Internal Standards for GC Analyses

Chromatography Forum: GC Archives: Internal Standards for GC Analyses
Top of pagePrevious messageNext messageBottom of pageLink to this message  By Ted on Thursday, February 22, 2001 - 01:38 pm:

Can anyone refer me to a listing of internal standards commonly used for gas chromatography? EPA, CLP, etc.? I am interested in a selection of aqueous soluble compounds to serve as internal standards for various GC analyses. Low boiling and/or high boiling compounds can be useful.

Top of pagePrevious messageNext messageBottom of pageLink to this message  By bill tindall on Thursday, February 22, 2001 - 06:50 pm:

I have been looking for an excuse to dust off my soap box and the misuse of internal standards is a tempting opportunity.........

Internal standard methods are not inherently better than external standard methods. In fact, the opposit is true. There is a price to pay for using an internal standard, so they sould not be used unless they are proven to be beneficial. Becaue two areas are used in the calculation, the precision of an internal standard method is 1.4 ( the square root of 2) worse than an external standard method. It remains to be demonstrated whether the benefit of using an internal standard overcomes this inherent disadvantage. In addition, the chance of an interference is doubled, a not insignificant risk in a complex sample.

So, what is a valid use for an internal standard? Measuring volume when it can not be easily measured conventionally is one use. A slurry where the solids are a significant volume fraction is an example. Correcting for a varying detector response might be another case. But, one must choose an internal standard that responds to detector variables in exactly the same way the analyte responds to detector variables. DDE was used as an internal standard for the MS analysis of PCB's. In my hands the response of DDE to changes in the MS source did not at all match the response of the PCB analytes, so using it was worse than not. Some use an internal standard to compensate for injection variables. A working injector should not be presision limiting.

So, to answer your question, there is no list of internal standards. (In cases there may be accepted internal standards for published methods, but one should question if they imporve the presicion). If you think you need an internal standard to fix a problem, one should be picked that has the chemical properties to potentially fix the problem anticipated. And then test to see if it helped. Data systems will cheerfully report results with and without the use of the internal standard. Collect some data and compare the precision of each approach.

What are some other valid uses for an internal standard?

Top of pagePrevious messageNext messageBottom of pageLink to this message  By H W Mueller on Friday, February 23, 2001 - 01:25 am:

A good place to start may be the chromatography catalogs.

How about dusting some more? I did some dusting here also, but darned, I didnīt find the derivatization of this factor 1.4. It seems, though, that there was some skepsis on this, the first time I saw it. The trouble was with not seeing a statistical difference between measuring an ext. standard + sample and measuring an int. standard + sample. Maybe you can jolt me out of being hung up?
Now the use of int. st. appears to have more to do with accuracy than precision. It is supposed to compensate errors in workup: extractions, derivatizations, etc. Also, one does not have to transfer (to another container, filter ....)samples quantitatively after addition of an int st.
Whereas I loath workup yields under 95%, one has to work with this sometimes. Often lower yields are accompanied by large variations (control of the workup is too imperfect), an int. st. comes in handy then.
Now, nearly perfect int. st. exist in the form of compounds with a radioactive isotope. To bad that radioactivity has been tarnished byond reason.


Top of pagePrevious messageNext messageBottom of pageLink to this message  By Anonymous on Friday, February 23, 2001 - 07:16 am:

Speaking of soap boxes, I work for a soap company that makes some OTC drug products and we most often use external standards for our FDA/cGMP analyses and obtain excellent precision and accuracy with our HPLC assays because we use autosampler-equipped HPLCs (Agilent). Why use an internal standard in cases where external standard works so well? Most of our GC assays are also done using external standards with nice precision and accuracy (also Agilent); in one product which is over 50% alcohol we do use n-propyl alcohol as internal standard following the lead of the AOAC.

Top of pagePrevious messageNext messageBottom of pageLink to this message  By Ron on Friday, February 23, 2001 - 02:28 pm:

Some methods require the use of internal standards simply because of historical reasons, i.e. instruments today are better than they were in the past. If internal standard methods were used in the past, it doesn't hurt to use them to maintain continuity with old data. Most modern instruments will do as well with external standard as with internal standard. Some standard methods require use of internal standard, but that is more of a compliance issue than performance.

Some techniques, static headspace for example, often give better precision with internal standards because of technique issues such as vial sealing. Adding internal standard before extraction can help compensate for recovery losses, although as noted above the chemistry must be the same for the results to be meaningful.

In regards to lists of internal standards, check the EPA Methods Web sites to see what compounds they have used for internal standards. If you can get deuterated analogues for your analytes of interest they are ideal, although some times pricey, internal standards.

Last, it is a good idea as suggested above to use external standardization if possible. Addition of internal standards is one more place where errors can be made. I do not agree that precision is affected significantly if internal standards are added consisently with high precision, but that is a big if in some cases.

Top of pagePrevious messageNext messageBottom of pageLink to this message  By bill tindall on Monday, February 26, 2001 - 04:06 pm:

whence the "1.4 worse factor". Many smart people read the forum, so if what I remember from statistics is incorrect I would be grateful for a correction.......

In an external standard method, one peak is measured to calculate an amount. In cases where sample preparation is simple weighing and dilution , and the autosampler is working properly, it is reasonable to assume that the precision of measuring the peak area of the analyte will be about equal to the precision for the calculation of amount of analyte. That is to say, integrating the analyte peak is the least precise part of the analysis. However, in the case of internal standard, two peak areas are measured. Assuming the precision for measuring each is about the same, then from the theory of error propogation, the square of the standard deviation of the amount of analyte will equal the sum of the squares of the standard deviations for measuring the analyte peak and internal standard peak. (wow, a simple equation takes a lot of words) From the above asuumptions, the square of the standard deviation of amount equals 2 times the square of the standard deviation of measureing peak area. Hence, the inherent precision of an internal standard method is the square root of 2(=1.4) worse than an external standard method where only one peak is measured.

Now, if something other than measuring peak area is precision limiting, the above discussion is irrelevant.

Top of pagePrevious messageNext messageBottom of pageLink to this message  By H W Mueller on Tuesday, February 27, 2001 - 07:42 am:

Thanks Bill for delinating this, I thought thatīs how the 1.4 came to be. There lies my problem. It was already hinted at this above: One can proceed in an external st. fashion with the internal st. method and compensate for deviations which are evident via the internal st. In other words, the error probagation is not necessarily intrinsic"? Or more clearly?: It seems that one can think of the internal st. technique as an external st. method with a built-in check. Now you can see why I have enormous problems with statistics, sometimes.
Something funny: it was observed, here, many times that overlap at the internal st. compensated the overlap at the unknown. (Had a systematic, not random, overlap).


Top of pagePrevious messageNext messageBottom of pageLink to this message  By guru on Sunday, June 17, 2001 - 04:42 pm:


I have a great trouble in the understanding of
using internal standard and external standard
method for a chromatographic analysis.

This discusion cleared many of my dbouts. However,
i would be greatful if anyone give me the exact
differnces in these protocols. I understand from a
chromatography book that for acurate quantitation
by internal standard method, one should make a
calibration curve ( with differnt composition of
the analyte and internal standard). This confuses
me a lot!

Top of pagePrevious messageNext messageBottom of pageLink to this message  By Spencer on Tuesday, June 19, 2001 - 07:03 pm:

Perhaps you are confused because the word "standard", practically speaking, has a different meaning when used in the phrases "internal standard" and "external standard".

An external standard quantitation method is one where you compare the response of your analyte in a sample to known concentrations of analyte in a "synthetic" matrix. e.g. Say you expect your sample to contain about 1 to 10% of your analyte. You proceed to build a calibration curve of maybe 4 or 5 levels of your "standard" surrounding the expected levels and then...y=mx+b.

An internal standard method is one where to all of your calibration "standards" AND to your sample, you add the SAME amount of a compound that is not in your real sample. This compound is referred to as the internal standard. Now your Ys and Xs are ratioed to this added compound and you proceed with your calculations. If you want more detail then that, check out some good quantitative analytical textbooks (Harris comes to mind) or more specialized books like Grob's "Modern Practice of Gas Chromatography".


Top of pagePrevious messageNext messageBottom of pageLink to this message  By Guru on Thursday, June 21, 2001 - 09:07 am:


Thanks for you clarifications.


Posting is currently disabled in this topic. Contact your discussion moderator for more information.