Chromatography Forum

is it possible to have 2 external calibration curves (in order to quanify two separate peaks) in a sequence? if so, then how would one normally run the sequence?
assuming both sample solutions are stable at running conditions (for the entire sequence run time), would the sequence below be ok? and also could this method be transferred for method validation?

Sequence example:
2 diluent blank injections
6 check standard inj sample 1
6 check standard inj sample 2
5 calibration inj sample 1
5 calibration inj sample 2
2 check standard 1 injections
2 check standard 2 injections
2 sample 1 injections
2 sample 2 injections
2 check standard 1 injections
2 check standard 2 injections
2 diluent blank injections

many thanks for any replies!

Why not just put the two compounds in the same standard?. Am I misunderstanding your post?.

You may have to perform one initial injection of each peak solution solution at the highest concentration used to show there was no trace impurity interference, but after than you should be able to mix the two.
That reduces your injection numbers greatly.

Bruce Hamilton

yes that does sound like a better solution, thank you for the suggestion! i may try that. but, if you are running two external calibration standards like in the sequence shown above, then do you actually have to run, for example, 6 check standard injections of sample 1 and 6 check standard injections of sample 2 for hplc validation? could you just run 6 check standard injections of sample 1 and then 2-4 injections of check standard 2? that was what i was basically wondering.. if you had to treat both check standards from each sample the same throughout the entire sequence.
sample 1 is the main analyte peak, sample 2 was prepared for an excipient degradant peak that we want to quantify (for trending, information only).

Sequence example:
2 diluent blank injections
6 check standard inj sample 1
2 check standard inj sample 2
5 calibration inj sample 1
5 calibration inj sample 2
2 check standard 1 injections
0 check standard 2 injections
2 sample 1 injections
2 sample 2 injections
2 check standard 1 injections
2 check standard 2 injections
2 diluent blank injections

My brain wouldn't cope with that. I would run two separate sequences to separate the analyses.

In your sequence, any failure during one analysis would be visible by an auditor reviewing the second analysis, and two standalone shorter sequences mean less issues with potential carryover etc.

Please keep having fun,

Bruce Hamilton

thanks bruce! we are going to run the calibration with 2 compounds in the standard like you suggested. makes more sense overall.

Sunny -you're making this all too complicated, at least for those with ChemStation. Set up Calibration Table with two lines, one for analyte 1 and one for analyte 2. Yes, make up both standards in one autosampler vial.