sancheer]

Does anyone out there have any idea what the rule of thumb might be when it comes to the recovery of a theoretical internal standard analyte value, percent wise?

[Bruce Hamilton]

I've no idea. Rules of thumb may be suggested by anyone, even me, but their relationship to reality can be tenuous, at best. I think you have to be more specific about what you want....

I have a feeling that there is a general guidance floating around the Internet somewhere for analytical methods, but I can't recall where I encountered it. Each organisation that provides published standard methods may have some published guidance on acceptable precision and IS recoveries for any submitted methods. A WWW search may locate the information relevant to your application.

Internal standard recovery will depend on where the IS is introduced into the sample preparation, the analysis method, and analyte concentration.

An internal standard recovery used to determine the sequence:-
crude sample extraction, SPE cleanup, on-column injection, MS detection, for analyte concentrations of pg/g in original sample, will be very different to:-
addition to GC solvent dilution of an accurately-weighed sample, split injection, FID determination of hydrocarbon components at %.

In general, I'd guess that, for simple dilution and measurement of % concentrations by FID, that >98% could be reasonable. As the analysis sample preparation complexity increases, and the original analyte concentration decreases, then > 75% could be reasonable.

I've seen in-house methods that allow > 50%, but they usually are for recovery of trace concentrations, and include all sample preparation.

Please keep having fun,

Bruce Hamilton

[Peter Apps]

The "recovery" of an internal standard is set at 100 % by definition.

Peter

[Peter Apps]

The "recovery" of an internal standard is set at 100 % by definition.

Peter

[HW Mueller]

That 100% "recovery" for an IS is a relative recovery (relative to itself). It also has an absolute recovery (possibly relatively to an external standard, or definately relative to an injection of this standard without a column, if air interference and other artifacts are under control, etc.). I would feel like having blinders on if not at least the range of absolute recovery was known.

[Peter Apps]

Hi Hans

Of course you are right. With an internal standard that closely matches the behaviour of the analyte, "recovery" does not come into the calibration or calculation of amount of analyte, but the fraction of the analyte and standard that the sample preparation, injection and separation passes to the detector needs to be high enough for LOD and LOQ to be at the required levels. Sometimes that fraction can be very small indeed - e.g. of the 50 mg of analyte in 5 ml of 1% ethanol an eqilibrium headspace analysis could put only 5 ng onto the column and into the detector - a fraction of 0.1 ppm - to yield a respectable peak.

Peter

[HW Mueller]

Peter, interesting example. I have great respect for those who have to do headspace, as I certainly would have nightmares about Raoult´s and Henry´s laws and deviations of/from them. (Hopefully I still remembered the names correctly).

[AICMM]

Sancheer,

In the US environmetnal business a 50 to 150% recovery window is applied to internal standards. Surrogates are added at the beginning of the process, internal standards right before injection into the instrument. Responses for analytes are pegged to the response of the internal standard. Below 50 or above 150 the sample has to be re-run after identifying why the IS failed. I have often seen the later eluting IS's fail due to dirty liners or matrix effects.

Best regards.

[Peter Apps]

Hi Hans

The great thing about headspace analysis is that you just calibrate peak area (or area ratio vs an internal std) against amount of analyte under identical conditions to those under which the samples are run, and forget about all the laws !

AICMM, the "internal standards" in the example you give are only used to correct for (or just to detect deviations in) the performance of the analytical instrument e.g. injection volume, split ratio, adsorptive or catalytic losses etc.

Where does the term "recovery" come from ? In chromatography we hardly ever "recover" anything - the analyte ends up in an HPLC mobile phase waste bottle, combusted in an FID or fragmented in an MS ! If we put 1 ml of a sample into a vial and inject only i microliter, is the recovery 0.1 % ?

Peter

[HW Mueller]

Peter,
The implication above was that the laws would indicate that robustness may be troublesome.
You know, of course, what I mean when I use the word recovery. I would think that an analysis which "sees" 99% of the analyte present in the aliquot is to be trusted more than one which "sees" only 1%. One would be inclined to think that the analyst who has 1% "recovery" has some skills lacking. If he doesn´t even know how much material gets lost in his analytical process (1% lost? 99% lost) it could be carelessness in the true sense of the word.

[Peter Apps]

Hans

Right again; headspace is far from robust - with the exception of liquid sample volume when the analyte is very soluble in the sample (ethanol in water for example) all the conditions have to be controlled within VERY narrow limits to get good repeatability.

I thnk that we all (nearly all anyway !) know what recovery means, but it struck me as a strange term to use and I was wondering where it came from - I suspect the analysts picked it up from synthetic chemistry where recovery is the fraction of the expected yield that you actually manage to produce. Probably it came into instrumental analysis via the classic wet chemistry gravimetric procedures, which are really synthetic chemistry used for an analytical purpose - e.g. by weighing how much barium sulphate you can "syntesise" from a sample you can calculate how much sulphate was there in the first place.

Peter

[HW Mueller]

Well, I can speak only for myself on this one. If I had lost all analyte in an unknown sample for weeks, because of clumsy workup trials, I would be inclined to shout after finally getting a bump: I recovered some! Finally I recovered some!