Notes on a Lab Experiment

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MATERIALS
Bromophenol blue, mixture of bromophenol blue and methyl orange solutios, micropipette, cuvette, test tubes, tips. PROCEDURE
Part 1: Determination of Amax of bromophenol blue
1. The wavelength of spectrophotometer is set to 470nm
2. Distilled water is filled into a 1cm cuvette and the smooth surface of the cuvette is cleaned using a paper towel. The cuvette is placed into the spectrophotometer and was re-zeroed. This cuvette is used as a blank. 3. The cuvette containing distilled water is removed and replaced with another cuvette which contains bromophenol blue. 4. The absorbance is read and recorded in Table 1.1 and the experiment is repeated with the wavelength 500, 530, 560, 590, 620, 650 and 680nm. Each experiment is re-zeroed using the blank cuvette after setting each wavelength. 5. A graph of absorption spectrum is plotted (absorbance readings versus corresponding wavelength) and the wavelength with maximum absorbance reading from the graph is determined. Part 2: The effect of concentration on absorbance of bromophenol blue solution 1. The mixture of tubes 1-6 is prepared and labelled according to Table 1.2a by using a micropipette and a vortex mixer to mix the contents. 2. The spectrophotometer is set to the Amax wavelength for bromophenol blue obtained in part 1. Tube 1 is used to zero the absorbance reading. 3. The absorbance reading of tubes 2-6 are measured by using the same cuvette used for tube 1.The absorbance readings are recorded in Table 1.2a The samples are returned to the original test tubes 4. The concentration of the bromophenol blue solutions in the test tubes 1-6 are calculated and the concentrations are recorded in Table1.2a 5. A standard concentration curve of absorbance versus concentration of bromophenol blue is plotted. The molar absorptivity of bromophenol blue at Amax of bromophenol blue (in unit L mg-1 cm-1 as described in Beer-Lambert’s Law) by using the standard concentration curve. Part 3: Determination of the concentrations of the bromophenol blue solutions of unknown concentration 1. The absorbance of two bromophenol blue solutions (Tubes A & B) of unknown concentration at the Amax of bromophenol blue is measured. The cuvette containing distilled water is used as a blank to zero the absorbance reading before measuring the absorbance of A and B. 2. The results are recorded in 1.2A and the concentrations of both unknowns are determined by using the (1) standard concentration curve used from Part 2 and (2) using the formula of Beer-Lambert Law. The calculations are shown and the results obtained from these two methods are compared. Part 4: The effect of concentration on absorbance of methyl orange solutions 1. The mixtures of tubes 1-6 are prepared according to Table 1.2b and the contents are mixed using a vortex mixture. 2. The spectrophotometer is set to the Amax wavelength of methyl orange which is 460nm and the absorbance reading is zeroed using the content in tube 1. 3. The absorbance reading of tubes 2-6 is measured by using the same cuvette used for tube 1. The absorbance readings are recorded in Table 1.2b 4. The samples are not discarded but returned to the original test tubes. The concentration of methyl orange solutions in tubes 1-6 are calculated and recorded in Table 1.2b. 5. A standard concentration curve of absorbance versus concentration of methyl orange is plotted. The molar absorptivity coefficient of methyl orange at 460nm (in unit L mg-1 cm-1 as described in Beer Lambert’s Law) is determined.

Part 5: Determination of the concentrations of two different solutes, bromophenol blue and methyl orange, in mixture C 1. The Amax of methyl orange is 460nm. The absorbance of bromophenol blue solutions in Tubes 1-6 is measured in Table 1.2a at the Amax of methyl orange. The results are recorded in Table 1.2a. 2. A standard concentration curve of bromophenol blue at Amax of methyl orange is plotted. The molar absorbtivity coefficient of bromophenol blue...
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