Lab 2 Solutions and Spectrophotometry
Version: Jan 2022
MED3008 CLINCIAL CHEMISTRY AND IMMUNOCHEMISTRY
Lab 2 Solutions and Spectrophotometry
1. Purpose
To determine the concentration of a copper sulfate (CuSO4) solution, and to
duplicate its concentration by two methods.
2. Objectives
2.1.
2.2.
2.3.
2.4.
2.5.
To learn how to use a pipette properly.
To learn how to dilute a stock solution.
To learn how to do the serial dilution.
To learn how to use a spectrophotometer.
To gain practice plotting a calibration curve and use it to determine the
concentration of an unknown solution.
2.6. To learn how to make a solution from a solid reagent.
2.7. To learn how to make a solution by diluting a stock solution.
3. Introduction
3.1. For this experiment, assume that you are working at a clinical chemistry lab
and you will use copper (II) ion solutions for the protein assay. The laboratory
manager has sent you a small sample of the copper (II) ion solution they use
and told you to make more at that specific concentration. Unfortunately, the
production manager did not tell you what the concentration was, and she just
left for vacation. Your job is to determine the concentration of the solution, and
to generate more solution of that same concentration.
3.2. In Part A of this experiment, you will make several solutions with known
concentrations of copper(II) ions, then make absorbance measurements on
them, and develop a calibration curve. You will then measure the absorbance
of the unknown solution and determine its concentration from that calibration
curve.
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Lab 2 Solutions and Spectrophotometry
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3.3. In Part B, you will make a copper (II) ion solution with a concentration
identical to the unknown by dissolving solid copper sulfate, CuSO4 in water.
3.4. In Part C, you will make a copper (II) ion solution with a concentration
identical to the unknown by diluting a concentrated, stock copper sulfate,
CuSO4, solution. You will compare the accuracy with which you can make the
solutions by the two methods.
4. Materials
4.1. Spectrophotometer
4.2. 2x Cuvettes
4.3. 2x Serologic glass pipettes (2mL and 10mL)
4.4.
4.5.
4.6.
4.7.
4.8.
4.9.
4.10.
4.11.
1x pipette bulb
5x 4ml test tubes
1x test tube rack
2x 50 mL tube
1x 25 mL volumetric flasks w/ stoppers
1x waste bottle for copper waste
Autopipette P1000 with tips
Vortex
5. Reagents
5.1. 20 mL 0.5 M CuSO4(aq) stock solution (You prepared in the last practical)
5.2. 1.5ml CuSO4(aq) unknown solutions A
5.3. Solid CuSO4
6. Safety
CuSO4 is listed as toxic and an irritant. Toxic substances are hazardous to health
when breathed, swallowed or in contact with the skin. An irritant may have a
temporary irritating effect on skin, eyes, respiratory tract, etc. If you come in contact
with the solid, you should gently brush off the affected area with a paper towel and
then flush the area with water. If you come in contact with a solution of copper (II)
sulfate, you should flush the affected area with water.
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Lab 2 Solutions and Spectrophotometry
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7. Waste disposal
Solutions containing copper ions must be placed in the waste bottle in the lab.
Designate a "waste copper" beaker and set it aside for use during your lab. You can
put the small samples of copper solution you will make in this and empty it into the
waste bottle at the end of class, instead of going back and forth to the waste bottle.
Always remember not to overfill the waste bottle. If your waste bottle is full, please
alert your lab instructor.
8. Lab procedure
8.1. Part A: Determination of the Concentration of a Copper (II) Ion Solution
8.1.1. Obtain about 20 mL stock Cu2+ solution. Write its exact concentration in
Data Table A.
8.1.2. Once the spectrophotometer is warmed up, add 1ml of deionized water
and take a blank spectrum in a cuvette.
8.1.3. Refill the cuvette with 1 mL stock solution and take an absorbance
spectrum. Identify the wavelength of maximum absorbance near 630 nm.
8.1.4.
8.1.5.
8.1.6.
8.1.7.
Record the wavelength and absorbance at this wavelength in Data Table
A. Absorbance values are reported to the 0.001. For all remaining
absorbance measurements in this experiment, be sure to use the same
wavelength you have identified as the maximum.
Dispense 1.5 mL Cu2+ solution and 1.5 mL deionized water into a test
tube for Solution 1 using a serologic pipette.
Mix the solution well by pipetting up and down a couple of times.
Make up a serial dilution of the solution from 1/2 down to 1/16 with
deionized water. Record your volumes used in Data Table A.
Measure the absorbance and calculate the Cu2+ concentration for
Solution 1. Record your absorbance and concentration in Data Table A.
8.1.8. Wash your used cuvette with distilled water three time and drain off the
water containing in the cuvette.
8.1.9. Repeat the above procedures 8.1.7 to 8.1.8 for solutions 2 to 4.
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8.1.10. Plot the absorbance of the Cu2+ solutions with their concentrations.
Draw the trendline and calculate the R2 value. If your plot is linear with
an R2 value of 0.9 or greater, continue the experiment. If your R2 value
is low, repeat your experiment.
R=
𝑛 ∑ 𝑥𝑦−(∑ 𝑥)(∑ 𝑦)
√𝑛(∑ 𝑥 2 )−(∑ 𝑥)2 √𝑛(∑ 𝑦 2 )−(∑ 𝑦)2
8.1.11. Safely dispose of the solutions in your copper waste bottle.
8.1.12. Obtain 1mL of unknown solution A.
8.1.13. Measure the absorbance of the unknown Cu2+ solution of sample A and
record it in Data Table A.
8.2. Part B: Preparation of a Copper (II) Ion Solution from Solid CuSO4
8.2.1. Carefully weigh the desired amount of CuSO4 on a weighing boat
according to the calculation you obtained in Question 4 on page 7.
Record the exact amount that you used in Data Table B.
8.2.2. Transfer the CuSO4 (s) into a 25 mL volumetric flask and dilute to the
mark with deionized water.
8.2.3. After the solution is well mixed, condition a cuvette with it. Then refill
the cuvette, measure and record the absorbance of your solution in Data
Table B.
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Lab 2 Solutions and Spectrophotometry
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8.3. Part C: Preparation of a Copper (II) Ion Solution by Dilution of a Stock
CuSO4 Solution.
8.3.1. Record the exact concentration of the stock copper (II) solution in Data
Table C.
8.3.2. Make sure you obtain a little more of the stock copper (II) solution than
you calculated in Question 6 on page 9.
8.3.3. First condition a serologic pipette, then use it to transfer the volume of
copper (II) solution you calculated into a 25 mL volumetric flask. Record
the exact amount that you transferred in Data Table C. Dilute to the mark
with deionized water.
8.3.4. After the solution is well mixed, condition a cuvette with it. Then refill
the cuvette, measure and record the absorbance of your solution in Data
Table C.
8.3.5. When you have finished taking measurements, collect all your copper
solution waste and place it in the waste bottle in the lab, making sure not
to overfill it. Rinse and dry all your glassware with water and return it to
the set-up area where you found it.
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Lab 2 Solutions and Spectrophotometry
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MED3008 CLINCIAL CHEMISTRY AND IMMUNOCHEMISTRY
LAB 2 Solutions and Spectroscopy Worksheet
Name: _________________ Student ID: __________________ Grade: __________
As you work through the steps in the lab procedures, record your experimental values
and the results on this worksheet.
Spectrophotometer used: _______
Table A: Calibration Curve of Cu2+ Solutions and Unknown
Stock Cu2+ solution concentration: _____________M
Wavelength: _______mm
Solution#
[Cu2+]
Absorbance
Calculated, (measured)
M
Stock
Dilution
factor
Volume of
Cu2+, mL
Volume of
distilled
water, mL
1
1
2
3
4
R2
Equation of
trendline
Unknown#:
Absorbance (measured):
[Cu2+] (calculated):
A
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Lab 2 Solutions and Spectrophotometry
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1. What is the concentration of Cu2+ in Solution 1 that you prepared? Show your
calculations neatly.
2. Would you predict the absorbance of solution 2 to be greater or less than that
of solution 1?
3. What is the concentration of Cu2+ in your unknown solution?
Show your calculations neatly. Record this concentration in Data Table A.
4. You desire to make a copper (II) solution at the same concentration as the
unknown A you just determined in Part A. How many grams of CuSO4 are
required to make 25 mL of this solution?
Show your calculations neatly. Record the result as the target mass in Data
Table B.
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Lab 2 Solutions and Spectrophotometry
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Table B: Preparation of a Cu2+ Solution from solid CuSO4
Target [Cu2+]
from Part A
(unknown A),
M
Target Mass
CuSO4, g
Actual of Cu2+
solution you
prepare, mL
Absorbance of
Cu2+ solution
[Cu2+] calculated
from absorbance, M
5. Comment on how the absorbance of your solution made from solid compares
to the unknown solution's absorbance in Part A. Do you expect them to be the
same?
Why or why not?
Table C: Preparation of a Cu2+ Solution from stock Cu2+ solution
Stock Cu2+ solution concentration: _____________ M
Target [Cu2+]
from Part A
(unknown A),
M
Target
volume Cu2+
solution, mL
Actual of
Absorbance of
2+
Cu solution Cu2+ solution
you prepare,
mL
MED3008 Clinical Laboratory Chemistry and Immunochemistry
[Cu2+] calculated
from absorbance,
M
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6. You desire to make a copper (II) solution at the same concentration as the
unknown you determined in Part A. How many mL of the copper (II) stock
solution are required to make 25 mL of this solution?
Show your calculations neatly. Record the result as the target volume in Data
Table C.
7. Comment on how the absorbance of your solution made by dilution of a stock
compares to the unknown solution's absorbance in Part A. Do you expect them
to be the same?
Why or why not?
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Lab 2 Solutions and Spectrophotometry
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Lab 2 Solutions and Spectrophotometry
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Lab 2 Solutions and Spectrophotometry
Pre-Lab exercise
Name:__________________ Student ID: _________________ Marks: __________
1. Which hazards are associated with copper(II) sulfate?
A. Flammable
B. carcinogenic
C. corrosive
D. toxic
E. irritant
2. Absorbances should be recorded to which place value?
A. 1
B. 0.1
C. 0.01
D. 0.001
3. What is a monochromator?
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4. Explain / define the absorbance maxima and for what it is used. (2 points)
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5. If an instrument provided only %T readings, state two ways you could obtain
the concentration of the substance being tested.
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6. What is the advantage of the double beam system?
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7. In your own words, briefly summarize Beer's Law.
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8. Why must standardized cuvettes be used with a spectrophotometer?
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9. Define “range of linearity.”
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