Colorimetric Analysis Lab Report
OBJECTIVE The objective of this experiment was to analyze and determine the unknown concentration of a protein solution by utilizing two different colorimetric techniques; Biuret and Lowry. The Biuret method was used with unknown #2 and the Lowery method was used for unknown #1. After the concentration of each unknown was analyzed (by Biuret or Lowry method), the alternate objective was to compare the results achieved by each method and to determine if the results from the approaches were consistent in contrast to each other.
THEORY
Biuret and Lowry methods use colorimetry as a tool to analyze protein concentration quantitatively. While both methods are respected and widely used, the Lowry method has a sensitivity level that is
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Increased sensitivity is achieved by biuret complexes that react with phosphomolybdotungstate acid reagent (Folin & Ciocalteu’s Phenol reagent) to produce an altered color that is absorbed at 750 nm. Greater molar extinction coefficients are achieved in the resulting compound, which lends to the increased sensitivity to lesser amounts of protein. This method characteristically has an even more increased vulnerability than the Biuret method to ammonium salts, reducing agents, and detergents which can alter the results for similar reasons. For this reason, the Lowry experiment should be conducted first to reduce the likeliness that the glassware would be contaminated with high concentrations of protein …show more content…
The 2.5 mL of Albumin Standard (200µg/mL) was distributed among test tubes 0 through 5 (0.00, 0.25, 0.50, 0.75, 1.00, and 0.00 mL respectively). Then, 1.00, 0.75, 0.50, 0.25, 0.00, and 0.00 mL of water was added to test tubes 0 through 5 respectively. One mL of the unknown was added only to test tube #5. Four mL of the biuret reagent was added to each test tube and allowed to stand for 30 minutes at room temperature. Then, the absorbance was determined for each test tube at 545 nm after tube 0 was used to tare the colorimeter to zero absorbance. Excel was used to format the data for
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