Chromatography Forum

Hi,

I would like to know whether there is any requirement that internal standard need to elute at the end (i.e. after the analytes elute) during an analysis?

Thanks for your time.

No requirement, but often considered "better", particularly with MS detection because it minimizes the possibility that something will change the ionization efficiency after the IS and before the analyte. My personal opinion is that the IS should elute as close as possible to the analyte; whether just before or just after is not that big an issue.

I would add that what is most important is that the internal standard accomplish the purpose for which it is added. Using an internal standard is not "free". the price paid is an inherent decrease in precision (because two areas are used in the calculation) and a doubling of the chances for coelution in complex samples. So there needs to be some demonstrated justification for using the internal standard. In most cases where I have seen an internal standard used in LC it didn't add to precision and I can quote cases where it made precision greatly worse. If you are using the internal standard to compensate for detector response drift, demonstrate that the compound chosen responds to detector changes like the analyte. Ditto for what ever other purpose an internal standard is used.

It is very easy to calculate results from data files with and without internal standard calculation. Do it both ways for a while to prove that the internal standard is doing something useful. You may find that it is not.

That´s the key, do it both ways, it doesnt cause any extra work (if you start out with the Int. St. method anyway), . . . maybe a little extra calcs. With the Int. St. there, you will at least have an extra assurance that things are going oK, during real analyses.
On the calcs, any book on chrom, or any integration software instructions will tell you.

adding to Bill's post -- and if you find the internal standard is not helping (or is making things worse, then that gives you some insight into the source of error (in your procedure after the IS and analyte have begun to separate).