Introduction Every chemical compound absorbs, transmits, or reflects light electromagnetic radiation over a certain range of wavelength. Depending on the range of wavelength of light source, it can be classified into two different types: UV-visible spectrophotometer : uses light over the ultraviolet range - nm and visible range - nm of electromagnetic radiation spectrum. IR spectrophotometer : uses light over the infrared range - nm of electromagnetic radiation spectrum. Devices and mechanism Figure 1 illustrates the basic structure of spectrophotometers.
Figure 1: Basic structure of spectrophotometers illustrated by Heesung Shim A spectrophotometer, in general, consists of two devices; a spectrometer and a photometer. Spectrometer : It produces a desired range of wavelength of light. First a collimator lens transmits a straight beam of light photons that passes through a monochromator prism to split it into several component wavelengths spectrum.
Then a wavelength selector slit transmits only the desired wavelengths, as shown in Figure 1. Photometer : After the desired range of wavelength of light passes through the solution of a sample in cuvette, the photometer detects the amount of photons that is absorbed and then sends a signal to a galvanometer or a digital display, as illustrated in Figure 1.
Figure 2: A single wavelenth spectrophotometer You need a spectrometer to produce a variety of wavelengths because different compounds absorb best at different wavelengths.
Figure 3: Absorbance of two different compounds Looking at the graph that measures absorbance and wavelength, an isosbestic point can also be observed. Figure 4: An example of isosbestic point Referring back to Figure 1 and Figure 5 , the amount of photons that goes through the cuvette and into the detector is dependent on the length of the cuvette and the concentration of the sample.
Figure 5: Transmittance illustrated by Heesung Shim. Beer-Lambert Law Beer-Lambert Law also known as Beer's Law states that there is a linear relationship between the absorbance and the concentration of a sample.
Example 1 Guanosine has a maximum absorbance of nm. Solution To solve this problem, you must use Beer's Law. Solution Using Beer-Lambert Law, we can compute the absorption coefficient. Example 4 In example 2 above, what is the molar absorption coefficient if the molecular weight is ? Solution It can simply obtained by multiplying the absorption coefficient by the molecular weight.
Example 5 The absorption coefficient of a glycogen-iodine complex is 0. Solution It can also be solved using Beer-Lambert Law. References Atkins, Peter and Julio de Paula. Physical Chemistry for the Life Sciences. New York: Oxford University Press, Chang, Raymond. Physical Chemistry for the Biosciences.
Currently, there are several major sources of water pollutants: chemicals and animal wastes from farms, oil and plastics, as well as industrial and untreated sewage discharges. These affect the quality of water in rivers and groundwater. Air pollutants come from burning agriculture and fossil fuels such as oil, gas, and coal. Specific pollutants or aggregates can be easily analyzed qualitatively and quantitatively by UV-vis spectrophotometers to control and monitor the quality of water and air.
Spectrophotometry is used as an analytical technique to find failures in the aerospace, chemical, oil, and gas industry by analyzing the metal alloys, such as iron and aluminum. For example, aluminum alloys are used in structural components in aerospace, so spectrophotometry can detect faults and weaknesses in structures.
Spectrophotometry is also used in the quality control of paint systems and cement. UV-vis spectrophotometers are suitable for colorimetry applications in industries that use pigments, such as printing, textiles, or ink production. Several sensors, devices, probes, or instruments use spectrophotometry to analyze biochemical and physical characteristics of solids and liquids.
There is a wide and growing range of spectrophotometers on the market today. For example, the CIs SpectraVue Leaf Spectrometer is a NIR spectrophotometer used to study many physiological processes and detect stress in plants and whose novel application is the non-destructive quantification of chemical concentrations and color analysis in plants.
Ecology and Environmental Science, B. Sc Agriculture. Feature image courtesy of Neal Fowler. Abushattal, A. What is different between spectroscopy and spectrophotometry? Bagai, E. How Is Spectrophotometry Used in Forensics?
Bassiri, E. Diaz, I. Re: What is different between spectroscopy and spectrophotometry? Graybeal, J. Hughes, V. The practical application of reflectance spectrophotometry for the demonstration of haemoglobin and its degradation in bruises. Journal of clinical pathology, 57 4 , — Morawski, R.
Keynote Lecture. Spectroscopy: Reading the Rainbow. Omoniyi, O. Rojas, F. Science Direct. Vo, K. Electromagnetic energy, collected from the sample, enters the device through the aperture yellow line and is separated into its component wavelengths by the holographic grating.
Simply put, the grating acts to separate each color from the white light. The separated light is then focused onto a CCD array detector where the intensity of each wavelength or each color if in the visible region is then measured by a pixel of the array. The CCD is then read-off to a computer and the result is a spectrum which displays the intensity of each wavelength of light. An example would be a spectral measurement of the visible range which we perceive of as color.
White light would enter the monochromator and be separated into a rainbow of each color. This rainbow, with blue light on one end and red on the other, would be focused on to the CCD.
Each pixel of the CCD would then measure the intensity of a color. The results would be a spectrum such as the one shown below. As shown, the blue pixels emit blue light, the green pixels emit in the green portion of the spectrum and the red pixels emit red light.
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