Chromatography Forum

Hi, dear i really need your help for the sake of humanity .

i asked many people no one give me knowledge in internal standard

i have five different soils from India and i want to measure the adsorption of my analyte into this soils !

my method :
0.5 g from the soil in 50 ml solution ( 20 ppm analyte ) with 24 shaken then filtered and analysed by RP-HPLC

Question :

1- Here i use only my analyte without internal standard and i am using manual injection , do you thing it is acceptable because my friend said don't use internal standard because it iwill adsorped to the solid , some said use it after when you filter your samples ( i.e before injection )

2- if i use it do i need to spike in all my sample the same concentration of internal standard like ( 1 ppm ) or it doesn't make any different if i change internal standard concentration .

i really want to see step by step internal standard adding and calculation

Thanks in advance

An internal standard can compensate for losses during sample preparation, and for variations in measurement efficiency, so if your method is likely to suffer from either, you would benefit from including an internal standard. If your method does not, you can get away with leaving it out.

Yes, an internal standard would bind to the soil so you cannot include it until the soil is gone. As soon as the soil is gone, you can put it in, and it will compensate for losses from this step onwards.

The conventional procedure is to add an internal standard that is chemically as similar as possible to the compound you are measuring, and to add it in similar quantities to those you expect to measure. When you prepare your calibration curve of the actual compound, add the internal standard to this in the same final concentration as is present in the samples.

The best way to see a calculation in action is to get a decent book, but you might find a tutorial by Googling.

In this situation (extraction etc.) I’d apply the standard addition procedure - at least prior to validity justification of the results.

Best Regards

One should know, not assume, what is going on, so one thing that can be done is to add the IS at different steps and check what happens.

There is a lot of material in this forum on internal standards. I preferred to use the external standard and IS on each sample simultaneously.

Imh, how do you know that the IS will bind to the soil?

Take a look at the US EPA methods. These methods include standards early on and then close to the end of sample prep. The standards added at the end of the procedure are used to compute the recovery of those added earlier.

If you are adding known quantities of material to soil and can control losses through preparation steps, the critical internal standard for you is the internal standard added later so that you can compute recovery. If you are analyzing material in soils, you need to add a standard early on and at the end to allow you to guage recovery issues.

The US EPA methods are on the EPA web site. Accustandard has links to the methods from their web site http://accustandard.com/ - which saves the trouble of digging through the EPA web site.

HWMueller, the method as described appears to be to add something to soil, shake, and analyse how much is left, in order to discover how much has bound to the soil.

If the internal standard doesn't bind to the soil, it must be so chemically different to the analyte that it will be a poor internal standard. For example, if the analyte and internal standard differ in their ability to bind to soil, they will probably differ in their ability to bind to filters, glassware, etc.

Danko, I'm slightly worried by the method of standard addition in this case. If you add the standard while the soil is swishing around in 20ppm analyte, effectively you are doing the binding assay in a range of different concentrations of analyte. If you do it in 20ppm (normal) and 40ppm (standard addition), and you get back 10ppm and 25ppm, this could mean (1) that the efficiency of measurement of standard is only 75% (40-20=20, we got 25-10=15, 15=75% of 20). If so, our 10ppm reading was actually 13.3ppm (75% of 13.3=10) and the soil bound 6.7ppm of the original 20ppm. But it could also mean (2) that in the presence of 20ppm, soil binds 10ppm, and in the presence of 40ppm it binds 15ppm, and there is absolutely nothing wrong with our assay at all; we're just looking at the binding chemistry of soil...

But the bottom line as HWMueller and Don_Hilton write, is to use as many controls as you can. You can never have too many standards.

A few years ago I helped someone with this sort of problem, the compounds being nonylphenols. We did this via synthesizing nonylphenols from 14-C phenol. Obviously, we looked at all components, not just the aqueous part. An IS was not invoved as one can easily see whether radioactivity is lost somewhere, etc.
If the use of a IS is advantageous, one better proof that it "mimicks" the analyte, not assume that it does.

Lmh, the standard addition will show weather or not the soil is being saturated with the compound. It might be useful to know the upper limit for adsorption……
It can be done by adding different amounts of soil to the same volume of analyte solution, but it can also be done by adding identical amounts of soil to a constant volume of different analyte concentrations.
A pitfall (if not knowing the adsorption capacity limit of each soil sample) could be in a case where all samples show zero analyte concentration (i.e. the analyte stays in the soil). Which of the soil samples is most adsorptive?

Best Regards

oh absolutely. If you don't manage to measure anything at all, you are not much the wiser... There is a lot to be said for studying the adsorption at multiple starting concentrations, but unfortunately none will help you understand whether anything went wrong with the analytical side of things afterwards.