Lab _1 - Spec. Quantification

Lab _1 - Spec. Quantification - Objectives To develop a...

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Unformatted text preview: Objectives: To develop a familiarity with spectrophotometry and operations of spectrophotometer. - Beer-Lambert Law- Operate the spectrophotometer To construct two standard curves (using linear regression model)- The importance of a standard curve- Standard curve for o-NP - Standard curve for Bradford protein assay Spectrophotometric Quantification Principle of Spectrophotometer 1. White light separated by prism to different wavelengths. 2. Wavelength at which absorbance is at a maximum is selected. 3. Transmitted light is collected by a photoelectric tube. 4. Transmitted light energy is converted to electricity. 5. A galvanometer reads the electricity. %T = light intensity transmitted by sample (I 1 ) x 100 light intensity transmitted by blank (I o ) %T is NOT directly proportional to the concentration of absorbing substances. Absorbance is directly proportional to absorbing substances. The relationship between absorbance (A) and %T is given by: A = log(100/%T) or A = 2 – log(%T) Relationship between %T and Absorbance (A) The concentration of a solution is determined using the relationship known as Beer’s Law : A = ε l c A: absorbance (no unit) ε : extinction coefficient (L/mole*cm) l: path length (1 cm) c: concentration (mole/L) This relationship states that the absorbance of light at a given wavelength is directly proportional to the concentration of the substance absorbing the light. Beer’s Law c,ε Standard curve eliminates the need to know ε Beer’s Law : A = ε l c y = mx + b (linear equation) A: measured (y) m: slope of the line ε : not known (m) b: y-intercept l: 1 cm c: to be determined (x) Unknown known concentrations Why do many biochemical assays need...
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Lab _1 - Spec. Quantification - Objectives To develop a...

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