Chromatography Forum

Hi guys ,

I am not chemist that why i struggle

I have very simple question as i search the net but nothing give me step by step how to do internal standard calculation ( because i am not chemist originally and this is my first time with this CALCULATION AND PREPARING THE CURVE ? so please help me otherwise i will cry ) .

i have three unknown analytes ( analyte 1 , 2 and 3 ) in my filtered solution of methanol with volume of 10 ml ( so that 10 methanol contain 3 unknown concentration ) .The most important analyte to me is ( analyte 1 ) so it was been found that chlorpyrifos compound is the best internal standard for my analyte 1 . But chlorpyrifos is my analyte number 2 ! ( the idea is to study the adsorption of the three compounds in five indian red mud , i thing it is better not adding internal standard as analyte number 2 and just spike it not sure i need advice ? )

now i need to prepare internal calibration curve for analyte 1 with chlorpyrifos ( but i thing i cann't add IS to analyte 2 or 3 ? because they are completely different in the property ,so only analyte 1 with chlorpyrifos ) :

what i did ( is it right or wrong ? )

1- 0.01 - 100 mg/L mixtures of the three analyte 1,2 and 3 were prepared in methanol as external standard .
( i've been told after that i must use internal standard so >>>go step 2 )

2- 0.01 -100 mg/L analyte 1 , contain 1 ppm chlorpyrifos in all the standards .

Result of step 2 :

at 0.01 ppm analyte 1 ( contain 1 ppm IS )the peak areas are :

analyte 1 = 7000
IS= 4000

( peak area of analye1 / IS ) Vs. concentration of analyte 1 )
so :

( 7000/4000) vS. 0.01

and i did the same thing for other concentration , is it correct my calculation ? TO make full internal standard calibration

Thanks

Several things you need to be aware of about internal standards:

1. The internal standard must *never* be naturally present in the sample

2. Internal standards compensate for certain types of errors, but they aggravate other types of errors. Specifically:
- any error that affects all peaks in the same way will tend to cancel. These include dilution and injection volume errors.
- any error that affects different peaks in different ways will tend to accumulate (i.e., use of an internal standard will make thinks *worse*). These include adsorption errors, sample degradation, baseline noise/peak shape/integration errors.

3. Computationally, internal standardization and external standardization are very similar. The difference is:
- in "external" standardization, you work with peak areas as a function of concentration
- in "internal" standardization, you work with area ratios as a function of concentration ratios. In most cases, the concentation of internal standard is constant in all calibrators and samples, so that you usually work with area ratios as a function of analyte concentration.

As I understand what you are trying to do, I think that an internal standard is not necessary and may well be counterproductive.

Thanks Tom i really appreciate you great help .

I am just now have doubt about the ( x) in the calibration curve is it the ratio of the concentration of the analyte to internal standard or just analyte concentration alone without internal standard concentration .

Example : 2ppm analyte contain 1 ppm internal standard give us peak area 7000 ( analyte which its concentration 2 ppm ) and 4000 ( internal standard which its concentration 1 ppm ) .

to plot this the Y = 7000/4000=1.75 mV
x= analyte conc./internal standard conc.

i am doubt about x is it the ration or just analyte concentration ?
is it acceptable to put x = analyte conc. only

Thanks

For another explanation of Internal Standard calculations, you can read "EPA Method 8000C - Determinative Chromatographic Separations"; specifically, sections "11.4.3 - Internal standard calibration" and "11.5 - Calibration acceptance criteria". Good definitions of terms, calculations are laid out well, and the advantages and disadvantages are laid out as Tom explained above.

http://www.epa.gov/osw/hazard/testmetho ... 00c_v3.pdf

Dear bisnettrj2,

Thanks indeed this looks a very helpful regulated method , i will read it .

Thank you very much

is it acceptable to put x = analyte conc. only

Yes, so long as you use the same concentration of internal standard in all calibrators and samples. If you have different concentrations of internal standards, then you must use the ratios to take that into account.

Yes the calibration was prepared with 1 ppm IS for all samples and claibrators .Tomorrow i will use 10 ppm internal standard and after two days i will use 5 ppm IS so i will prepare three calibration curve contain 10 ppm and then 5 ppm wawoo it is very time consuming .

The technician in the lab said to me use this equation ( do you thing it is correct he said without preparing calibration curves !! )

[ Analyte ] in the sample = peak area of Analyte in the sample * [Analyte] in the standard * peak area of IS in the standard /peak area of Analyte in the standard *peak area of IS in the sample

That's correct, if a bit convoluted. Beware, though; it assumes that the calibration plot goes through zero, which may or may not be true (that's why it's a good idea to run a full calibration!).