Chromatography Forum

Hello,

We use chemstation B.03.02 and have been quantifying analytes and creating calibration tables using external standards. I would like to switch to using an internal std for quantification, but am not exactly sure how to set that up in chemstation.

Currently we are looking at three separate analytes, we make a 5 point calibration. The external standards are prepped for 5 concentrations ranging from 7.3ug/ml to 149.4mg/ml


If using the ISTD method in chemstation would we spike each calibration level at the same concentration as the analyte, or would the concentration of the ISTD be constant at all levels? The response of the analytes we are looking for are linear, thus I would think that the ISTD concentration would match that of the analyte for each calibration level, but I am not very familiar with chemstation, and want to make sure i'm setting up the calibration correctly.

internal standard should be at a constant concentration in all samples and standards. It won't work properly if you use a range of standards containing equal concentration of internal standard and the compound you are trying to analyse. The reason is that in internal standard calibration instead of plotting PeakArea versus concentration, the software plots PeakAreaOfCompound/PeakAreaOfInternalStandard, and if the two are equal in all your standards, all your points end up as "1" (+/- errors)! (i.e. you have 2000/2000, 4000/4000, 8000/8000 etc. etc., whereas really you want something like 2000/3000, 4000/3000, 8000/3000 etc.)

I haven't used Chemstation for ages, so get someone else to help, but as I remember, you need to go to the bit where you specify report, and specify that your report is ISTD (instead of ESTD or area % or the various other options), even if you're not generating a report. You'll also need to indicate in the compound table which compounds go with which internal standards. There are some very good Agilent documents on the process if you Google.

Thank you for your suggestions. I am still having issues with chemstation. I created a 5 point calibration point for the compounds. In each level I spiked the reference std with the same amount of itsd. I set the report in chemstation to quantify based off the istd, and have added the same amount of istd to each sample ran. To check the calibration I reran one of the original calibration levels which contained 83.3 ug/ml of each analyte, and 333.33 ug/ml of the istd. The result was 62-63 ug/ml for the three analytes. I feel like I must not be doing something correct. My qc check should not have been so far off.

I've tried the above ie 2000/3000 , 4000/3000 , 6000/3000 , 8000/3000 etc and it works if you select the correct report format. It throws up some error message in that it doesn't like the curve for the internal standard within the curve information. I don't think you need to specify the internal standard amount in the sequence when running calibrations but you do for unknown samples. The analyte % and internal standard %s for the calibration samples can be set in the data analysis tab.
Regards
WK