Juliana Park Mayumi Tamada CHEM 111B LAB/ M-F 1-4PM 15 August 2012 Spectroscopy Lab Introduction In this lab, the molar absorptivity of the complex FeLn2+ will be determined by using the absorbance of the complex and its concentration. The absorbance will be found by using a spectrophotometer. For the next part of the lab, the formula of the complex will be determined by also using the volume of ligand and the absorbance again. Experimental There are two different parts to the experiement. In the first part, five 100 mL flasks of 5 mL ligand solution, 5 mL 2 M sodium acetate, 4 mL 3 M NH2OH, and 1-5 mL Fe2+ solution are diluted with water.
Then, 3.4 g of ammonium sulfate was slowly added to the supernatant 1 as it was stirred for 15 min to achieve 50% saturation (85g/L of solution). The supernatant was then centrifuged at 9000 x g and 40C for 15 min and 5 ml of the second supernatant was transferred to a conical tube. The obtained second pellet was resuspended in 4 ml of distilled water and transferred into another dialysis
The known nutrient solutions were used to create a base-line for protein, starch, and sugars. As listed in Table 1; protein (5g/L), Starch (0.2g/L), and sugar (20g/L) were separated in to 9 different test tubes at 2ml a piece, 3 per nutrient solution and tested for colormetry with the 400ug of the three reagents Lugol’s, Biuret, and Benedicts. Further steps were taken with the nutrients treated with Benedict’s reagent and they were heated in a water bath until they reached a constant 65 degrees C for 7 minutes and let cool to see color change. Distinguishing Organic Molecules in Unknown Dietary Supplements The same reagents used in setting the baseline were used to test the unknowns for nutrient content. Each of the 3 unknowns was distributed by dispensing 2ml of sample solution in to three test tubes.
The total sample volume was made up to 13 μL by adding water. The reaction vial was placed on ice, and was added 2 μL each of 10x NTP labeling mixture, 10x transcription buffer, and T7 RNA polymerase. 1μL of protector RNase inhibitor was also added, and the contents in the vial were mixed gently and incubated for 2 hours at 37 degree Celsius. To remove the template DNA after transcription, 2 μL of DNase I was added and incubated for about 15 minutes at 37 degree Celsius. The reaction was stopped by adding 2 μL of 0.2 M EDTA at pH 8.
Review the effect of pH on enzyme function. Students should understand that enzymes function best at specific pH levels (which vary depending on the enzyme). Non-optimal pH levels can affect the shape of the enzyme, thereby decreasing its effectiveness as a catalyst. Extreme pH levels can permanently denature the enzyme protein, whereas less extreme pH conditions may only temporarily alter . Lactase is effective at pH 2 – 7 (including dH2O) and therefore breaks down the lactose sugar in milk into glucose and galactose.
Read the absorbance at 20 second intervals from the start of the mixing. Then record your measurements on the table. After two minutes remove the tube from the spectrometer and visually note the color change. 6) Now mix the contents of tubes 4 and 5, transfer to a cuvette, and repeat your measurements for two minutes at 20 second
I added varying levels of substrate to the test tubes in each experiment. The amount of substrates were .5 grams, 1 gram, 2 grams, 4 grams, and 8 grams. The output of the experiment (the dependent variable) was the number of molecules of product formed per minute at (106). RESULTS Test Tube # | pH Level | Amount of Substrate | Number of Molecules of Product Formed per Minute (106) | Test Tube #1 | 3 | .5g | 19 | Test Tube #2 | 3 | 1.0g | 39 | Test Tube #3 | 3 | 2.0g | 82 | Test Tube #4 | 3 | 4.0g | 96 | Test Tube #5 | 3 | 8.0g | 96 | | | | | Test Tube #1 | 5 | .5g | 39 | Test Tube #2 | 5 | 1.0g | 81 | Test Tube #3 | 5 | 2.0g | 168 | Test Tube #4 | 5 | 4.0g | 198 | Test Tube #5 | 5 | 8.0g | 198 | | | | | Test Tube #1 | 7 | .5g | 72 | Test Tube #2 | 7 | 1.0g | 145 | Test Tube #3 | 7 | 2.0g | 300 | Test Tube #4 | 7 | 4.0g |
Repeat steps 1-5 for trial 2. Variables and Control test: Independent Variable: Amount of each reactant poured into the test tubes Dependent Variable: Height of each solution Constant variables: Amount of time waiting for solution Data Table: Test tube # | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | Trial 1 | 0.5 cm | 1.0 cm | 1.1 cm | 1.5 cm | 1.5 cm | 1.5 cm | 1.3 cm | 1.5 cm | Trial 2 | 0.5 cm | 0.7 cm | 1.0 cm | 1.0 cm | 1.3 cm | 1.7 cm | 1.9 cm | 1.5 cm | Observation/Analysis: Solution turns yellow when a separate product is added, solid of the solution leaks down to the bottom after 20 minutes. Conclusion: The group tried to find the excess or limiting for the reaction between KI and Pb. In the end the hypothesis was supported by the data. We found that little amounts of each product led to a greater height of solids.
Then allow test tubes to remain at various temperatures for 5minutes, remove the test tubes and add 2cm of catechol and shake to allow them mix. Finally the record the results. As temperature increases the rate of enzyme activity increases until it reaches its optimum point, in this graph the optimum point is 20-60 oC, This is the point where enzymes works best if temperature increases above the optimum point the rate of enzyme activity decreases until the point where the enzymes are denatured, the color intensity gets darker as temperature increases. Effects of pH on Enzyme Activity pH directly has an effect on the active site, and is therefore important to have proper pH,however high or lower ph. affect the enzyme activity, to get enzymes working there should be proper ph.