Spectrophotometry Lab Report
Spectrophotometry
Prepared for: Dr. Joseph Dasso
By: Lucy Onsarigo
Biology 1406 C5L
September 23rd, 2014
Introduction
Spectophotometry is the ability of molecules to absorb and transmit light energy for determining the concentration of substances in a solution. (Mark Garcia 2014). The instrument used is called spectrophotometer to distinguish different compounds since they absorb light at different wavelength. Some have wide range of wavelength and the shorter the wavelength the higher the energy. For one to know the absorbed light one has to put a cuvette into a sample holder with a solution and record the amount of light transmitted and absorbed through the solution. A concentration of protein is used, the reaction between
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We added 1 ml of distilled water to test tubes labelled 2, 3,4 and 5. . To tube 1 we added 1ml of standard protein solution and recorded the concentration of the standard protein as 10mg/ml. To tube 2 we added 1ml of standard protein solution and shake it so that it can mix well. With a fresh pipette we removed 1ml from tube number 2 and added it to tube 3 then gently shake the tube. With another fresh pipette we removed 1ml from tube 3 and added it to tube 4, shake well. With a fresh pipette we removed 1ml from tube 4 and added it to tube 5 and gently we shake the tube and another fresh pipette we removed 1mlfrom tube 5 and discarded this fluid. Tube number 1to 5 had the same amount of fluid in them but the concentration of protein was becoming weaker as we progressed to tube number 5 and this is what we used to develop our standard curve.
In the tube that we labelled “U” we added 1ml of unknown protein and into the tube labelled “B” we added 1ml of water. At this point we had six tube with water and protein solutions, the “U” with unknown protein and water but tube 1 to 5 with a standard protein solution and water. The tube “B” had only water in it so it was acting as our blank, a blank in our experiment was used to calibrate the spectrophotometer machine, it was identical to other tubes only that it did not have the solute that we were
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Our independent variable in this experiment was the change in concentration, so my hypothesis of the lower the concentration will have the lowest absorbance due to less molecules was right. We did the experiment correctly, since we wiped off our finger prints and set the machine to zero. In all the tubes apart from the one marked “B” we added protein which was the substance being determined, so “B” was our blank used to calibrate the machine. It contained everything apart from the solution that was absorbing
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