Ethanol Lab Preparation
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Arizona State University *
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Chemistry
Date
Dec 6, 2023
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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
൬
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
ଵ
𝑉
ଵ
= 𝐶
ଶ
𝑉
ଶ
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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