Lab Report: Ethanol Determination Experiment
Objective: “To Determine the Ethanol content in several beverages using the enzyme alcohol dehydrogenase.”
The Chemistry behind this experiment is simple enough to understand. In the reaction carried out in the experiment, Nicotinamide Adenine Dinucleotide (NADH) is formed which can be analyzed quantitatively to find the concentration of Ethanol. The reaction involves oxidation of Ethanol to Acetaldehyde. This oxidation takes place with concomitant reduction of NAD+ to NADH. This formation of NADH can be seen in the Δ absorption at 340 nm micro level. At this wavelength, the reduced co enzyme absorbs at a very fast rate than the oxidized one, so the observance of NADH is possible.
The equilibrium constant in this reaction is a source of concern while calculating the Ethanol content in this experiment. The equilibrium is in the general direction of the Ethanol production rather than the intended reaction we want to do. Not only this, the substrate (Ethanol) must all be converted into Acetaldehyde in order to make this a success. Since this is not going to happen spontaneously, we will have to induce some artificial conditions in order to make this happen. We can influence the reaction kinematics in the following ways:
- The concentration of the reactant Ethanol should be at least 5 times the amount of NAD as it will tilt the reaction direction in or favor and help us determine the Ethanol amount. Also, the NAD+/NADH concentration will also be greater than 5 even after the reaction of all of Ethanol involved.
- Since the production of Acetaldehyde will slow the reaction and if the reaction is slowed, all of Ethanol will not be converted so we will introduce Semicarbazide that can react with Acetaldehyde spontaneously and thus the reaction will keep occurring at a fast paced rate. This s very common in biochemical reactions.
Methods Section:
At first, the assays for known and unknown solutions must be made:
- Solution 1 contains 0.15 Sodium Pyrophosphate and 0.16 semicarbazide. Solution 2 contains NAD+ 3mM. Solution 3 has a standard Ethanol solution 2mM. Solution 4 has yeast alcohol dehydrogenase. Then there are several unknown solutions you have to use. Dilute them with distilled water.
Procedure is as follows:
- Obtain 10mL buffer containing .022M glycine
- .15M Na pyrophosphate, and .16M semicarbazide.
- Take a 96 well plate (UV-transparent) and fill wells 1-12 on row A with 250µL of buffer. Blank the spectrophotometer with just the buffer. All pipetting will be done using a multichannel pipet.
Considerations:
- Keep volumes low
- Perform each reading in duplicate
Results and Calculations:
Standards are done according to Table 1 below in rows A, B & C for a total of three standard trials to be measured at 340nm. ADH enzyme will be added directly before measuring in the spectrophotometer. This standard serves as a positive control.
A sample of beer with known alcohol concentration is obtained. The sample of ethanol content is tested for reduction at 340nm according to Table 2. The volumes will be pipetted into rows D, E, and F for a total of three trials.
A negative control is conducted without the ADH enzyme to verify what the data should like if the enzyme proves unsuccessful in catalyzing the reaction. The volumes for the negative control are below in Table 3. A negative control is done for two trials, pipetting into rows G and H. Measurements are taken at 340nm using a spectrophotometer.
In order to compare the data, a standard curve of absorbance vs. ethanol will be made based off of the standard samples. This standard curve will be used to estimate the concentration of ethanol in the unknown sample. The concentration found through the standard curve of the sample will be compared to the value on the label of the beer.
1 0 0.218
2 5.0 0.44
3 10.0 0.79833
4 20 1.26133
5 30.0 1.793
6 40.0 2.453
7 50 3.267
8 60 3.269
Formulas:
∆G= -RT ln Keq
Keq=[ Product][Reactant]
Discussion:
Using this formula, we can find the required values. There are other matters to consider in this experiment too. We have to keep the associated costs in mind. So, in order to conserve the lab resources, minimum amount of reagents and chemicals are used for the experiment. The absorbance can be easily measured with the 250 µl of solution. I have also done 3 trials to be sure of the results of the equation. Since we want to know the exact amount of Ethanol in all these reactions, it is necessary for us to consider it as a limiting reactant. The limiting reactant is also fully utilized in the reaction and none of it is left behind. As it can be seen in the samples, their value has been varied in order to cover an appropriate range of Ethanol concentration to work with.
References
N. O. Kaplan and M. M. Ciotti, “Methods in Enzymology III,” 253-256 (1957).
H. U. Bergmeyer, “Methods in Enzymatic Analysis,” 2nd Edition, 3, 1499 (1974), 3rd Edition, 6,
598 (1986).
Pollak N, Dölle C, Ziegler M (2007). "The power to reduce: pyridine nucleotides – small molecules with a multitude of functions". Biochem. J. 402 (2): 205–18.doi:10.1042/BJ20061638. PMC 1798440. PMID 17295611
