Spectrophotometry
Spectrophotometry is a branch of electromagnetic spectroscopy concerned with the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength.
Spectrophotometry is a tool that hinges on the quantitative analysis of molecules depending on how much light is absorbed by colored compounds.
A spectrophotometer is commonly used for the measurement of transmittance or reflectance of solutions, transparent or opaque solids, such as polished glass, or gases.
[3]: 65 However, they can also be designed to measure the diffusivity on any of the listed light ranges that usually cover around 200–2500 nm using different controls and calibrations.
[2] Within these ranges of light, calibrations are needed on the machine using standards that vary in type depending on the wavelength of the photometric determination.
To determine the respective concentrations of reactants and products at this point, the light transmittance of the solution can be tested using spectrophotometry.
The use of spectrophotometers spans various scientific fields, such as physics, materials science, chemistry, biochemistry, chemical engineering, and molecular biology.
[6] They are widely used in many industries including semiconductors, laser and optical manufacturing, printing and forensic examination, as well as in laboratories for the study of chemical substances.
[3]: 65 Ultimately, a spectrophotometer is able to determine, depending on the control or calibration, what substances are present in a target and exactly how much through calculations of observed wavelengths.
In the words of Nobel chemistry laureate Bruce Merrifield, it was "probably the most important instrument ever developed towards the advancement of bioscience.
A single-beam spectrophotometer measures the relative light intensity of the beam before and after a test sample is inserted.
In such systems, the grating is fixed and the intensity of each wavelength of light is measured by a different detector in the array.
Most instruments will apply a logarithmic function to the linear transmittance ratio to calculate the 'absorbency' of the sample, a value which is proportional to the 'concentration' of the chemical being measured.
The spectrophotometer then converts the transmission ratio into 'absorbency', the concentration of specific components of the test sample relative to the initial substance.
[3]: 21–119 Spectrophotometry is also a helpful process for protein purification[18] and can also be used as a method to create optical assays of a compound.
[20] In addition to the traditional Beer-Lamberts law model, cuvette based label free spectroscopy can be used, which add an optical filter in the pathways of the light, enabling the spectrophotometer to quantify concentration, size and refractive index of samples following the hands law.
Additionally, Spectrophotometers are specialized to measure either UV or Visible light wavelength absorbance values.
The concentration of a protein can be estimated by measuring the OD at 280 nm due to the presence of tryptophan, tyrosine and phenylalanine.
These curves can be used to test a new batch of colorant to check if it makes a match to specifications, e.g., ISO printing standards.
Largely, spectrophotometry is best used to help quantify the amount of purification your sample has undergone relative to total protein concentration.
[3]: 21–119 Following this testing the sample at 420 nm for specific interaction with ONPG and at 595 for a Bradford Assay the amount of purification can be assessed quantitatively.
[3]: 21–119 In addition to this spectrophotometry can be used in tandem with other techniques such as SDS-Page electrophoresis in order to purify and isolate various protein samples.
One major factor is the type of photosensors that are available for different spectral regions, but infrared measurement is also challenging because virtually everything emits IR as thermal radiation, especially at wavelengths beyond about 5 μm.
Applications may include evaluation and categorization of lighting for sales by the manufacturer, or for the customers to confirm the lamp they decided to purchase is within their specifications.
