Hi, I am studying HPLC quantitative analysis. For the standard addition method, based on my study, I need to make a calibration curve for each sample. For each curve, I need to prepare several solution series. That is a huge load of work for all of my samples.
1. What should I do to minimize the work?
2. My pure analyte standard goes with the whole process (hydrolysis and derivatization). Do I still need to calculate the recovery rate of the analyte?
3. Still a tiny question for internal standard(IS). Why should the IS have similar physical and chemical properties with the analyte?
We are not doing that much standard additions but here is what I would do:
Run a classical calibration (preferably with internal standards) as 7 point calibration before each series (a series could contain 20-200 samples).
Run each sample with and without standard addition (also with IS). Run a calibration at the end of the series.
prerequisite of this is of course that You know Your sample concentrations with a precision of 2 orders of magnitude, otherwise You will work with too much standard added (cross contamination to Your samples) or too little standard addition (You will not be able to quantify the addition).
It is always good to know the recovery rate.
IS can be used for very different purposes. a) The most challenging is to level out matrix effects (pretty dirty samples and vulnerable ESI sources such as waters). for this the IS needs to elute as close as possible to the analyte and behave chemically as similar as possible. b) the least challenging: compensate for volumimetric issues (such as condensing not exactly to the point etc. this can be done by nearly anything. c) recovery IS (different to matrix effects but similar demands) in a multistep procedure You might need to control for SPE losses, condensation losses etc....
Success
Kai
You can run several molecules at the same time if the separation is good enough.
You already have good answers. Also, I would like to add a few, following the order of your questions:
1. What should I do to minimize the work?
As Catherine M G C Renard said “you can run several molecules at the same time if the separation is good enough”, and if you have a quality control system in place.
2. My pure analyte standard goes with the whole process (hydrolysis and derivatization). Do I still need to calculate the recovery rate of the analyte?
As Kai Bester said “It is always good to know the recovery rate”, but sometimes it is not required in official controls (unfortunately).
3. Still a tiny question for internal standard(IS). Why should the IS have similar physical and chemical properties with the analyte?
IS should have similar physical and chemical properties of the analyte because we simulate the behavior of the analyte in our analysis. Ideally it should be the isotope-labeled analyte.
I agree with Roberto Molteni. However, it is sometimes not possible to have isotope labelled IS for all analytes, the best possible match in terms of structurally related compound should be incorporated.