STEWART WANI

RII4504F

BIOCHEMISTRY PRACTICAL 2 05.10.2011

INTRODUCTION A beam of light passing through a substance can be absorbed if the energy of the photons coincides with an energy transition available in the absorbing substance. Therefore, if light with many wavelengths passed through a substance, the absorption of that light will rise to a peak value and then fall again as the wavelength coinciding with an energy transition is reached and passed. MATERIALS AND METHODS Reagents: 0.00625mM Fast Green 0.2M Cobalt Chloride 0.12M HCl Experimental Procedure 1. Set zero and infinity absorbance on the Spectronic 20 as given in instruction on the instrument. Always put the Spectronic tube in the instrument with the perpendicular alignment mark in line with the mark on the instrument. Always wipe the outside of the tube carefully to remove spillage and finger prints before placing it in the instrument. A minimum of 3ml solution must be placed in the tube. 2. Place a 0.00625mM solution of Fast green in the instrument. Read the absorbance against water at 25nm intervals over the wavelength range 400-650nm. Take extra absorbance readings around the absorption maximum so as to determine accurately the wavelength of absorption maximum. Plot your values against wavelength to obtain the absorption spectrum of Fast Green. 3. Dilute the 0.2M cobalt chloride solution provided with an exactly equal volume of 0.12M HCl to give a 0.1M concentration. Obtain the absorption spectrum and maximum of CoCl2 using 0.12M HCl as your blank. 4. Use a 5.0ml graduated pipette to add to 4 test tubes the following volumes of 0.2M CoCl2 solution: 0.50, 1.00, 2.50, 3.50ml. Rinse the pipette with distilled water and add the exact volume of 0.12M to give a total of 5.0ml in each tube. Calculate the molarity of COCl2 in each tube. Measure the % transmission of each solution and of 0.2M COCl2 at the peak absorption obtained above, using 0.12M HCl as a blank. With the aid of log tables convert your % values to absorbance values. Plot absorbance and % against the molarity of the solution TABLE OF RESULTS Molarity (M) 0.00 0.02 0.04 0.10 Absorbance 0.00 0.0731 0.189 0.472 % Transmission 100 84.5 64.7 33.7

0.14

0.664

21.7

CALCULATIONS % Transmission = I x 100 IO IO = 100_____ I % Transmission Absorbance = log (IO/I) = log (100/% Transmission) QUESTIONS 1)

2) 475nm to measure concentration of K2Cr2O7 513nm to measure concentration of CoCl2 3) The light absorbed by K2Cr2O7 is green The light absorbed by CoCl2 is blue 4) The colors of the light absorbed are complementary to the colors of the corresponding solutions. The solutions take on the color of the light that was reflected by the solute. 5) % T against molarity is a curve, while absorbance against molarity is a straight line. The most useful graph is the absorbance against molarity, because it allows the determination of constants, and it is easier to tell whether or not the cobalt chloride conforms to the Beer-Lambert Law. 6) The solution obeys the Beer-Lambert Law, because the equation: log (IO/I) = bc, taking log (IO/I) as a function of concentration c, is an equation for a straight line. It is verified by the straight-line nature of the absorbance against molarity graph. CONCLUSION Substances have the ability to absorb and reflect light. The color of a substance is dependent on its absorption spectrum. If its peak absorption value lies within the range of visible light, the substance takes on a color that is the compliment of the color of light that it has absorbed.

REFERENCES http://firstyear.chem.usyd.edu.au/calculators/colour_wheel.shtml http://www.chemguide.co.uk/analysis/uvvisible/theory.html http://faculty.weber.edu/nokazaki/Comparative%20Animal%20Physiology/Laboratory/S pectrophotometry04-1.pdf David L. Nelson, Michael M. Cox, Lehninger Principles of Biochemistry 4th Edition, Freeman and Co., 2005