BL4820 Biochemistry Techniques -- Lecture 1 -- Protein Assay
2. Standard Curves
For substances where the extinction coefficient is not known, but a pure sample or a "standard" sample is available, Beer's law can still be used by constructing a STANDARD CURVE. This is done by measuring the absorbance of several concentrations of the pure substance or the "standard" substance for which unknowns will be compared. This can even be done for substances which do not strongly absorb light, if the substance can be reacted with a dye which will produce a unique color when combined with the substance of interest.
For Example
- Reactant + Dye (in excess) = Colored Complex or Compound
A STANDARD CURVE can be made using known amounts of the Reactant:
Plot Used to Make the Standard Curve
The slope of the linear part of the curve can be used like an extinction coefficient to determine the amounts of reactant in an unknown solution. There are two ways to do this:
- Use the Absorbance of the Unknown and going along the Standard Curve find the corresponding Amount of Reactant. This is illustrated in the above Standard Curve Plot example by the dashed lines starting on the Y-Axis or Absorbance Axis and going out to intersect the Standard Curve (must be in the linear portion of the line) and then continuing down to the X-Axis or Amount of Reactant Axis. After finding the Amount of Reactant, the concentration of the reactant in the original solution can be found by dividing the amount of reactant in the sample by the volume of the sample used in the reaction that gave the absorbance of the unknown.
- Determine the slope of the line formed by the points in the linear part of the standard curve and use the slope to determine the Amount of Reactant using the absorbances found for the unknowns. This approach is best when done using a statistical program to calculate the linear regression fit for the equation of a line to the data set used to make the Standard Curve. The linear regression draws the "best-fit" line to the data and gives you the equation of the line. The way to apply this method is discussed at the very end of the Help Notes on Units.