Spectrophotometry relies on the varying capabilities of molecules to absorb photons of light. This absorbance is characteristic of specific compounds, and can therefore be utilized to quantify and classify the presence/quantities of various compounds. (Harris, 2007) With the use of standard calibration curves, the concentrations of various samples can be graphically interpreted as a function of the absorbance. The relation between absorbance and concentration is governed by the Beer-Lambert law, which states that the absorbance changes proportionally with the concentration. (Harris, 2007) This fundamental principle allows for a direct correlation between the examined absorbances and the corresponding concentrations. Some samples require preparation prior to direct examination, as they may be "invisible" to the spectrophotometer initially. These colorimetric analysis techniques utilize a dying reagent, which acts to increase the absorbance of the sample. (Congdon, et.al 1993)
[...] In order to determine the concentrations of various proteins, this investigation utilized several assays, as a method of both preparing and analyzing protein samples. The Coomassie Blue assay relies on the Colorimetric interactions, between the additional compounds and the protein analyte. This technique allows samples with little or no absorptivity to become “visible” either by chemically altering the component, or producing alternate compounds in proportional quantities. (Congdon, et.al 1993) This highly utilized technique is often employed for its ease of use, as well as its high sensitivity to analyte. [...]
[...] This discrepancy may be explained simply by a matter of elapsed reaction time. Colorimetric assays utilize the reactivity of components to essentially reveal a compound in solution. If this reaction has not run to completion, the values obtained may be inaccurate, as lower concentrations require much more time to react. (Gore, 2000) This condition may account for the inefficiencies observed, but may also be related specifically to poor sampling practices and the inclusion of foreign compounds. Variations within the path length seemed to follow a similar trend, as an increased path length should yield an increased molar absorptivity. [...]
[...] Calculated extinction coefficients for BSA and Gamma Globulin proteins by means of UV absorption spectrophotometry Protein Concentratio Path Absorbance Extinction Wavelength Average n (ug/ml) Length E-Coef. Coefficien t bulin bulin bulin Sample Calculation: Extinction Coefficient Beer Lambert: Absorption = (Extinction Coefficient) (Concentration) (Path Length) 0.608 = E (600 ug/ml) (1cm) E = 0.608 / 600 ug/ml = 0.00101 Figure 03. Standard curves for BSA and Gamma Globulin proteins derived by Lowry assay spectrophotometric analysis Table 04. Interpolated protein concentration values by means of BSA standardization Protein Dilutio Assay Absorbance Diluted Stock Avg Conc n Concentratio Concentratio (ug/ml) Factor n (ug/ml) n (ug/ml) Blue Blue lin Blue lin Blue Blue Blue lin lin lin lin Table Interpolated protein concentration values by means of Gamma Globulin standardization Protein Dilutio Assay Absorbance Diluted Stock Avg n Concentratio Concentratio Conc Factor n (ug/ml) n (ug/ml) (ug/ml) Blue Blue lin Blue lin Blue Blue Blue lin lin lin lin Table 06. [...]
[...] (Harris, 2007) With the implementation of distance, and absorptivity, the absorbance can be proportionally related to the concentrations of various compounds in solution. This relationship, defined by the Beer-Lambert law relies on two fundamental principles. As light passes through a sample, it is absorbed proportionally throughout the entire medium, and is therefore independent of the experimental spectrum. Secondly, the quantity of light absorbed by a sample relates directly to the quantity of analyte molecules present in solution. (Gore, 2000) This reaction implies that as the number of molecules increases within a sample (concentration), so does the overall absorbance of light. [...]
[...] Each protein ranged by upwards of 1000ug/ml, suggesting a major design flaw within analysis. The Gamma-Globulin standard resulted in an overall lower variability, although was still not within acceptable levels. 500 ug/ml) (Table 05) The hemoglobin protein also presented an especially discrete deviation from the other proteins. As previously stated, UV spectrophotometry is susceptible to the inclusion of interfering molecules. Molecules such as oxygen readily absorb UV radiation, and can potentially skew the results. (Aitken & Learmonth, 2002) Hemoglobin contains ferrous components, and thereby reacts to bind oxygen within the blood. [...]
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