Bio-Rad Microplate Manager Software - Manual (Page 110)

Microplate Manager User Guide
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variation, and the ratio of the explained variation to total variation becomes 1.000.
Or, more simply, the closer the correlation coefficient is to 1.000, the better the fit of
the regression.
Since the cubic spline is a local function, the r value is meaningless and no goodness
of fit may be assigned. (The cubic spline forces the curve through all the data points.)
Limitations of the Methods
Results calculated from mean absorbance data outside the range of the standard curve
may be unreliable. (For the purposes of this discussion, the range of the standard
curve is defined as the absorbance range between the highest and lowest mean
absorbances of the standards used to form the standard curve.) Calculated results that
are determined from mean absorbance results outside the range of the standard curve
are extrapolations, and should be treated as such when determining the final results of
your analysis. Microplate Manager indicates in the Unknown Concentration
Report when the standard curves are extrapolated to determine the concentration of
an unknown.
Results calculated from mean absorbance results at the extremes of the range of the
standard curve may also be unreliable. The linear regression, when properly fitted,
generally produces reliable results at the extremes of the range of the standard curve.
However, the quadratic, cubic, and logistic methods, by their mathematical natures,
can produce unreliable results at the extremes of the standard curve. None of the
methods will give a unique concentration for a given absorbance if the calibration
data is not monotonic in x (i.e., there is more than one x value associated with a
unique y value).