Question about ISTD calibrations and chemstation

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Im hoping someone can look over my process and let me know if Im making any errors.

I have set up a 6 point curve for three analytes, the curve ranges from from from 10 ppm to 250 ppm. Each std contains an internal std at 100 ppm.

Using chemstation I added each level of the curve as normal, I selected the istd peak and labelled it as the istd and entered in the amount 100 ppm.

When I prep samples I have been spiking each sample with 100 ppm of the istd.

My first question I have is inragrd to my dilutions. Due to the concentration of the three analytes I have to run two dilutions for each sample.

The sample is first mixed with 20ml of solvent. this mixture is injected for the first run.

For the second run I make a 1:20 dilution from the 20mls. (950 ul of solvent, 50ul sample)

When spiking either the first dilution or second what is the most straight forward way to account for any additional dilution caused by adding the istd?

For instance if I were add 100ul of istd to my 1:20 dilution how is that extra volume accounted for? What I have been doing is adding 900ul solvent, 50 ul of istd, and 50ul of sample, this way my sample is still a 1:20 dilution as the total volume is the same as it would be if I used 50ul of sample into 950ul of solvent.

For the sample that is not further diluted though I still have to "spike" it with 50ul the istd, am I correct that the addition of the istd would technically be a dilution, albeit a small one?

Second Question

Would it be ok to dissolve my istd in the extraction solvent, rather than spiking each sample? If I were to do that and used the same spiked solution to make my 1:20 dilution should still be at the same 100ppm concentration.

third question

When using chemstation and quantifying based on an istd what happens if the technician prepping the sample is slightly off with either the spiking, or if in the original solvent slightly over or under the desired volume? On the chromatogram that prints out the ISTD always says 100 ppm as that is the quantity entered in the curve, but what would happen if a syringe was off and only 50ppm of teh istd was actually put in the sample?

Thank you in advance for your help, it is very important that I use best practices and fully understand the rationale and any possible errors that may occur.
my curve is below


At one point you make it sound like your sample is soluble in your solvent and then later you talk about an "extraction solvent". Which is it?

If your sample is really "dilute and shoot", I would mix my internal standard in the diluent at the correct concentration. Then, it's pretty much not going to change.

First dilution (say you make your IS at 100 ppm in the dilution solvent):

[IS] = 19*100/20 = 95 ppm

For the second dilution (50 µL to 1,000 µL)

[IS] = (50*95 + 950*100)/1,000 = 99.8 ppm (more or less 100 ppm)

In this situation, you can truly make it the same across all dilutions if you add it to your sample at 100 ppm. Then, no matter how many dilutions you make, it will always be 100 ppm. This will only work if it's truly, "dilute and shoot". If it's an extraction, completely different ballgame. Please clarify, is it an extraction or dilute and shoot?

I don't run much automated with the Agilent Chemstation so I'm not well versed on how that part of the software works.
The sample is is extracted with solvent in a sonicator, or using the soxhlet method.
The solvent from the extract is the first gc run. The second run is is a 1:20 dilution. 50ul of extract to 950 ul of solvent.
Generally, the more information you can give about what you are actually trying to accomplish, the better we will be able to help you with your problem. There are a lot of experience folks who offer good advice on this forum. Chances are quite good that someone has actually done what you're trying to do and they can save you a lot of time.

What is the nature of your sample (synthetic polymer, natural product, etc.)? How long do you carry out the soxhlet extraction? Are there any other cleanup steps after the extraction? What are your analytes?
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