Determination of Cytochrome C: Preparing a Calibration Graph Introduction This is an experiment to determine the concentration of a colored compound in a solution by comparison of its light absorption with the light absorption of a standard solution of a known solution.
This experiment will be done by using spectrophotometer, as it will determine the amount of light that will be absorbed by the solution. Also by using the Gilson pipette it enables you to accurately make sure solutions have been measured correctly, also when using the Gilson pipette you should change the tips to stop contamination of the solution.Objectives Learn to use a spectrophotometer Determine the degree of light absorption by a colored compound Plot a spectrum of light absorption against wavelength is best to use for peak wavelength Make a standard solution of known concentration, and then plot them to a graph Understand calibration graphs Measure the concentration using a graph Writing a experimental report Method Refer to manual Results (Nm) (ABS)The table above shows the results for the determination of peak wavelength. Tube No. 1 2 3 4 5 6 Stock Solution (0.1mg/ml) (ml) 0 2 4 6 8 Deionized Water (ml) 10 8 6 4 2 0 Final Concentration [cytc] (mg/ml) 0 0.02 0.
04 0.06 0.08 1 ABS at peak = 405 nm 0 0.012 0.220 0.281 0.
649 0.661 Corrected ABS (-blank) 0 0.012 0.220 0.281 0.649 0.661 The above information shows the results of the Stock Cytc Solution 0.
1 mg/ml. The determination of Unknown Cyt C Concentration. ABS of Unknown = 0.410 ABS Units Concentration from Graph = The Sources Of error 0.02 = 4 % 0.410 Mass Structure of Cytochrome C 13,300 ï¿½ 0.
1 = 7.5 X 10-6 m Cytochrome C 12500 x 1 = 0.938 7.5 x 10-6 Purity of Cytochrome c: 0.661 x 100 = 70.46 % 0.
938ConclusionThe aim of the experiment was to determine the light absorbed by the solution, hence it was achieved as the results show the amount of absorption at different wavelength, the peak absorption shows me that there is a rise in absorption until it hits the peak at 405 nm, and then there is a sudden drop in absorption. The results of the calibration of cytochrome c show me that it goes up by each test tube, as more ml of stock solution is used. However, the results should show on the graph as a straight line, in this case there must have been an error or the solution was not prepared and measured correctly. The sources of error show a 4%. Overall the experiment was a success as the objectives were met and the absorption of the known solution were recorded.