The Light Energy Absorbed By Colored Compounds

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Spectrophotometry is the process by which the light energy absorbed by colored compounds, is determined and used for quantitative and qualitative analyses. We use this process to often produce a standard curve, and use that to compare absorbance with different factors. In Experiment 3.1-3.4, spectrophotometry is used to produce the absorbance spectrums for the basic and acidic forms of PNP, to determine the pKa of PNP, and to develop standard curves for determination of unknown concentration and pH of unknown samples. Quantification of proteins is needed to determine the progress of protein purification. As the protein becomes more purified, its specific activity will increase as well. In Experiment 4.1, dilutions of PNP are prepared, and the Bradford Method was used to measure protein concentration. There are different types of chromatography, and we would be using gel filtration and affinity. Chromatography is defined as the separation of molecules based on properties such as molecular mass, charges or solubility. In Experiment 5.1, gel filtration chromatography was used to separate molecules based on their molecular mass. The stationary phase in the column contains tiny pores, those that are unable to fit into the pores are eluded first; thus, the small molecules are eluded last. We are able to witness a visual demonstration of gel filtration chromatography, the separation of three different colored compounds: blue dextran, cytochrome c, and potassium chromate. In

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