Spectrophotometry uses visible light, near-infrared light, and near-ultraviolet light to measure the reflection and transmission properties of solutions and cultures. The light intensity measurement is then converted to a function from which researchers can determine concentration or purity of solutions, as well as cell density. The quantitative analysis provided can be applied to forensic examinations as well as clinical studies.
Spectrophotometers are the most important instrument required for spectrophotometry and can measure different frequencies transmitted or reflected:
• UV-Vis spectrophotometers (ultraviolet-visible) measure the UV (190 to 380 nm) and visible (380 to 760 nm) spectrums of light
• Visible spectrophotometers measure visible light (380 to 760 nm).
• UV-Vis near-infrared spectrophotometers (UV VIS NIR) measure near-infrared frequencies (2600 to 3300 nm).
Single beam spectrophotometers require a standard to set the blank measurement before reading the test solution. On the other hand, double beam spectrophotometers, split the beam of light so that one hits the standard and the other the test sample. The ratio of intensities is measured, averting discrepancies in the light source or detector. However, this variant is bulkier and has a lower dynamic range.
NanoDrops™ are also classified as spectrophotometers; able to measure the concentration of DNA, RNA or protein in a single drop of solution.
Colorimeters measure light absorbance at specific frequencies, i.e. different colors, from a colored sample. Also able to gauge the concentration of a solute, the transmitted wavelength must match the absorbance frequency of the sample. Applications include pharmaceutical analysis, environmental testing, and biochemistry.
Microplate readers (plate readers) use pre-defined protocols and standard filters to measure, absorbance, luminescence, and fluorescence from solutions in a 96-well plate or larger. They are particularly suited towards ELISA and protein quantitation.