Chromatography Forum

In developing a new GC method requiring an internal standard, how does one select a chemical compound to use? I know the obvious requirements that the internal standard does not interfere with the peak of interest, and that it does not react with the sample matrix. I have used various phthalates in the past used in other methods. Case in point, I have a method where using one diethylphthalate elutes early in the run but my second analyte of interest is near the end of the run. Is there any rule regarding where to place the internal standard peak to get good quantitation? For example before the first analyte of interest or after the second analyte of interest given that all are on clear sections of baseline?

Thanks for all input.

I did develop a procedure where I had two components of interest; I chose an internal standard that eluted between the two, so just used that internal standard for each analyte.

I did develop a procedure where I had two components of interest; I chose an internal standard that eluted between the two, so just used that internal standard for each analyte.

What compound was your internal standard? What was the nature or your analyte(s) of interest? Basically how did you go about choosing which compound for your internal standard?

I don't think there is a "rule" that an internal has to be before your first or after your last analyte. I mean obviously you want it well after your solvent peak, and not so late that it carries over from one sample to the next. It's nice if the RT is decently close to the analyte. Some other things to consider:

Something Stable:
I don't want a dirty injection port eating my IS, making it look like my sample concentration increased. I don't want it to breakdown in the solution vial either.

Something with good solubility in the solvent you're working with, and not crazy volatile:
I don't want it to evaporate, or get concentrated because the solvent is evaporating.

If you're doing MS, something with a similar primary ion mass to the analyte:
I want any changes in the MS to effect my IS and Sample similarly.

If MS, something with a few good secondary ions:
So I can compare the ratios to check the MS performance (unless you have a surrogate or tune check for that).

Something you're 100% sure is not going to be present in your samples or standards.

You could always add a couple Internals and see which one gives better results

I did develop a procedure where I had two components of interest; I chose an internal standard that eluted between the two, so just used that internal standard for each analyte.

Please tell me what compound you used which eluted between the two compounds of interest. I am seeking information on how to choose a compound. My intuition is telling me that any phthalate or high purity alkane could be best first candidates for internal standards... but my intuition is wrong sometimes.

The most general rule is that the standard must behave like the analyte at all stages of the sample prep and analysis. That means structures as similar as possible - hence the populaity of isotope-labelled standards, as long as both analyte and standard produce a clean peak.

I did develop a procedure where I had two components of interest; I chose an internal standard that eluted between the two, so just used that internal standard for each analyte.

Please tell me what compound you used which eluted between the two compounds of interest. I am seeking information on how to choose a compound. My intuition is telling me that any phthalate or high purity alkane could be best first candidates for internal standards... but my intuition is wrong sometimes.

I used octacosane as internal standard, after seeing it used in this publication: Journal of Surfactants and Detergents volume 8, pages 199–202 (2005)

With regards to the elution point of the internal standard it is often seen that it should be after the target analyte though I've always understood this to be desirable rather than necessary. If it comes out later it can give you additional information about the separation that might not be possible otherwise. I'm working on a method at the moment and am using toluene as the internal standard. It elutes after some of the things I'm interested in but before others. To date I've not observe any problems with using this.

The issue of the ITSTD being chemically similar to the analyte is an interesting one. Clearly you often go for this due to the fact that similar materials tend to behave the same in terms of extraction, stability etc. but ultimately if you have something that has all the other necessary characteristics it might not be necessary to have something that's structurally related.

The issue of the ITSTD being chemically similar to the analyte is an interesting one. Clearly you often go for this due to the fact that similar materials tend to behave the same in terms of extraction, stability etc. but ultimately if you have something that has all the other necessary characteristics it might not be necessary to have something that's structurally related.

I worked in - surprise !!! - the consumer products industry for decades. For the first 30 years, we used n-propyl alcohol as internal standard for ethanol assay, as had similar properties, well-resolved on GC, available for reasonable cost in high purity.

Then USP procedure for whatever reason used acetonitrile as internal standard, and management made us adapt and re-validate to use that for regulatory "safety".

What you choose will also depend on what detector you are using. Phthalate can be good for FID, but maybe not as good for ECD. Hydrocarbons are good for FID but don't show up at all on ECD and sometimes give negative peaks. Highly halogenated will respond well on ECD but not on FID.

MS is different, you want something that will give at least a few good fragments, but not something like pentachlorophenol which has many closely spaced fragments because of so many chlorines.

I prefer something of similar functional group when possible and similar boiling point.

The great thing about the internet (didn't have that when I started out) is you can usually find someone who has already ran a similar test and have already figured out what works best

Using an internal standard with a similar functional group to your analytes can help ensure similar behavior during the analysis, leading to more accurate quantification.
fnf

My experience has been that I choose a standard that elutes where nothing else does AND I can sample it reliably (precisely and reproducibly). You certainly don't want your IS to react with the medium - at least in the timeframe of the sampling/analysis.

I analyze a lot of beer and methyl heptanoate fits the bill very well for most samples. My sample prep generally involves just addition of the IS (in a methanol solvent) to the sample so it's pretty straightforward. As a rule, headspace-SPME is my method of sampling for the analysis.