Question

How can one determine unknown concentration given the standard curve of concentration versus absorbance?

Answer 1

The calibration curve is a method widely used in analytical chemistry to determine the concentration of a substance (analyte) in an unknown sample, especially in solutions. The method is based on the proportional relationship between the concentration and a certain analytical signal (for our case, absorbance). Knowing this relationship, it will be possible to know the concentration in a given sample by measuring that absorbance signal. Absorbance (light absorption) usually maintains a linear relationship with the concentration of solutes in solutions. Being a linear relationship, it can be graphically represented by a line in an absorbance versus concentration graph.

From the standard graph or calibration curve absorbance versus concentration, measuring the absorbance of the sample of unknown concentration, its concentration can be estimated by extrapolation on the calibration curve. (the measurement of the absorbance of the unknown sample is located on the axis of the ordinates, it is carried horizontally until the line is cut and then vertically lowered to the axis of the abscissas (concentrations))

Another way to obtain more accurately the concentration of the unknown sample is through the explicit equation of the line that, applied to our calibration curve, is given by:

Equation of a calibration line is:

Where:
b: is the ordinate at the origin (intersection of the line on the ordinate)
m:   is the slope of the line
C_A: is the concentration of the analyte, represented on the abscissa axis
A: is the measured absorbance signal

The slope m is calculated, taking two points of the line, this is:

the concentration of the unknown sample is obtained by measuring the absorbance signal, according to:

Concentrated sample unknown: