biochem report
Common Introduction:
-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
-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