The amount of absorbed light, however, is not a direct measurement. Instead, absorbance is a calculation based on the % light transmittance using the following formula:
- Absorbance = 2.00 – log % light transmittance
The % light transmittance for a particular concentration of an analyte in solution can be calculated by measuring the amount of transmitted light detected in a spectrophotometer and dividing that by the amount of known incident light that enters into the analyte in solution (see the diagram to the right).
- % light transmittance = (Transmitted Light)/(Incident Light)
In practice, the spectrophotometer provides the absorbance values for the user.
Because the molar absorptivity coefficients and path lengths are constant for a particular analyte and the concentration is directly proportional to the absorbance measured by the spectrophotometer, the absorbance of a single standard solution of a known concentration analyte can be measured and then used to calculate the concentration of an unknown sample. The following equation can be used as the working formula for Beer's Law:
- (Standard Absorbance) / (Standard Concentration) = (Sample Absorbance) / (Sample Concentration)
An example of this calculation is as follows:
A creatinine assay creates a chemical reaction with creatinine, producing a colored complex. A calibrator with a standard concentration of creatinine at 2.5 mg/dL is subjected to the chemical reaction and produces a measured absorbance calculated by the spectrophotometer of 1.68.
An unknown patient sample is prepared in the same way as the standard and subjected to the chemical reaction in the assay and the absorbance calculated by the spectrophotometer is 0.96.
The known values are as follows:
- (1.68) / (2.5 mg/dL) = (0.96) / (unknown sample concentration)
Completing this calculation by solving for the unknown sample concentration, a result of 1.43 mg/dL creatinine is obtained.
