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- Posts: 1861
- Joined: Fri Aug 08, 2008 11:54 am
Me: "... so we use internal standards to compensate for losses during extraction, and changes of ionisation efficiency. But because the ionisation efficiency of a target analyte can be affected by other things that coelute, it's really important our internal standard coelutes with the analyte. If it elutes somewhere else, it can't compensate for disturbing things that elute with the analyte. That's why an isotopically-labelled version of the analyte is the best internal standard. We put the internal standard in all the samples, and the same amount in all the standard samples we use for the calibration curve, and we plot the ratio of peak areas, instead of just the peak area of the target analyte"
Student: "why?"
Me: .... starts explanation again, wishing he hadn't gone so fast.
Student: "No, why do you do the calibration curve?"
Me: "because you need a calibration curve to know how much stuff you've got. Different analytes will have different calibration curves."
Student: "no they won't. If you use an isotopically labelled internal standard identical to each analyte, then it'll ionise exactly the same. So the ratio of peak areas will be exactly the same as the ratio of amounts. All your calibration curves will be perfectly linear with a gradient of 1"
Student and I look at a couple of calibration curves. They are perfectly linear with gradients ranging from about 0.95 to about 1.05, even when the corresponding external calibration curves are very curvy.
Student: "you see, all you're measuring is how accurately you made up the internal standard and the analyte stock! What's the point of wasting all those runs to make a calibration curve?"
I rather feel the student has a point. I understand that if the internal standard isn't chemically identical to the target analyte, we probably need to do a calibration curve, as we can't guarantee they'll have the same response. But why am I doing internal standard calibration curves when I went to the trouble of getting a deuterated version of my target analyte? Surely isotope dilution on its own is enough?