Spectrophotometers, as the name suggests, are a piece of equipment that measures the light absorption of a sample by using a focused beam of light going through the sample at a specific wavelength.
As this beam passes through, the different compounds in the sample absorb some of the light and once it exits the sample and hits the detector, the machine gives us a number representing how much of the initial light beam has been absorbed by the sample.
One of the major factors that determine the amount of absorbance a sample has is the concentration (c) of analyte inside.
As the concentration of compounds inside the sample increases, the space for light to pass through uninterrupted decreases, leading to higher absorption.
The other factor is path length (b), which means the longer the light beam has to travel through a sample, the higher the chance of it encountering a compound and being absorbed.
Usually, spectrometry cuvettes are standardised, and the path is 1 cm, which simplifies future calculations.
The last factor that should be considered is molar absorptivity, also known as extinction coefficient (ε).
This coefficient is a way to measure how good the analyte of interest absorbs light from the specific wavelength that has been shined on them.
This factor is also directly proportional to the absorbance.
As these three factors are directly proportional to the absorbance, the Beer-Lambert law can be used:
A= εbc
This equation is for a straight line, which has a y-intercept at 0.
