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Scientists have a magnitude of ways to determine the composition of various biological substances.
One of those ways is through the use of spectrophotometry. The field is still expanding rapidly in the instruments and methods. This paper will go through some of the processes that can be conducted when applying spectrophotometry to a broad range of biology. While several methods are available for use in biology, there are some advantages and limitations to each, which will be explored in this essay. Spectrophotometry has come a long way to incorporate computers to determining the various settings that are required to monitor the biological matter (Baker, 2010). Everything from the signaling the pathways in which proteins act, to the behavior of nucleic acids are explored during the application of spectrophotometry in biology.
Spectrophotometry allows scientists to investigate substances without having to touch them. Each object that is being investigated is held in a glass container that is sealed. As long as there is enough light shining through, the substance can be identified. When a scientist is inspecting a piece of matter that is toxic or otherwise dangerous, the technique is vital. The technique is particularly adept at identifying gases that are sealable. In biology, spectrophotometry can be used to tell the difference between pigments in plant cells, for example, but its uses extend far beyond that (Vodopich and Moore, 2002).
The spectrophotometry method when applied to biology can measure the amount of radiant energy contained in a substance and multiple wavelengths of light. Plant material, such as chlorophyll and various other substances suck up the energy. The material absorbs at various wavelengths. The type of light used is also absorbed at different lengths. For example, ultraviolet is absorbed at a shorter wavelength than infrared. It is possible with this technique to place a compound in a class and identify it. This is determined by measuring the wavelength by which peak absorption occurs, which is also known as the absorption maximum. This process is vital to identifying a substance that is not known. After looking at a series of standards, the scientist is able to determine how much concentration is contained in a substance, based on its sample. For example, the UV range can quantify nucleic acids (Lewiston, 2002).
In order to make use of spectrophotometry, scientists must utilize a spectrophotometer, which is the instrument that is used to measure how much light at a certain wavelength is passing through, usually a glass container. Beer’s law indicates that the amount of light that is absorbed by a substance is equal to the amount of the substance’s concentration. It is also determined by the amount of solute that is present. The amount of concentrated coloured solute that is in a solution can be ascertained in a scientific laboratory by measuring how much light can be absorbed at a specific wavelength. The spectrophotometer comes into play by allowing a selection of the wavelength to move through the solution. The wavelength that is chosen is usually corresponding with the absorption maximum of the salute (Fankhauser, 2007).
Measuring fluorescence signals is a very fine way of determining a substance. It is extremely detailed and often provides a very accurate reading of biochemical environments. This type of reading has been used to provide measurements for spectrum, lifetime, polarization and fluorescence intensity. The latter term measures the presence of fluorophores and various other micro-elements. Most fluorometers have a light source, detectors that are highly sensitive and a specimen chamber with built-in optical components. A lamp is the most common tool used to light a fluorometer. Xenon arc lamps are a common lamp that is used alongside the tool because it gives off a consistent intensity over a large area. They can emit either ultraviolet or infrared light. The orthogonal arrangement guarantees a lack of leakage of the light through to the detection area. Commonly, a high sensitivity photodetector is used alongside the device, usually a charge-coupled device camera or a photomultiplier.
In order to measure the spectral area, a bandpass filter or monochormeter is put into the path of the exiting light paths. This excitation area is the fluorescent intensity taken as a function of the wavelength at a consistent wavelength emission. This is the fluorescent intensity that is measured as part of a function of emission wavelength taken as a consistent excitation wavelength.
The fluorometer is also a mechanism that can measure the fluorescence lifetime because it can utilize processing electronics and signal these electronics with the subnanosecond resolution. While most fluorometers don’t have the capacity to function in the frequency and time domains, certain specially-made fluorometers are capable of operating with both factors considered. Both femtosecond or picosecond lasers are compatible in the time domain and these are commonly used to operate as excitation light sources. In this operation, a time-conscious single photon adding method is operational. A high-speed electronic is able to measure the time delay between the resultant fluorescence photon and the excitation light pulse. When using the frequency domain, the subject matter is turned on by the light source with a high level of frequency. The fluorescence signal is prompted at the same frequency. It is, however, amplitude-demodulated and phase-delayed. Heterodyning or homodyning can be used to as detection techniques to measure the fluorophore lifetime information.
In the polarization measurement, a scientist would insert a polarizer into the subject matter and into the emission light paths. These polarizers can then be rotated to calculate the perpendicular and the parallel parts of the fluorescence emission.
Measuring the fluorescence spectrum can play a particularly vital role in measuring the polarization, spectrum and lifetime of biological structure and function. Calcium and pH metabolites change the spectra of fluorosphores. This change occurs when the fluorescent amino acids cause the solvent to relax. For example, tryptophan and tyrosin can determine the levels of folding and protein structure. A renonance energy transfer can allow for spectal monitoring. In this process, the energy transfers between the two fluorophores. The donor fluorophore must go over the absorption spectrum of a secondary fluorophore, which is referred to as the acceptor. A scientist can then confirm the protein by labelling the correct substances with a fluorenscence resonance energy transfer. The scientist can determine the distance between each fluorophore by resolving the intensity of the relative fluorescence of the acceptor and of the donor. (So and Dong, 1996).
The stark spectroscopy application is perhaps the simplest form of spectrophotometry. It can be applied to various biological molecular materials and systems. The method is useful at determining vibrational Stark spectra in a manner that doesn’t need advanced equipment. By simply using frozen glasses, almost any molecular system can be studied, as well as proteins and ions. The application of spectrophotometry offers information about the change in polarizability that is associated with a transition, as well as the change in dipole. The dipole change is a reflection of the degree of charge separation occurring in a transition. In the polarizability change, a description of the transition’s sensitivity to an electrostatic field found in places such as ordered synthetic material or a protein, is available.
There are limitations to the application of stark spectrophotometry. Uncertainties are found while attempting to determine the magnitude of an electric field and the absorption lineshape. When attempting to measure the absorption lineshape, there is a systematic failure because the method only accommodates single beams. However, an absorption spectra that contains thicker samples, which are optically denser, can be put into a cuvette without the electrode coatings. This can be done with a dual-beam spectrophotometer to record the absorption lineshape. To attain the Stark sample, only the magnitude needs to be determined. The Stark spectrum has various derivatives of absorption, so both the accuracy of the wings of the absorption lineshape and a decent signal-to-noise ratio is needed.
The external electric field is contingent on the thickness of the sample, as well as the applied voltage, which can be determined mechanically by using precision calipers. A scientist could instead observe the empty sample cuvette to inspect the interference fringes. These measurements need to be done at room temperature in order to be effective. It should be noted, however, that the electric field’s exact magnitude is not exact, due to a disconnect between the internal field and the externally applied field.
Some of the applications don’t address all interactions in biology. For example, not every gene can be cloned successfully and each protein can’t be purified. Various components of protein are even lost because of the purification process and they can never be identified. The study is more about making trade-offs than anything else. The ability to decide which factor is more important than the other is a key skill. Over the years, the technology has grown, and it will continue to do so (Baker, 2010). Scientists have even made recent steps to determining how protein interactions, modifications and abundances change in cell cycle and environmental conditions, and this skillset continues to grow.
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