Solutions & Dilutions
Introduction
This lab will utilize various types of measuring glassware to demonstrate appropriate techniques for making a primary
solution and for making serial dilutions from the primary solution. The strengths of the dilutions will be checked by
measuring the absorbance of each diluted solution on a spectrophotometer (See Page 37 for Spectrophotometer
instructions). Also, proper pipette and burette procedures, as outlined on pages 46-47 of this manual, will be utilized
throughout the lab exercise.
A serial dilution is the stepwise dilution of a substance in solution. In this lab, you will be diluting Copper II Sulfate.
Many areas of science use serial dilutions to accurately create highly diluted solutions for different experiments. Serial
dilutions are usually made in increments of 1000, 100 or 10. By using these increments, a technician can ensure that the
final data points will result in concentration curves on a logarithmic scale. Serial dilution may also be used in some labs
to reduce the concentration of microscopic organisms or cells in a sample, as with bacterial colonies in water samples, to
make it easier to enumerate the individuals.
Materials
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Copper II Sulfate Pentahydrate [CuSO4•5H2O] - Crystals
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Distilled-Deionized Water (DIW)
Scale
Spectrophotometer
Spatula
Plastic Weigh Boat
Stirring Rod
Beakers
Volumetric Flask
Volumetric Pipette, Pipette Pump
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** Before beginning this lab you should read pages 37-38, 45-46, and 132 in this lab manual. **
Procedure
Make a Solution
1. Using a weigh boat and a scale, weigh out 2.00g CuSO4•5H2O
2. Transfer the crystals to a 100mL beaker, rinsing the weighboat into the beaker assuring that no chemical is left
behind. Use deionized-distilled water (DIW) at all times when mixing reagents.
3. Add enough DIW to the beaker to dissolve the Copper II Sulfate. Do not use more than ~60mL.
4. Transfer the dissolved Copper II Sulfate to a 100mL volumetric flask. Rinse the beaker into the flask. Add DIW to
the mark, placing the meniscus on the line.
5. Stopper the top of the flask and mix well (See Page 44 for “Mixing Water Samples”). Do Not use your finger as a
stopper! This is Solution #1, the Primary Standard, LABEL appropriately.
6. Fill a cuvette with the Primary Standard (Solution #1). Take this cuvette to the spectrophotometer and read its
absorbance at the wavelength of 810nm. Remember to blank the spectrophotometer before taking this
reading.
7. Record this absorbance in your lab notebook. The absorbance should be between 0.910 and 0.980, if it is not,
check with the Instructor.
Make Serial Dilutions
Dilute Primary Standard
Make Solution #2
1. Using a clean 1mL volumetric pipette, transfer 1.0ml from your Primary Standard (Solution #1) to a clean 100mL
volumetric flask. Add deionized-distilled water (DIW) to the mark.
2. Stopper the top of the flask and mix well (See Page 44 for “Mixing Water Samples”). Do Not use your finger as a
stopper! This is Solution #2, LABEL appropriately.
Dilute Primary Standard
Make Solution #3
3. Using a clean 10mL volumetric pipette, transfer 10.0mL from your Primary Standard to a clean 100mL
volumetric flask. Add DIW to the mark.
4. Stopper the top of the flask and mix well. This is Solution #3, LABEL appropriately.
Dilute Solution #3
Make Solution #4
5. Using a clean 10mL volumetric pipette, transfer 10.0mL from Solution #3 to a clean 100ml volumetric flask. Add
DIW to the mark.
6. Stopper the top and mix well. This is Solution #4, LABEL appropriately.
Measure the Absorbances (ABS)
7. Fill cuvettes with Solution #2, Solution #3, and Solution #4.
8. Measure the absorbances of the solutions at 810 nm. Remember to blank the spectrophotometer before taking
this reading.
9. Record the absorbances in your lab notebook.
Note: Solutions #2, #3, & #4 are serial dilutions of the Primary Standard; Solutions #2 & #4 are of the same
concentration and should have the same absorbance when read on the spectrophotometer.
Data
The following data table is included only as an example. You MUST record your data in your laboratory notebooks if you
would like to receive credit for your work!
Solution
Primary Standard (#1)
Secondary Standard (#2)
Tertiary Standard (#3)
Quaternary Standard (#4)
Blank
ABS
Calculations
Both the theoretical concentration and the actual concentration of the Primary Standard are known; they are both
100%. You must calculate the concentrations of each dilution (Solutions #2, #3, & #4).
Theoretical Concentrations (%):
Solution #2 Concentration =
mL of Primary Standard used to make Solution #2 x 100 =
%
Total mL made of Solution #2
Solution #3 Concentration =
mL of Primary Standard used to make Solution #3 x 100 =
%
Total mL made of Solution #3
Solution #4 Concentration =
mL of Solution #3 used to make Solution #4 x 100 =
%
(Total mL made of Solution #4) x 10
Actual Concentrations (%):
Solution #2 Concentration =
ABSsolution #2 × 100 =
ABSprimary standard
%
Solution #3 Concentration =
ABSsolution #3 × 100 =
ABSprimary standard
%
Solution #4 Concentration =
ABSsolution #4 × 100 =
ABSprimary standard
%
Create a Standardization Graph
Obtain graph paper and construct an Absorbance vs Actual Concentration (%) graph using the calculated data. Make
sure to label your graph correctly. Absorbance will be on the Y-axis and Concentration will be on the X-axis. Be sure to
space the numbers appropriately to use the entire sheet.
The spectrophotometer blank is the 0% solution and the Primary Standard is the 100% solution. Plot the five data pairs
on the graph. Use only the 100% and the 0% points to draw the straight line. These standards are correct. The other
points are your dilutions and their positions relative to this line will show how accurate you were in their creation.
Name ___________________________
Group ______________________ Date ______________
Pre-Lab Questions—Solutions & Dilutions
To be completed prior to the beginning of your lab. Record your answers in your lab notebook only.
1. According to the MSDS for this substance, is Copper II Sulfate Pentahydrate (Cupric Sulfate) acidic, basic, or
neutral? What could result if this substance comes in contact with your skin? (Information about MSDS’s can be
found in the Appendix of this manual.)
2. What is the wavelength range of the digital spectrophotometer? (Hint: read the instructions for the
spectrophotometers in this manual.)
3. “DIW” is an abbreviation that is used during this lab. What does it mean?
4. Define meniscus. (Hint: read “Using Graduated Glassware” in this manual.)
5. True or False. You should handle a volumetric pipette with one hand on the pipette pump and the other hand
on the bulb of the pipette. (Hint: read “Using a Volumetric Pipette” in this manual.)
6. What wavelength will you use to read the ABS of your solutions created in this lab procedure?
7. What should the absorbance of your Primary Standard be?
8. What information will you be graphing for this lab?