Chromatography Forum

When I inject 5 micro litres and 20 micro litres of the same sample and then calculate the results using my calibration curve, my results tell me that I have more concentration with 20 micro litres. This can't be so as it's the same sample and therefore should be the same concentration - just the amount is different. Can someone please explain.

Difficult to explain without knowing *exactly* what you did.

I have more concentration with 20 micro litres

How much more? 4x ? a few percent?

How did you generate the calibration curve?
Were the units on the amount axis mass or concentration?
Did you use a constant volume with individual concentrations or one concentration and different volumes?
Is the problem sample near the top or bottom of the linear range (or is it in the middle)?
How repeatable is the concentration difference you are seeing?

Concentration does not change of course. The amount on column changes. Example: If your solution is 100 ug/ml, and you inject 0.005 ml (5 micro liters) then 0.5 ug (micrograms) is being injected. If you inject 0.020 ml then 2 ug is being injected. With different volumes you are looking at mg on column only.

It would've been scary if it wasn't the case i.e. more stuff on column when injecting larger volume
The solution is simple --> Normalize the result with the injection volume.

Best Regards

It could be that you are overloading your column, depending on your concentration. Are you seeing any tailing on the 20 mcL injection? THat can make integration difficult and lead to higher values. It would help for us to see the two chromatograms.