Ethanol Lab Preparation

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Arizona State University *

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367

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Chemistry

Date

Dec 6, 2023

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pdf

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3

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Lab 8 Preparation Fill in the sample table for the standard curve samples. The unknown ethanol samples and controls are also given in this document. We will also go over the unknown alcohol samples and the control samples (other than no ethanol) in class before starting the experiment. You are bring your only samples of this lab. Please follow the rules for samples provided in the lab. Write down AND taking a picture of the alcohol content (in %) of the container. Notes on Standard curve samples: Calculation all sample to have a final volume (V 2 ) of 250 l Well concentration of buffer is consistent thus volume added is consistent o 0.15 M or 150 mM stock concentration (of sodium pyrophosphate in buffer stock, there are more components but it is the sodium pyrophosphate that we are paying attention to) o Desired sample concentration is 75 mM Well concentration of NAD+ is consistent thus volume added is consistent o Stock concentration: 3 mM o Desired well concentration: 1 mM Amount of enzyme is consistent thus volume added is consistent o Stock concentration: 5000 units/ml o Desired concentration: 25-50 units/ml Choose an enzyme concentration in this range The well concentration of ethanol is DIFFERENT thus the volume of each sample will be different o Stock concentration of ethanol: 2 mM or 2000 M o 9 concentrations in the range of 0 to 200 M is desired (see table below) You make adjust the concentrations in your standard curve as long as 200 M remains the top concentration used. Water to make up the total volume once all other components are accounted for.
Sample table: Well concentration of Ethanol ( M) Volume of buffer (0.15 M) Volume of NAD+ (3 mM) Volume of Enzyme (5000 units/ml) Volume of Ethanol Standard (2 mM) Volume of water Total volume ( l) 200 250 175 250 125 250 100 250 75 250 50 250 10 250 0 250 Remember that 200 M is 0.2 mM If any ethanol volume is less than 2 l plan to make a dilution (10x, 25x, or 50x) of the stock solution to raise the volume in the well above 2 l Background samples: The 0 μ M ethanol concentration can be used for the background for the standard curve since the known ethanol solution is diluted in water. For the unknown samples background, I suggest replacing the enzyme volume with water and keeping all other components of you. This way there will be no reaction that occurs in your sample. Allowing you to consider the buffer, NAD+, any absorbance from your unknown ethanol sample (that isn’t the NADH product of the reaction), and any absorbance that could come from the microplate. o Note: this background would not consider any absorbance of the enzyme.
Ethanol Lab Unknown Calculations: Determine the theoretical concentration of your Unknown Samples 1. Start with the % ethanol listed on the container. Ex: 6.5% 2. Relate the % to a volume of ethanol a. 6.5% means there are 6.5 ml of Ethanol per 100 ml of the samples b. The calculations assume a total volume of 100 ml 3. Determine the # of moles Ethanol in that volume of ethanol a. Ethanol Density: 0.789 g/ml b. Ethanol MW: 46 g/mol c. Calculation: 6.5 𝑚𝑙 0.789 𝑔 𝑚𝑙 ൰ ൬ 𝑚𝑜𝑙 46 𝑔 ൰ = 𝑚𝑜𝑙 𝐸𝑡ℎ𝑎𝑛𝑜𝑙 4. Divide by the 0.1 L assumed total volume 𝑚𝑜𝑙 𝐸𝑡ℎ𝑎𝑛𝑜𝑙 0.1 𝐿 𝑇𝑜𝑡𝑎𝑙 𝑉𝑜𝑙𝑢𝑚𝑒 = 𝑀 𝐸𝑡ℎ𝑎𝑛𝑜𝑙 5. The resulting value is the theoretical concentration of the sample in M Note: red values above are dependent on your sample %. Calculate an ~2 mM Unknown Ethanol Intermediate The known stock solution in the lab this week to create the standard curve (and the associated calculations to create the wells) is a 2 mM solution. If you plan to make an intermediate of your unknown ethanol sample that is ~2 mM in concentration (based on the % listed on the container/calculation above). Then you can just substitute the volume of known ethanol in two of your values on the standard curve with unknown ethanol without having to do additional calculations. Use: 𝐶 𝑉 = 𝐶 𝑉 Where, C 1 is the approximate concentration of Ethanol in mM, V 1 is the volume you want to pipette of the stock unknown ethanol (I suggest using 2 μ l as this is the lowest volume I advise you to use), C 2 is the 2 mM solution you want to make, and calculate the total volume of your dilution with is V 2 . *You will be back calculating the actual/experiment concentration of the ethanol sample you bring. The volumes you use for the intermediate dilution and the theoretical concentration of your alcohol (in M or mM) will be used in your calculations for the lab report*
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