Nitrophenol Lab

The preparation for the experiment started by gathering the solutions of enzyme Peroxidase, substrate hydrogen peroxide, the indicator guaiacol and distilled water. Two small spectrometer tubes and three large test tubes with numbered labels. In addition, one test tube rack, one pipet pump and a box of kimwipes were also gathered. Before the experiment, the spectrometer must be set up to use by flipping the power switch to on. Following, the machine was warmed up for 10 minutes and the filter lever was moved to the left. In addition, I set the wavelength to 500 nm with the wavelength control knob. Before the experiment, I had to create the blank solution by pipetting 0.1 ml of guaiacol, 1.0 ml of turnip extract and 8.9 ml water into tube #1. Following the creation of the blank, a control 2% solution was created.

Tables 2,3,4,5 and 6 show that as duration increased the absorbance also increased for each pH. The solution in the conical flask became darker (yellow) in time this is because the substrate, p-nitrophenyl phosphate was catalysed by acid phosphatase, releasing Nitrophenolate anion. It was the Nitrophenolate anion giving off the yellow colour; the presence of this feature increases the absorbance rate. The addition of sodium hydroxide distorted the shape of the enzyme making it no longer effective in its function.

The isosbestic point of the acid (pH6) and basic forms (pH10) of para Nitrophenol (PNP) was expected at 350nm. As you can see in figure 2, the graph shows the intersection of 2 curves at ~350nm, which is matched with the literature value. Also, the pKa of PNP was expected 7.15 at room temperature. Refer to figure 3, the pKa is estimated to be 7.15-7.2, which very close to the literature value. In addition, the lab was succeeded in illustrating the use of a spectrophotometry to analyze concentrations of chemical substance. The absorbances of 2 unknowns were felt on the standard curve as the expectation (refer to table 4). The minimum absorbance of the known standards was 0.193 and the maximum is 1.830. The absorbance of the unknown

Lab six requires students to observe the effects of pH and enzyme concentration on catecholase activity. Enzymes are organic catalysts that can affect the rate of a chemical reaction depending on the pH level and the concentration of the enzyme. As pH comes closer to a neutral pH the enzyme is at its greatest effectiveness. Also at the absorbance of a slope of 0.0122 the enzyme is affected greatly. The pH effect on enzymes can be tested by trying each pH level with a pH buffer of the same pH as labeled as the test tube and 1mL of potato juice, water, and catechol. This is all mixed together and put in the spectrophotometer to test how much is being absorbed at 420nm. As the effect on enzyme concentration can be tested almost the same way. This part of the exercise uses different amounts of pH 7-phosphate buffer and potato juice, and 1mL of catechol mixed together in a test tube. Each substance is put in the spectrophotometer at a wavelength set tot 420nm. The results are put down for every minute up to six minutes to see how enzyme concentration affects reaction rate. The results show that the pH 8 (0.494) affects the enzyme more than a pH of 4 (0.249), 6 (0.371), 7 (0.456), and 10 (0.126). Also the absorbance is greatest at a slope of 0.0122 with test tube C that has more effect on the reaction rate, than test tube A, B, and D.

In the first part of the enzyme lab, we mixed a substrate and an indicator with an enzyme. There was also a neutral buffer in each of the chemical mixtures. The neutral buffer regulated the pH to around 7. We got a color palette and once we mixed each together, we observed and saw a change in the color of the substance. The darker and more brown the substance got, the more oxygen produced by the reaction. Our results showed that amount of oxygen produced increased about 10% a minute until it sort of equilibrated at 4 minutes and didn’t change to the fifth minute mark. If we were to change anything we did in the experiment, we would make our comparisons to the chart more precise. Overall we thought it

Before the start of this experiment, the theoretical yield was calculated. Based off the data, it was found that the limiting reagent is 3-nitrobenzaldehyde. The theoretical yield is determined by relating the moles of the limiting reagent to the moles of the anticipated product by a ratio obtained from the overall equation. The theoretical yield was calculated to be 1.3 g. However, the actual yield obtained was greater than the theoretical yield; in other words, the actual mass of the product was higher than the theoretical mass. This led to an abnormally high yield of 320% and an impure product.

To improve the results from the experiment buffer solutions that were not whole pHs could have been used e.g. pH 4.5, 5.5 etc. This would have provided more reliable results as a wider range of results would have been produced. Using pHs with decimals would also help to more accurately determine the optimum pH as the optimum may have been above or below the pH stated in the hypothesis; 8. In this experiment however the optimum is taken at 8 because the graph does not rise again.

To begin, three sets ofabout 0.3000g of KHP are weighed out on an analytical balance. Put the three sets of KHP into three separate, labeled flasks. All three sets of the KHP is then dissolved with approximately 50mL of deionized water. Next, a buret is used to start the actual titration. Buret is initially filled to 0.00mL mark with the NaOH solution, this is recorded as initial volume. Next, add 2-3 drops of phenolphthalein indicator into each of the three flasks containing KHP. A magnetic stir bar is then added to the first flask, and placed above a stir plate. Everything is positioned under the buret. Stirrer is put on medium speed and the titration can start. Slowly release the NaOH into the KHP flask. As the end point is reached, a pink color will be seen in the flask. When the lightest pink possible remains in the solution for more than 30 seconds titration is complete. The final volume is recorded, and the same steps are taken for the other two sets of KHP solution. Finally, blank titration is completed to determine deviation.

When making the absorption graphs for the yellow 5 and yellow 6, the lines of best fit were giving numbers that were extremely small. After help from the TA, it was found that our concentrations used where twice as small of a concentration compared to what was actually used for Full Throttle. To correct this the molarity’s were multiplied by ½. For example when finding the x value in our equation y=2.044x+0.2067 the answer is x = 7.28-5 moles. To correct this, that number is multiplied by 0.5 to get the correct value of 3.64*10^-5. This new number is what was used in the equation c1v1=c2v2. After the solution was made to mimic Full Throttle it was discovered that the beverage only had one food dye. The experiment was intended to have a beverage with two food dyes present. To make up for this, orange PowerAde was used and the experiment was repeated for orange PowerAde. The solution was made for the orange PowerAde and its absorption graph was compared to that of the diluted orange PowerAde solution. Comparing the two graphs, have the same peaks although these peaks are at different absorption levels. Since the two graphs have the same peaks it is safe to assume that the same food dyes are present in both solutions. It is important to note that there are two peaks in each graph despite only seeing one peak. This is because both peaks are located so close to each other that they merge as one overall peak. When comparing the two

First, it was downloaded the spreadsheet from eLearn provided by the instructor titled as “Experiment I data”. The first thing, it was calculated the slope for each Time point and Response set of data. It was noticed that inhibitor concentration [I] was constant, and substrate [S] concentration was increasing. So, it was used the Response vs. Time data to allowed to measure the initial velocity [Vo] of the enzyme converting substrate to product. It was typed the formula “=slope(“ in the shift cell called “slope”. When it was typed the formula, it was selected the Y-values which is the Response column dragged down until the last response. Then, put a comma to separate the X-values which is the Time column followed by a closed parenthesis. Since, slope should be calculated for all of the others, it can be save time by doing

Graphs will be used with the enzyme alkaline phosphatase of the unknown in the enzyme solution to Determine Km and Vmax

First, we can talk about error in the standard curve made which could be related to measurements of the absorbance leading to the equation of the straight line that could affect all of the calculations. Another source of error, as mentioned before, could be in the choice of the alkaline phosphatase concentration in B. Choosing a concentration that is not very accurate would have affected part C in the experiment. As of what concerns future improvements of this experiment, more standard dilutions could be used to generate the standard curve so that there is a wider range of values of concentrations of PNP for a more accurate conversion from absorbance values to PNP concentration values along the experiment. Furthermore, in part B and C more alkaline phosphatase dilutions and more PNPP dilutions could be prepared for the same reason: providing a wider range of values and therefore more accuracy in calculations. An addition area of improvement could also be related to the preparation of alkaline phosphatase in the first, trying to make a preparation that is as pure as possible for a higher value for the specific activity.

There was a steep drop in the curve around an average pH of 8, this information may be due to a random error. The test tube with a pH of 8.0 was analyzed to have an absorbance of

By removing one milliliter of the stock solution to the first test tube, and repeating the same process for the other two tubes allowed us compare concentrations and know what the dilution factor would be when placed into the dialysis tubing. All three tubes had the same amount of water (4mL of H2O) added. Once the solution for each test tube was mixed well, we transferred 2 mL of each concentration into the dialysis bag. We secured each piece (3 in total) of the dialysis tubing to ensure that once the concentration was added that it would not leak out of either end. Three stir plates were used, all set at the same speed, and the dialysis bags filled with different concentrations were added. Before we began to measure the absorbance rate, we did take a sample of the water immediately after placing the dialysis tubing into each beaker. We observed each beaker over a period of 25 minutes. At each five-minute interval, we removed one milliliter of the solution and placed it into a cuvette. The cuvette sample was then placed in the spectrophotometer to calculate the absorbance rate of each sample. After each sample was measured, all data was recorded in the lab manual. Once all measurements had been recorded we used the absorbance rate to help calculate the concentration. The light that is absorbed when using the spectrophotometer is usually relative to the concentration of a specific

In this experiment, the pKa, dissociation constant, of 2-naphthol was determined by measuring the UV-visible absorption spectra of solution of the acid at different pH values.