A method used to measure the light absorption of a solution and quantitatively determine the solution's concentration.
Physics, chemistry, biochemistry, and molecular biology.
Spectrophotometry is the quantitative study of electromagnetic spectra that is used to measure the light absorption as well as the diffusion or specular reflectance.
Spectrophotometry involves the use of a spectrophotometer. A spectrophotometer is a photometer that can measure intensity as a function of the color, or the wavelength of the light. Other important features of spectrophotometers include the spectral bandwidth and linear range. The most common spectrophotometers are used in the UV and visible regions of the spectrum, as well as into the near-infrared region.
There are two major classes of spectrophotometers; single beam and double beam. A double beam spectrophotometer measures the ratio of the light intensity on two different light paths, and a single beam spectrophotometer measures the absolute light intensity. Although ratio measurements are easier, and generally more stable, single beam instruments have advantages in a larger dynamic range with more compact results.
The spectrophotometer measures the fraction of light passing through a given solution. In a spectrophotometer, light is guided through a monochromator, which picks light of one particular wavelength out of the continuous spectrum. This light passes through the sample that is being measured. After the sample, the intensity of the remaining light is measured with a photodiode or other light sensor, and the transmittance for this wavelength is then calculated.
Based on the obtained transmittance, the concentration of the solution can then be determined using the Beer-Lambert law, in which,
Here, the distance the light travels through the material is l, E is the molar absorptivity of the absorber, and c is the concentration of absorbed species in the material.
- Sample solution
- Buffer where the sample is suspended in
A video for the basics and operations of general spectrophotometry can be viewed on YouTube. Click Here to see it.
- Open up the spectrophotometer and warm it up for few seconds.
- Set up the range of wavelength the researcher would like to obtain the transmittance of the solution sample from.
- Put buffer into the cuvette, place the cuvette into the slot, and obtain the light absorbance of the buffer with no samples and use this reading as the baseline.
- Clean the cuvetter again.
- Dissolve and mix some samples with the buffer in a small tube outside the cuvette.
- Put the sample into the cuvetter, and obtain the light absorbance and concentration of the sample.
If automated softwares are available in conjunction with the spectrophotometer, use these to plot out the light absorbance of the sample across the designated range of wavelength of the light applied, and record the concentration of the solution.
If automated softwares are not available, manually record the light transmission through the cuvette at different light wavelengths, and calculate the concentratino using the Beer-Lambert Law.