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 uses photometers, known as spectrophotometers, that can measure the intensity of a light beam at different wavelengths. Although spectrophotometry is most commonly applied to ultraviolet, visible, and infrared radiation, modern spectrophotometers can interrogate wide swaths of the electromagnetic spectrum, including x-ray, ultraviolet, visible, infrared, or microwave wavelengths.
Overview Spectrophotometry is a tool that hinges on the quantitative analysis of molecules depending on how much light is absorbed by colored compounds. Important features of spectrophotometers are spectral bandwidth (the range of colors it can transmit through the test sample), the percentage of sample transmission, the logarithmic range of sample absorption, and sometimes a percentage of reflectance measurement.
A spectrophotometer is commonly used for the measurement of transmittance or reflectance of solutions, transparent or opaque solids, such as polished glass, or gases. Although many biochemicals are colored, as in, they absorb visible light and therefore can be measured by colorimetric procedures, even colorless biochemicals can often be converted to colored compounds suitable for chromogenic color-forming reactions to yield compounds suitable for colorimetric analysis. 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. 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.
An example of an experiment in which spectrophotometry is used is the determination of the equilibrium constant of a solution. A certain chemical reaction within a solution may occur in a forward and reverse direction, where reactants form products and products break down into reactants. At some point, this chemical reaction will reach a point of balance called an equilibrium point. To determine the respective concentrations of reactants and products at this point, the light transmittance of the solution can be tested using spectrophotometry. The amount of light that passes through the solution is indicative of the concentration of certain chemicals that do not allow light to pass through.
The absorption of light is due to the interaction of light with the electronic and vibrational modes of molecules. Each type of molecule has an individual set of energy levels associated with the makeup of its chemical bonds and nuclei and thus will absorb light of specific wavelengths, or energies, resulting in unique spectral properties. This is based upon its specific and distinct makeup.
The use of spectrophotometers spans various scientific fields, such as physics, materials science, chemistry, biochemistry, chemical engineering, and molecular biology. 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. Spectrophotometry is often used in measurements of enzyme activities, determinations of protein concentrations, determinations of enzymatic kinetic constants, and measurements of ligand binding reactions. 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 astronomy, the term spectrophotometry refers to the measurement of the spectrum of a celestial object in which the flux scale of the spectrum is calibrated as a function of wavelength, usually by comparison with an observation of a spectrophotometric standard star, and corrected for the absorption of light by the Earth's atmosphere.
History Invented by Arnold O. Beckman in 1940 , the spectrophotometer was created with the aid of his colleagues at his company National Technical Laboratories founded in 1935 which would become Beckman Instrument Company and ultimately Beckman Coulter. This would come as a solution to the previously created spectrophotometers which were unable to absorb the ultraviolet correctly. He would start with the invention of Model A where a glass prism was used to absorb the UV light. It would be found that this did not give satisfactory results, therefore in Model B, there was a shift from a glass to a quartz prism which allowed for better absorbance results. From there, Model C was born with an adjustment to the wavelength resolution which ended up having three units of it produced. The last and most popular model became Model D which is better recognized now as the DU spectrophotometer which contained the instrument case, hydrogen lamp with ultraviolet continuum, and a better monochromator. It was produced from 1941 to 1976 where the price for it in 1941 was US$723 (far-UV accessories were an option at additional cost). In the words of Nobel chemistry laureate Bruce Merrifield, it was "probably the most important instrument ever developed towards the advancement of bioscience."
Once it became discontinued in 1976, Hewlett-Packard created the first commercially available diode-array spectrophotometer in 1979 known as the HP 8450A. Diode-array spectrophotometers differed from the original spectrophotometer created by Beckman because it was the first single-beam microprocessor-controlled spectrophotometer that scanned multiple wavelengths at a time in seconds. It irradiates the sample with polychromatic light which the sample absorbs depending on its properties. Then it is transmitted back by grating the photodiode array which detects the wavelength region of the spectrum. Since then, the creation and implementation of spectrophotometry devices has increased immensely. Design
There are two major classes of devices: single-beam and double-beam. A double-beam spectrophotometer compares the light intensity between two light paths, one path containing a reference sample and the other the test sample. A single-beam spectrophotometer measures the relative light intensity of the beam before and after a test sample is inserted. Although comparison measurements from double-beam instruments are easier and more stable, single-beam instruments can have a larger dynamic range and are optically simpler and more compact. Additionally, some specialized instruments, such as spectrophotometers built onto microscopes or telescopes, are single-beam instruments due to practicality.
Historically, spectrophotometers use a monochromator containing a diffraction grating to produce the analytical spectrum. The grating can either be movable or fixed. If a single detector, such as a photomultiplier tube or photodiode is used, the grating can be scanned stepwise (scanning spectrophotometer) so that the detector can measure the light intensity at each wavelength (which will correspond to each "step"). Arrays of detectors (array spectrophotometer), such as charge-coupled devices (CCD) or photodiode arrays (PDA) can also be used. In such systems, the grating is fixed and the intensity of each wavelength of light is measured by a different detector in the array. Additionally, most modern mid-infrared spectrophotometers use a Fourier transform technique to acquire the spectral information. This technique is called Fourier transform infrared spectroscopy.
When making transmission measurements, the spectrophotometer quantitatively compares the fraction of light that passes through a reference solution and a test solution, then electronically compares the intensities of the two signals and computes the percentage of transmission of the sample compared to the reference standard. For reflectance measurements, the spectrophotometer quantitatively compares the fraction of light that reflects from the reference and test samples. Light from the source lamp is passed through a monochromator, which diffracts the light into a "rainbow" of wavelengths through a rotating prism and outputs narrow bandwidths of this diffracted spectrum through a mechanical slit on the output side of the monochromator. These bandwidths are transmitted through the test sample. Then the photon flux density (watts per meter squared usually) of the transmitted or reflected light is measured with a photodiode, CCD or other light sensor. The transmittance or reflectance value for each wavelength of the test sample is then compared with the transmission or reflectance values from the reference sample. 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.
In short, the sequence of events in a scanning spectrophotometer is as follows: The light source is shone into a monochromator, diffracted into a rainbow, and split into two beams. It is then scanned through the sample and the reference solutions. Fractions of the incident wavelengths are transmitted through, or reflected from, the sample and the reference. The resultant light strikes the photodetector device, which compares the relative intensity of the two beams. Electronic circuits convert the relative currents into linear transmission percentages or absorbance or concentration values.
In an array spectrophotometer, the sequence is as follows:
The light source is shone into the sample and focused into a slit The transmitted light is refracted into a rainbow with the reflection grating The resulting light strikes the photodetector device which compares the intensity of the beam Electronic circuits convert the relative currents into linear transmission percentages and/or absorbance/concentration values
Many older spectrophotometers must be calibrated by a procedure known as "zeroing", to balance the null current output of the two beams at the detector. The transmission of a reference substance is set as a baseline (datum) value, so the transmission of all other substances is recorded relative to the initial "zeroed" substance. The spectrophotometer then converts the transmission ratio into 'absorbency', the concentration of specific components of the test sample relative to the initial substance.
Types of spectrophotometers Some common types of spectrophotometers include the following: UV-Vis spectrophotometer: Measures light absorption in UV and visible ranges (200-800 nm). Used for quantification of many inorganic and organic compounds. Infrared spectrophotometer: Measures infrared light absorption, allowing identification of chemical bonds and functional groups. Atomic absorption spectrophotometer (AAS): Uses absorption of light by vaporized analyte atoms to determine concentrations of metals and metalloids. Fluorescence spectrophotometer: Measures intensity of fluorescent light emitted from samples after excitation. Allows highly sensitive analysis of samples with native or induced fluorescence. Colorimeter: Simple spectrophotometers used to measure light absorption for colorimetric assays and tests.
Applications in biochemistry Spectrophotometry is an important technique used in many biochemical experiments that involve DNA, RNA, and protein isolation, enzyme kinetics and biochemical analyses. Since samples in these applications are not readily available in large quantities, they are especially suited to be analyzed in this non-destructive technique. In addition, precious sample can be saved by utilizing a micro-volume platform where as little as 1uL of sample is required for complete analyses.