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Since the advent of sciences until now, scientists in the forms of chemists, physicists, biologists etc have developed the field in an extensive manner that has resulted in scientific explanations of almost every living and non-living object or subject, one of the greatest achievements of human society. In this process, scientists have been successful in several laboratory methodologies to prove hypothetical concepts. In specific, this paper will attempt to discuss and compare two of the techniques that will be significant in understanding the role of laboratory techniques in different scientific processes.
In order to understand the mechanics of spectrophotometry, it is very imperative to discuss underlying fundamentals associated with this technique. In physics, second law of thermodynamics has set a condition for organisms to maintain their existence: utilization of the energy. In other words, there is a rule of unvarying supply of energy that enables organisms to continue their lives. Studies (Walker, pp. 50-59, 2008) have indicated that although there are many sources of energy, however, sun is the ultimate source for all organisms. In this case, pigments become the important components that are responsible for casing the ultimate energy of sun in the form of molecules that convert into chemical energy through photosynthesis.
Scientists (Gaw & Murphy, pp. 25-27, 2004) have proved that light is made of wavelengths of different modes that cause human eyes to see different colors due to reflection of light at unlike wavelengths. In other words, pigment molecules respond to light ways in diverse ways depending on the structures of these molecules. When a human looks at an object, the eyes process the wavelength of light qualitatively while remaining incapable of processing the quantity of light. For this reason, scientists were successful in creating an electronic instrument that enables the experts to arrange assessment of light's reflection and absorbance qualitatively, as well as quantitatively. Although it is a creation of physics, however, it is an observation that a broad range of fields has been beneficial of this capability of spectrophotometer, such as biochemistry, molecular biology, etc.
In particular, this technique uses an identified wavelength to shine and pass the light ways through an object. Presence of a particular pigment for absorbing light of the specific wavelength in the sample will result in passage of less amount of light. During this process, experts use the light meter to assess the quantity of light that is able to pass through the object. It is hypothesis that the greater presence of pigment in the object will result in more amount of absorbance, and less light for the light meter. Consequently, experts (Gaw & Murphy, pp. 66-68, 2008) are able to acquire spectrum of absorption for the specific pigment after passing light ways of different wavelengths during the technique, and completion of this methodology enables experts to identify particular pigment based on its absorption spectrum.
With the utilization of spectrophotometer, experts are able to study objects and their pigments through a complicated process of diffraction that usually performs by the use of prism in conventional manner. One of the significant characteristics of the spectrophotometer is its control knob that carries out the task of rotating diffraction grating that allows a specific wavelength to pass through a thin opening in the object (Walker, pp. 11-17, 2008). Upon passing, the spectrophotometer allows measurement of wavelength on the dial. During this process, the departing light is a one-color light that refers as the incident beam and Io symbolizes it that then bypass a channel like a test tube enclosing the sample object. This passage in the spectrophotometer is cuvette, a passage made of uniformed walls of glass allowing a consistent path of 1 cm.
From this tube, the incident beam transforms into a transmitted beam that hits the tube of photocathode in the instrument, and this transformation changes 'Io' into 'I'. At this stage, radiant energy of the beam becomes the major focus of the technique (Demain, pp. 57-63, 2008). However, there remain two cases, if the sample object absorbed no light, energy will not reduce due to special construction of the tube. On the other hand, if the sample absorbed light, there will be reduction of radiant energy. In both cases, striking of 'I' beam on the photocathode tube results in the generation of electric current that will be relative to the amount of radiant energy in the beam (Jiang, pp. 19-25, 2007). In the spectrophotometer, a meter measures the amount of electric current present in the tube and shows it on two scales: linear and logarithmic scales.
The former scale shows the percent of transmittal denoted by %T, whereas, the later one shows the level of absorbance in the object that is often refers by the symbol 'OD' (optical density) (Marshall, pp. 20-24, 2008). In this way, the linear scale helps experts to acquire the measurement of passed light that hit the photocathode tube, and the measurement of light not passed from the sample with the help of logarithmic scale on the instrument (Hayes, pp. 38-45, 2007). The use of scales does not end here, as both values enjoy a relationship with each other that is another significant characteristic of spectrophotometry. However, user of the spectrophotometer should take into consideration the fact that solvent used during the process has the ability of interacting with light wave, and thus, the methodology should involve deducting solvent from the measured absorbance (Boylan, pp. 41-47, 2002). With this step, the spectrophotometer is an efficient electronic instrument that provides an accurate reading of the transmitted, as well as absorbed amount of light. Regarding the relationship of both scales, the logarithmic scale allows experts to measure concentration of solute molecules in the object based on the principles of Lambert-Beer law.
Regarding the utilization of this technique, spectrophotometry is very useful in infrared regions, as well as ultraviolent-visible regions (Thomas, pp. 28-34, 2007). However, in infrared regions, studies have indicated that experts confront complications due to existence of glass and glass that causes some absorption of light (Linn, 1452-54, 2005). Still, comparing it with other instruments such as microplate reader, spectrophotometer has a number of advantages. For instance, it ensures results that are more accurate, allows measurement of molecules at any wavelength, enables experts to record an absorption spectrum, and lastly, carries out the measurement process of kinetic in a constant manner.
Now the paper will make efforts to discuss another laboratory technique applicable in biochemistry and few other fields that will involuntarily create its comparison with the technique of spectrophotometry discussed earlier in the paper. In specific, immunochemistry is not a technique but a particular branch of chemistry that includes examination of reactions and constituents of immune system (Van Emon, pp. 61-68, 2007); however, in a chemical manner rather than biologically. Since its advent, there have been major advancements in this branch that resulted in its consideration in various fields, such as virology, biochemistry, neuroscience, diagnostic science, etc (Van Oss, pp. 23-25, 1994). As mentioned earlier that immunochemistry is not a technique but a branch, therefore, there are many techniques available in this branch, such as immunohistochemistry, Western blotting, ELISA, immunization, fluorescence assays, etc (Work, pp. 13-15, 2008).
In this regard, the paper will focus on one of the techniques of immunochemistry: immunoassay based on the fundamentals of immunology and has been able to prove their significance during their availability since last four decades (Turner, pp. 59-62, 1997). In addition, it has been an observation that immunoassays have been very successful in their implementation in the field of biological monitoring, as compared to spectrophotometry that dominated the field of environmental monitoring (Johll, pp. 20-22, 2006). In particular, biological monitoring is all about assessing the absorbed amount of chemical after a disclosure. One of the major factors of significance of immunoassays in the field of biological monitoring is due to bioavailable characteristic of the internalized chemical in the organic system (Diamandis, pp. 48-57, 1996).
Principally, immunoassay is a term in broad perspective that refers to any technique that carries out the detection of response of any antibody to a foreign body, or in technical terms, antigenic analyte (American Society for Microbiology, pp. 500-641, 2006). Interaction of the two molecules allows experts to recognize, confine, filter, and quantitate the foreign molecule from the immune system (Arrhenius, pp. 54-55, 2008). Analysis of the material related to immunochemistry indicated that detection of macromolecular bodies such as bacteria was the major objective of creating technique of immunoassay (Turner, pp. 69-79, 2008); however, the technique developed, and now, it allows detection of other molecular bodies, such as chemicals in industries, drugs, etc (Wild, pp. 33-37, 2001).
Although immunoassays are dominant in biological monitoring, however, analysis has shown that experts are now putting efforts to use for environmental monitoring as well for identifying pollutants in the environment while spectrophotometry is focusing on pigments to assess infrared regions (Maisano, pp. 54, 2010). While comparing immunoassays with spectrophotometry, it is an observation that some of the significant factors of utilization of immunoassays in biological monitoring are sensitivity and simplicity that are quite less in spectrophotometry (Gore, pp. SPECTROPHOTOMETER & IMMUNOCHEMISTRY
Further scrutiny of the process of immunoassay methodology in immunochemistry has indicated that it develops in four specific phases that "antibody production, antibody characterization, method validation, and final field studies" (Wheelis, pp. 37-42, 2007) that often takes months and even years to complete. One of the basic reasons of such long period is due to extensive amount of time during the production of antibody that includes its extraction, purification, and categorization for immunoassay, a very expensive and prolonged process (Alexander & Ansell, pp. 106-80, 2006). In this first stage, experts vaccinate animals with the identified antigen and monitor levels of antibody, and upon high measurement of antibody, experts segregate producer cells that allows production of antibody, essential for carrying out immunoassays (Philips, pp. 35-38, 1992).
Subsequent to production of antibodies, process involves their characterization that involves assessment of specific characteristics of antibody, as well as its similarities with other antibodies. It is observation that experts (Butler, pp. 11-19, 1991) often avoid similarities that create complexities for the process, and thus, it is very imperative that experts should report every similarity of antibody with other chemical structures (Cermilli & Ardizzoni, pp. 1067-75, 2009). Evaluation of different arrangements of immunoassay in terms of sensitivity and preciseness is an essential step required before the stage of method validation. Later, verifying linear range for analyses is an important task of the third stage that follows with the determination of recovery studies, as well as connection with different chemical techniques that allow the experts to validate the method in a significant manner (Platts-Mills, pp. 255-58, 1998).
Lastly, final field studies involve identification of agent of the exposure, as it will determine the specificity and sensitivity of immunoassay. It is an observation that immunoassays are ideal for few conditions, such as public health issues that involve recurring evaluations, and thus, ideal for immunoassays due to their expensive costs (Brennan, pp. 1-10, 2003). In addition, agents needing lower limits of measurement and objects with matrix interference are also perfect for immunoassays. Studies (Holmes & Reed, pp. 22-24, 2007) have indicated that developing countries in African continent are benefiting extensively from antibody analyses of immunoassay due to unavailability of other forms of complicated methods.
One of the other differences between immunoassay and spectrophotometer is their nature, as the former is a methodology of analyses, whereas, the later one is an electronic instrument. Moreover, there are different versions of spectrophotometer, and on the other hand, there are different examples of immunoassays, such as radioimmunoassay, fluorescence immunoassay, etc (Geddes, pp. 27-38, 2008). Lastly, in order to understand the practicality of immunoassay, experts usually carry out it in three steps: arranging immunological reaction, magnetic separation, and finally, color development. In the first step, experts put water sample, labeled pesticide sample, and magnetic particles into a test tube that initiates the reaction process taking approximately 5-10 minutes. Subsequently, experts create a magnetic field that isolates the non-labeled solvents in the test tubes that follows with addition of labeled pesticides' substrates, which causes the sample to acquire a color. These steps relates immunoassay, and on a broad perspective, immunochemistry with spectrophotometer, as experts then utilize this electronic instrument to measure the concentration of pesticides in the sample.
Conclusively, the paper has discussed some of the significant aspects of two specific techniques: Spectrophotometry and Immunoassay (Immunochemistry). Although paper discussed and compared the two techniques in light of numerous studies and researches, however, it is very imperative to continue research based on new outcomes that will allow a more updated and critical understanding of these two laboratory techniques. It is an expectation that the paper will be beneficial for students, teachers, and professionals in better understanding of the topic.