Purpose of Internal Standard?

[Troubleshooter]

Hi,

I want to know about role of internal standard. I know my question is very
simple but my concept is not clear yet although i know that internal standard is added in sample and in standard solution and compare the ratio ? but why we use it?

Treat me like a child as i am very new in this field.

Regards,
Kashif

[Consumer Products Guy]

Internal standard is most often used when there may be incomplete extraction, so the ratio of a similar, intentionally-added compound is used to quantitate the unknown. Internal standard also compensates for varying injection amounts, as can happen with manual injections. Today's autosamplers are so good that most internal standard assays have been "retired" by our company, and external standard quantitation using autosamplers is the norm.

[mbicking]

I want to add to CPG's comments:

First, select an internal standard that has similar chromatographic and detector response properties compared with your analytes, and alse elutes without interfering with other peaks. This compound is added at the same concentration (or mass) in both samples and standards.

For calculations, you will always use the ratio of your analyte peak area to the internal standard peak area: A(x)/A(IS).

The assumption is that any changes in peak response, caused by injection volume problems or detector changes, will affect the analytes and internal standard in the same way. But the ratio will still be constant. For example, if both A(x) and A(IS) decrease by 10%, the ratio will still be the same, and your analytical results would not be affected.

Use an internal standard method when you have a detector that shows changes in response over time, and also whenever you need better precision. Most pharmaceutical active ingredient analyses are still done by IS methods, because the precision is better than with an external standard method (not by a lot, but it is better).

I also will disagree slightly with CPG on the use of internal standards for recovery correction. Although there is no standardized terminology, I like to refer to such added compounds as "surrogates" because their role is to correct for recovery changes. I like to use "internal standard" only for those compounds used to correct for just instrumental problems.

[AdrianF]

To get a detailed answer to this question and many others you can go to the FAQ at the top of the page where LCR provides a free text book on all the basics of LC. I have checked and there is an extensive article on Internal Standards.

[tom jupille]

Thanks for the "plug" Adrian!

You can also do a search of past posts and find quite a bit of information.

Actually, you need to be careful with internal standards. They can improve precision as described, but only if the errors are correlated in your analyte and IS peaks (as in the case of dilution or injection problems). They can degrade precision of the errors are uncorrelated (as in integration or baseline noise problems). In the latter case, the percentage errors in the analyte and IS peaks accumulate instead of cancelling.

[HW Mueller]

Yes, if you check past chains you will also see that some of us suggested to do both methods, IS and ES. That way the IS will tell you if anything went wrong with every injection, but it will not add to imprecision.

Merlin, I don´t understand why one would use different terminology for standards which were added at different stages of the analysis. I have done tens of thousands analysis were the first act of sample workup was the addition of an IS. In the workout of the method I might have added IS at a later time to pinpoint any losses. This is the first time that someone pointed out that I may have used misnomers.
(Any recovery problems or HPLC problems can be seen immediatly if one combines IS and ES methods, especially if you know what the absolute area should be. "Absolute" is meant in contrast to relative peak areas and can fairly easily be determined by injecting standard without a column.)

[mbicking]

HWM:
My comment is more about terminology than practice. The distinction is borrowed from EPA methods, where surrogates are used in addition to internal standards. One is added for recovery/processing monitoring, and the other primarily for instrumental correction. Certainly, adding one at the beginning can perform both functions, but then you may not always know if there is a problem, or where it occurs.

The practice of comparing IS and ES results is a wonderful way to check on your method and instrument, and careful analysts like yourself are going to do this. My general concern is that too many people inherently trust the procedure and the result, without ever looking at the specifics, and they often will miss developing problems.

It was not my intent to imply that you are doing anything wrong. Rather, your comments indicate you are a careful analytical chemist who thoroughly reviews your information (not just results). I wish more people had this attitude.

I would simply like to propose a different terminology to distinguish the purpose of adding each compound. I am not necessarily fond of "surrogate" as a term, but it has some precedent. Perhaps we could come up with a better one?

[Bruce Hamilton]

All's well in chromatography when we have time to quibble about terms

I'm not sure I'd use surrogate for anything associated with "standard", as it's common use is to describe a substitute. In my world, a surrogate is used when a compound is not available, not because the compound would necessarily make a good standard.

I'd also like to mention that internal standards are often used for quantitative mass spectrometer detection because the analyte's behaviour, and thus detected quantity, is very sensitive to what else is in the sample.

In the case of mass spectrometer detection, one option is use deuterated forms of the analyte - if available, as they usually have similar properties. There is thriving industry in making deuterated standards of most pharmaceuticals and their metabolites, including those misused in sport and recreation.

Please keep having fun,

Bruce Hamilton

[Troubleshooter]

Thanx a lot again.Now i got clear about the use of internal standard.

Regards,
Kashif