Experiment title: Spectrophotometer and its function.
Measurement of the amount of light to the absorbance is called spectrophotometer. Spectrophotometer used to test the sample by passing through the light it’s worked to measure the light that passing through. Biological substances such bromophenol blue and methyl orange, are the common substances to be used in testing of interaction of substance with the light. These solutions called pigments where they usually can absorb the visible of light that passing through it. The cleared compounds or solution also can absorb the light that is not seen through the naked eyes. The wavelength specifically used to test the absorbance level is 400 to 600 those are in nanometer unit, in this experiment the wavelength to used is 470, 500, 530, 560, 620, 650, 680 nanometer. Measures the absorption of light by spectrophotometer with a specific wavelength used to determine the concentration of the pigments or solution.
Figure 1.1 shows the schematic diagram of the spectrophotometer. The instrument show the content of the light source which is come from the light source of a spectrophotometer. The arrow as in the Figure 1.1 to show the path of light from the light source. A prism refracting a light to the wavelength selector and transmit to the cuvette and the results sent to the recorder or computer.
1. To determine the wavelength of maximum absorption, Amax, of bromophenol blue. 2. To construct a standard concentration curve for bromophonol blue. 3. To determine the concentration of the unknown bromophenol blue solutions. 4. To determine the concentration of two different solutes, bromophenol blue and methyl orange, in a mixture. Materials:
1. Distilled water
2. Bromophenol blue,
3. mixture of bromophenol blue and methyl orange solutions, 4. micropipette,
5. 2 cuvettes.
6. test tubes (Labeled 1-6)
9. Tissue and glove
Part 1: Determination of Amax of bromophenol blue.
Before conducted experiment, safety precaution applied to prevent any circumstances. Wore lab coat and goggle, thoroughly wash hands and wore gloves. The wavelength of the spectrophotometer set to 470nm. Filled the cuvette with distilled water. Tap the cuvette with tissue until dry, avoided any interferences to sample such as finger prints, small drops of water to get the best results. If there’s any obstacle on the cuvette, there will be an error in reading the results. Put the cuvette inside the spectrophotometer, pressed ‘Auto Zero’ to zero the reading of the absorbance (ABS). Pour in bromophenol blue to the other cuvette. Took out the cuvette containing distilled water, replaced with the cuvette containing bromophenol blue solution, results recorded as it display on the spectrophotometer. Took out the cuvette content bromophenol blue, discarded the content into Discard Jar. Rinse thoroughly with the distilled water, make sure clean. The cuvette removed from the spectrophotometer.
1. Placed cuvette containing bromophenol blue.
2. Results recorded in Table 1.1.
3. Step repeated from 2 to 9 with different wavelengths of 500, 530, 560, 590, 620, 650, and 680nm. 4. Wavelength reset for different wavelength using the distilled water.
Wavelength (nm): 590nm
Absorbance (ABS): 1.122 ABS
Part 2: The effect of concentration on absorbance of bromophenol blue solutions 1. Micropipette set to the amount required.
2. Labeled and prepared mixtures to the test tubes provided accorded to Table 1.2a 3. Contents mixed well using the vortex mixer.
4. Spectrophotometer set to the Amax wavelength of 590nm for bromophenol blue that determined in Part 1. 5. Tube 1 solution used to zero the absorbance reading.
6. Same cuvette from solutions tube 1, used to measure the absorbance...
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