-Beer-Lambert Law found by Beer August and Johann Heinrich Lambert is the linear relationship between the attenuation of light and concentration of the material through which the light is travelling. It states the absorbance is proportional to the concentration when a parallel beam of monochromatic radiation of equal pathlength is passing through a homogenous concentration. A=εbc, where A is absorbance, ε is molar absorptivity (L molˉ1cmˉ1), b is the pathlength (1cm) and c is the concentration of the solution (L molˉ1)
At high concentration beer lambert law is not applied because of the electrostatic interactions between molecules and at high concentration, there are changes in refractive index. Spectrophotometer:
-A spectrophotometer is an instrument that measures the intensity of light absorbed after it passes through a sample solution. With the spectrophotometer, the amount of a known chemical substance (concentrations) can be known by measuring the intensity of light detected. It is used in quantitative analysis in various fields such as biology, chemistry. -ONPG is a compound structurally similar to lactose and thus is used as a substrate instead of lactose. Lactose Galactose + Glucose, however these products are not easily quantifiable. By substituting glucose with a compound that can be easily noticed with a distinguishable colour change, we can then assay for lactose activity. ONPG is colourless but gives Galactose and a yellow coloured compound, 0-Nitrophenol (ONP) Galactose-ONP (colourless) Galactose + ONP (yellow)
Image: Formation of ONPG (Paul.H.,2012)
Experiment 5: Quantitative Analysis of protein using a spectrophotometer Using the biuret test, we can quantify the concentration of proteins in fluid. And by adopting a spectrophotometer that measures the intensity of light absorbed after it passes through a protein solution, we can then determine the absorbance of the protein solution. Using the spectrophotometer we can also do an enzyme assay using different concentration of substrate and draw out a standard curve to find out the concentration of the unknown. Proteins are found in blood and other body fluids such as urine. We measure the concentration in urine so as to check for infection such as diabetes. In the laboratory, we measure the concentration of proteins for scientific and clinical investigations. Experiment 6: Stopped enzyme assay to study B-galactosidase The objective of this experiment is to find out how temperature affects substrate concentration on enzyme activity. Lactose is a sugar found in milk. However, many adults are deficient in lactase, an intestinal enzyme that catalyses digestion of the same substrate. Researched has shown that 70 to 90% of adults around the world are lactose-intolerant. Lactose undergoes enzymatic hydrolysis to give galactose and glucose. However, these products are not easily quantifiable therefore lactose is being replaced by OPNG which gives galactose and a yellow coloured compound ONP. The colour change is proportional to the concentration and thus is used as a direct signal of activity. In stopped enzyme activity, the reaction is not affected by the time as only the final absorbance when all activity has stopped is recorded. Experiment 7: Continuous enzyme assay to study B-galactosidase The objective of the experiment is to conduct a continuous assay of B-galactosidase for hydrolysis and to find out the velocity of B-galactosidase at different substrate concentrations of OPNG initially. In enzymatic reactions, the substrate binds to the active site of the enzyme forming an enzyme substrate complex and then it decompose to from back the free enzyme and a new product. The Kinetic data can be plotted as a Michaelis-Menten curve. The Michaelis-Menten curve shows how the velocity of a reaction varies with substrate concentration. Michaelis-Menten rate equation: E + S ES P + E
Assumption: concentration of...
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