HPLC calibration Curves

[tranch66]

Hi

Long time reader, first time poster...Lets see if anyone out there can help?

Does any one know what the guidelines are for day to day calibration curve acceptance?

We us r2 for linearity determination, which are all within accepted criteria. However, the slope and intercept values differ day to day. What are the limits of acceptance for them, as they will affect concentration calculations

[DR]

There are typically no set limits for slope and intercept for calibration curves. Generally speaking, they are expected to be quite linear (hence the common minimum of 0.99 spec for r²). This said, you should still pay attention to slope and (particularly) to intercept. If they wander significantly from the norm, something is up (usually either a detector issue or interference resulting from deterioration of column performance or mobile phase /sample prep. error).

[tranch66]

thanks for the reply

But what is "wander significantly from the norm"?

Do the calibration curve values need to be within a certain level of acceptance day to day?

[tom jupille]

Re the intercept: In principle, it should be statistically indistinguishable from zero (to oversimplify a bit, the standard error interval of the intercept should include zero). As long as that is true, you're OK.

Re the slope: there is no set rule. In principle, since you will be running your samples and calibrators on the same system on the same day, variations do not matter. In practice, it's a good idea to do some form of control charting so that you can get an idea of how much day-to-day variation you can actually expect and also catch trends in your slopes. In specific situations, of course, you may have some additional constraints (e.g., for trace analysis, a large standard error in the slope will impact LOD and LLOQ).

[Anthony_Ng]

I was working in a contract testing lab and they require R2>=0.995. Basically 5 points almost touch the calibration curve.

One day we found that the slope is not as steep as usual. But still R2>0.995.

Lastly we got the answer that one of our autopipette failed, making the stock solution of calibrator more dilute than usual.

So that's a good idea of keep tracking with the slope value.

[tranch66]

Thanks for the reply guys.