Chromatography Forum

It is common to use the same injection volume for calibration and test samples (unknown). But if you use internal standard, it should be possible to use different injection volumes, because Area ratio (substance to analyze/internal standard) is independent from injection volume.


In my case, the peak of the highest calibration point is very large (out of range, cap), so it could be convenient to inject a smaller volume for this calibration level. For the lower calibration points, the higher volume is better to achieve optimal sensitivity.


On the other hand, you could reduce the sensivity of the detector (adjust smaller gain) and inject always the same volume.

What is the best procedure?

In my case, the peak of the highest calibration point is very large (out of range, cap), so it could be convenient to inject a smaller volume for this calibration level. For the lower calibration points, the higher volume is better to achieve optimal sensitivity.

I would not recommend this. Besides possible accuracy issues , I assume the range of the calibration curve was set where it is because samples could be that high...

So you would either need inject all samples at both volumes or re-inject off scale samples... which is far from optimal!

On the other hand, you could reduce the sensivity of the detector (adjust smaller gain) and inject always the same volume.

If possible I would recommend this.

Doing this might give you a better signal to noise ratio at the low end despite smaller peaks.

Why are you using such a board calibration curve?

In my experience, when I have needed broad calibration curves maximum accuracy was not required at the low end.

I've not done much IS work, but when I have it's been for a relatively narrow calibration range.

I have always wondered how accurate the IS method would be when using a broad calibration even if you are in the linear range of the detector... Big differences in peak areas between unknowns and IS means potentially large measurement variability because on uncertainties in BOTH the numerator and the denominator.


- Karen