Weights, Volumes, Solutions and Dilutions
August 28th, 2014
Kathleen Nolan and Dylan Catterton
Purpose: The purpose of this lab was to gain experience using analytical balances,
micropipettors and mathematical expressions in order to make stock solutions and
dilutions; to understand the range of variability and to improve the accuracy of
experimental results (Sparace, 2014).
Data:
Table 1: The absorbance of each copper sulfate solution at 800 nm.
[CuSO4 • 5H2O], mg/mL
0.0
1.0
2.5
5.0
10.0
Absorbance @ 800nm
0
0.078
0.116
0.146
0.366
0.4
Absorbance at 800 nm
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
0.0
2.0
4.0
6.0
8.0
Concentration (mg/mL)
10.0
12.0
Figure 1: Absorbance data from Table 1 plotted as concentration (mg/mL) vs.
absorbance with a trend line.
Table 2: Amount of microliters pipetted into each reaction tube and the actual (measured
amount obtained).
Stock Solution
1 (red)
2 (blue)
3 (green)
Expected Volume
Measured Volume
Reaction Tube A
123
47
30
200
177
Reaction Tube B
26
84
90
200
163
Reaction Tube C
15
10
175
200
191
Table 3: Amount of microliters placed on weigh boat using 20µL
micropipettor and weight in milligrams with the percent error calculations.
Volume
Weight
% Error
5µL
2 mg
60%
10µL
8 mg
20%
15 µL
12 mg
20%
20 µL
17 mg
15%
Table 4: Amount of microliters placed on weigh boat using 200µL
micropipettor and weight in milligrams with the percent error calculations.
Volume
Weight
% Error
50µL
49 mg
2%
100µL
95 mg
5%
150 µL
143 mg
5%
200 µL
191 mg
5%
Table 5: Amount of microliters placed on weigh boat using 1000µL
micropipettor and weight in milligrams with the percent error calculations.
Volume
Weight
% Error
200µL
194 mg
3%
400µL
388 mg
3%
800µL
775 mg
3%
1000µL
970 mg
3%
Table 6: Using various dilutions method the dilution factor and amount of copper sulfate
and water in each test tube are shown, the percent concentration calculations and
absorbance at 800 nanometers are portrayed as well.
Dilution
4:5
3:5
1:1
1:3
1:8
Volume of
CuSO4 (mL)
4
3
5
1.67
0.6
Various Dilutions
Volume of
Concentration (%)
H2O (mL)
1
1.60%
2
1.20%
0
2%
3.3
0.67%
4.4
0.24%
Absorbance at
800nm
.839
0.608
1.032
0.348
0.127
Table 7: Using serial dilutions the dilution factor and amount of copper sulfate and water
in each test tube are shown, the percent concentration calculations and absorbance at 800
nanometers are portrayed as well.
Dilution
4:5
4:5
4:5
4:5
4:5
Volume of
CuSO4 (mL)
4
4
4
4
4
Serial Dilutions
Volume of
Concentration (%)
H2O (mL)
1
1.6%
1
1.28%
1
1.024%
1
0.8192%
1
0.65536%
Absorbance at
800nm
0.829
0.675
0.547
0.433
0.342
Absorbance at 800 nm
1.2
1
0.8
0.6
0.4
0.2
0
0.00%
0.50%
1.00%
1.50%
Concentration (%)
2.00%
2.50%
Figure 2: Concentrations (%) vs. Absorbance at 800 nm for various dilutions of 2 %
[CuSO4 • 5H2O].
Absorbance at 800 nm
1.2
1
0.8
0.6
0.4
0.2
0
0.00%
0.50%
1.00%
1.50%
Concentration (%)
2.00%
2.50%
Figure 3: Concentrations (%) vs. Absorbance at 800 nm for serial dilutions of 2%
[CuSO4 • 5H2O].
Calculations:
IV. Checking the Accuracy or Calibration of Micropipettors
Formula: % Error: measured – expected x 100%
expected
2mg - 5µL x 100 = 60%
5µL
8mg - 10µL x 100 = 20%
10µL
12mg - 15µL x 100 = 20%
15µL
17mg - 20µL x 100 = 20%
20µL
49mg - 50µL x 100 = 2%
50µL
95mg - 100µL x 100 = 5%
100µL
143mg - 150µL x 100 = 5%
150µL
191mg - 200µL x 100 = 5%
200µL
194mg - 200µL x 100 = 3%
200µL
388mg - 400µL x 100 = 3%
400µL
775mg - 800µL x 100 = 3.13%
800µL
970mg - 1000µL x 100 = 3%
1000µL
V. Exercise in Dilutions
Formula: C1V1 = C2V2
A. Various Dilutions
(2%)(4mL) = (5mL)(x)
8/5 = 1.6%
(2%)(3mL) = (5mL)(x)
6/5 = 1.2%
(2%)(5mL) = (5mL)(x)
10/5 = 2.00%
(2%)(1.67 mL) = (5mL)(x)
3.34/5 = 0.67%
(2%)(0.6 mL) = (5mL)(x)
1.2/5 = 0.24%
B. Serial Dilutions
(2%)(4mL) = (5mL)(x)
8/5 = 1.6%
1.6% x 0.8 = 1.28%
1.28% x 0.8 = 1.024%
1.024% x 0.8 = 0.8192%
0.8192% x 0.8 = 0.65536%
Solution Concentration Calculations (Q4):
Formula: C1V1 = C2V2
a) Sucrose [molecular weight: 342.3g]
342.3g sucrose = 1M
34.23g sucrose in 1000mL H2O = .1M
34.23g sucrose in 500mL H2O = 1M
3.423g sucrose in 500mL H2O = .1M
.3423g sucrose in 250mL H2O = .1M
.3423 x 3 = 1.0269g sucrose
b) Sodium Chloride
1gram in 100mL = 1%
2 grams in 200mL = 1%
.5grams in 50mL = 1% so
2.5grams in 250mL = 1%
c) Ethanol
250mL (.8) = 200mL ethanol
250mL – 200mL = 50mL H2O
d) Sorbitol
50g sorbitol / 200g H2O = 0.25 x 100% = 25.0% solution
Question 6,7,8 Calculations:
6.
M= (grams NaCl/molar mass of NaCl)/L of solution
0.10 x 1000 mL = 100 g of NaCl
58.44 g/mol=MW of NaCl
Molarity = (100 g NaCl / 58.44 g/mol) / 1 L
Molarity = 1.711 M
7.
M= (grams NaCl/molar mass of NaCl)/L of solution
Formula: C1V1 = C2V2
(2%)(3 mL NaCl)=(5 mL NaCl solution)(x)
x=1.2%
0.012 x 1000 mL= 12 g of NaCl
M=(12 g/ 58.44)/1 L
M= .205338809 M
.205338809 M x (1000 mM/ 1 M)= 205.34 mM
8.
.1 M= 100 mM
(100 mM) (x)=(3 mL) (2.5 mM)
x= .075 mL
Questions from Lab Manual (Sparace, 2014):
For calculations see data section.
4.
a. 250mL of 0.3M sucrose
Dissolve 1.0269g sucrose in 100mL of water then fill the beaker to the 250mL
mark.
b. 250mL of 1% (w/v) NaCl
Dissolve 2.5g NaCl in 100mL water of water then fill the beaker to the 250mL
mark.
c. 250mL of 80% (v/v) ethanol
Place 200mL ethanol in a beaker and fill the beaker with water to the 250mL
mark.
d.250g of 25% (w/w) sorbitol
Place 50g sorbitol in a beaker and add 200g of H2O.
6. Molar concentration of NaCl = 1.711 M
7. Millimolar concentration of NaCl= 205.34 mM
8. Use .075 mL of the 0.1 M stock solution of ATP and add 2. 925 mL of H20 to produce
3 mL of a 2.5 mM solution.
Discussion and Conclusion:
There was some error due in our micropipettor findings either due to human error
or because of calibration problems which was noticeable in our data for Table 1, 2, 3, 4,
5 and Figure 1. There was also trouble with the tips while measuring the substances in
the micropipettor. Also our absorbance is probably off because of the amount of time it
took to get the cuvette into the spectrophotometer. For Figure 2 and Figure 3 there is a
noticeable trend that as concentration increase so does absorbance. Our data collected in
Table 6 and Table 7 also could be off due to a mix up of pipettes at a couple points.
In conclusion, this lab was about reviewing and using quantitative methods in lab
such as measuring, weighing and diluting which was achieved. It was found that as
concentration increases so does absorbance by making dilutions and plotting the data on
graphs. The correct methods for micropipetting was learned and how to check its
accuracy as well as how to calculate the percent error. Different scales were used to
check the accuracy of the micropipettes by assuming one micro-liter is equal to one
milligram. The difference between various and serial dilutions by using the dilution
formula and other methods was also determined.
Literature Cited
Sparace, Salvatore, and Brandon Moore. "Weights, Volumes, Soultions, and Dilutions."
Biology 4341 Laboratory Manual: Biological Chemistry Laboratory Techniques.
Clemson: Clemson U, 2014. 1-10. Print.