11.1: Introduction
Introduction
Spectrophotometers are one of the most frequently used tools by scientists to determine both the presence and concentration of dissolved chemicals. As radiant energy (visible light) strikes matter, molecules will absorb certain wavelengths of light and transmit or reflect others based on the nature of their chemical bonds. For example, proteins and nucleic acids absorb wavelengths in the visible light range of 240-300 nanometers (nm), pigments and dyes absorb light in the 400-770-nm range, and other organic molecules absorb wavelengths above 770-nm. Each chemical has a distinctive atomic arrangement and bonding pattern, and thus absorbs or transmits different wavelengths of visible light in a pattern that is unique for that chemical. This unique pattern of light absorption and transmittance creates a “fingerprint” for that chemical. In this exercise you will determine the unique “fingerprint” for a colored molecule and use a spectrophotometer to measure the concentration of a chemical in a given sample.
Spectrophotometers are instruments designed to detect the amount of light energy that is absorbed or transmitted by molecules dissolved in a solution. Since molecules have wavelengths unique to their structure, different chemicals and their concentrations can be identified based on their absorbance or transmittance.
A spectrophotometer is an instrument used for detecting the presence of any light-absorbing particles dissolved in a solution and for measuring the concentration of those particles. A light source inside the spectrophotometer emits a full spectrum of white light towards a compartment where a sample liquid is placed. The samples are prepared in cuvettes that are made using specialized plastics or quartz so that they do not absorb any light and will not affect our measurements. Before the light passes through the sample in the cuvette, an adjustable prism and diffraction grating filters the light so that only a single wavelength of light can be selected and allowed to pass through the sample. All molecules differ in how strongly they absorb each wavelength of light in the visible spectrum because of differences in their molecular structure and composition. This allows us to use a specific wavelength of light to detect the presence of, and quantify, one molecular compound in a simple or complex liquid mixture. Spectrophotometers are also calibrated by using a “blank” solution that we prepare containing all of the components of the solution to be analyzed except for the one compound we are testing for so that the instrument can zero out these background readings and only report values for the compound of interest.
Light passing through a sample solution will partially be absorbed by molecules present in the sample. The amount of light unable to pass through a sample is measured as the absorbance value. Absorbance is directly proportional to the concentration of the molecules and is measured on a logarithmic scale from 0 to infinity. The amount of light that is not absorbed is transmitted or passed through the sample. Compared to the amount of light entering the sample, the amount that exits is measured as a percentage of the light transmitted. Percent transmittance is inversely proportional to the concentration of the molecules in the sample and is measured on a linear scale from 0% to 100%.
A photodetector on the other side of the sample compartment converts the intensity of the light it receives into an electrical signal. The instrument can then calculate and display the absorbance and % transmittance values by measuring the difference between the intensity of light of the selected wavelength entering and exiting the sample.
The absorbance scale reflects the measurement of the amount of light absorbed and converted into absorbance (\(A\)) units by the spectrophotometer. Absorbance units are calculated by using the following equation:
\[\text{Absorbance} (A) = \log_{10} \left(\dfrac{1}{T}\right) \nonumber\]
where “\(T\)” is the decimal form of “\(\%T\)”
\[T = \dfrac{\%T}{100} \nonumber\]
If a solution containing a given dye is found to transmit 10% of the light when placed in a spectrophotometer, its absorbance then would be calculated as follows:
Transmittance (T) = 10% = 10%/100 = 0.10
Absorbance (A) = log 10 (1 / 0.10) = 1.0