Chromatography Forum

Hi all, I am relatively new to HPLC but have been assigned to a very massive method development project- yey. I've been having trouble establishing response factors for external standards. The problem is, when my two standard curves have non-zero y-intercepts (which is always), I cannot establish a Response factor between the two substances except at specific concentrations, as the Response factor is specific to that concentration. This is because the Area/Concentration ratio varies based on the concentration. I am calculating the RF via (AreaA/ConcentrationA)/(AreaB/ConcentrationB)=RF. Am I misunderstanding the use of response factors or how to calculate them? Secondly, shouldn't the y-intercept be zero; and the fact that it isn't an indication of a poor run (ie. overloaded etc etc) or non-linearity?

Some examples:
If analyte A's standard curve is A=2x+1. At x=1 A/x=3/1, At x=2 A/x=5/2.
If analyte B's standard curve is A=3x+4. At x=1 A/x=7/1, At x=2 A/x=10/2.
At concentration 1, a response factor of 3/7
At concentration 2, a response factor of 2.5/5


Thanks!
Wes

Normally we try to find the source of problems with a non zero y-intercept (carryover, matrix effects, ...), but sometimes, there isn't much you can do. In this case you are forced to use at least 2 standards to prepare your calibration curve, where otherwise you can get away with only 1.

For your response factor question: divide the slopes of both regression lines to get the response factor, regardless of the concentration.

good luck

ace

What is the %RSD of each response factor over the calibration range calculated using the formula, peak area/concentration?

Are you trying to establish a relative reponse factor or are you planning to use one as an internal standard?