CHEM 1412 Lab – 4 Dr.

Pahlavan

Solutions Concentration
Preparing, Diluting, and Absorbance
Purpose:
a) To prepare solution and determine the amount of copper using a spectrophotometric method.
b) Determine the relationship between the concentration and the absorption of light by the solution
using the spectrophotometer.
c) To demonstrate the operation of a spectrophotometer and to determine whether Beer's Law applies
to a particular solution.

Equipment and Chemicals

Balance Plastic pipet Spectrophotometer - 20 Cuvets Graduated cylinder, (10-mL)

Spatula Test tube rack Test tubes Volumetric flask (100-mL)
Weighing paper
Copper (II) sulfate pentahydrate, CuSO4 • 5H2O

Introduction
Solutions are in an important part of chemistry and must be prepared with accuracy. In this laboratory a copper
(II) sulfate solution prepared, and then will be diluted to other solutions with different concentrations.

A a concentrated solution contains a large amount of solute in a given amount of solution and dilute solution
contains only a small amount of solute in a given amount of solution. To describe the concentration of a
solution expressed in molarity, M, which is defined as the number of moles of solute in one liter of solution
(Eq. 1).

Example 1 - Consider the preparation of 500.0mL of a 0.50M solution of sodium chloride:

g ( NaCl )= (100.0ml x1.0 L/1000ml) x ( 0.50moles NaCl) ) ( 58.5 g NaCl ) = 2.925 ~ 3.00 g NaCl
1L NaCl 1mole NaCl

To prepare 100.0mL of 0.50M NaCl solution. Using analytical balance to measure 3.00 g of NaCl and add it to
100 ml volumetric flask and dilute it with deionized water (D.I.) to the mark. For a 100-mL volumetric flask,
the mark on the neck indicated that when filled to the mark, the flask will contain exactly 100.0 mL at of 0.50M
solution of NaCl.

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CHEM 1412 Lab – 4 Dr. Pahlavan

Diluting Solutions
To make a dilution, the following equation is used: M1V1 = M2V2
In this equation, M1 is the concentration of the stock solution, V1, is the volume of the stock solution required
to prepare the dilute solution, M2 is the concentration of the desired dilute solution, and V2 is the volume of the
dilute solution needed. The dilution equation is commonly written as shown in Eq. 2. The subscripts 1 and 2
refer to the concentrated solution and the dilute solution, respectively.

M1V1 = M2V2 Eq. 2

Example 2 - Assume that the 0.50M sodium chloride solution prepared in the example above is in the
stockroom, but for another experiment, 100mL of a 0.10M sodium chloride solution is needed. In performing a
dilution calculation, M1, M2, and V2 are generally known and Equation 2 is rearranged to solve for the unknown
V1. Using equation 1 allows us to solve for the volume of the concentrated solution required to prepare the
dilute solution.

Proper analytical technique for preparing the diluted solution requires that the initial and final volumes (V 1 and
V2) must be accurately measured using a graduated cylinder or, preferably, a pipet and a volumetric flask.

Concentration and Absorbance, Beer’s Lambert Law

An important problem chemist encounter in the lab is how to determine the concentration of an unknown
solution. If the solution is colored, the concentration of an unknown solution can be determined by measuring
the intensity of the color. A colorimeter (spectrophotometer) is used to measure the absorbance of visible light
that gives the solution its color. Generally, the more intense the color of the solution, the greater the absorbance
of light will be. In using colorimetry, it is important to remember that the color of light transmitted by the
solution (the color we see) is complimentary to the color of light absorbed by the solution (the color we
measure). Since the color of light depends on its wavelength, the wavelength of light absorbed by a colored
substance in solution is complementary to the wavelength of light transmitted by the substance. Copper (II)
sulfate solutions, for example, are blue. The absorbance of copper (II) sulfate solutions is measured at a
wavelength of 635nm, corresponding to red light.

A spectrophotometer essentially enables one to isolate a narrow beam of nearly monochromatic (one
wavelength) light, pass it through a sample, and measure the radiant power, I, of the light transmitted through
the sample relative to the incident radiant power, Io. The ratio I/Io is called the transmittance, T, and is the
fraction of light of a given wavelength transmitted through the sample.
The percent transmittance is given by: %T = (I/Io) x 100

Beer's law states that, for a given solution, the absorbance is directly proportional to path length and solute
concentration. In the spectrophotometric measurement of color intensity, the light transmitted through the
solution is measured.
Another term that is more useful theoretically than transmittance is absorbance. It is a measure of the amount
of light of a given wavelength absorbed by the sample itself and is thus related to the interaction of the light

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CHEM 1412 Lab – 4 Dr. Pahlavan

with the atoms, molecules, and ions in the sample. As expected from their definitions, absorbance, A, and
transmittance are inversely related. The mathematical relationship is:

A = log (1/T) = -log T and A = 2 – log (%T)

Example 3 – Do the following conversions.

a) absorbance 0.015 to transmittance  T = 100 antiLog (-0.0150) = 96.6%

b0 25.5% transmittance to absorbance  A = - Log (25.5/100) = 0.593 OR A = 2 - Log (25.5) = 0.593

Spectrophotometer shows both percent transmittance (%T) and absorbance (A). The amount of light at a
given wavelength that a sample absorbs is often directly proportional to the concentration of the absorbing
species in the sample and to the distance (path length) the light must travel through the sample. Mathematically,
this relationship is given by Beer’s Law and is expressed as:

xample 4- A compound had a molar absorptivity of 2.17x103 L/cm.mol. What concentration of compound
would be to produce in solution having transmittance of 18.24% in a 2.50 cm cell?

a = 2.17x103 L/cm.mol , T% = 18.24% , b = 2.50 cm , c=?

Solution: A = -Log (T%) = -Log (18.24/100) = 0.735
A = abc  c = (A /ab) = ( 0.735 ) / ( 2.17x103 x 2.50)  c = 1.35x10-4mol/L or M
The following is the schematic presentation of spectrophotometer (Fig 1). and absorbance vs. concentration
graph (Fig. 2).

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CHEM 1412 Lab – 4 Dr. Pahlavan

Working Curve Analysis

The absorbance of a series of three to five standard solutions are measured and plotted on graph paper against
the concentrations of these standards. This is known as a Beer's Law plot. The absorbance of an unknown
concentration is then measured, and its concentration is determined directly from the plot. This method is more
commonly used than the absolute calculation, because experimental error will average out over the number of
standards. and absorbance vs. concentration graph (Fig. 2).

Fig. 1 Fig. 2

Safety Precautions
Copper (II) sulfate is moderately toxic by ingestion and inhalation and is a skin and respiratory irritant. Avoid
contact with eyes and skin. Wear chemical splash goggles, chemical resistant gloves, and a chemical- resistant
apron. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure
Part A. Preparing the Stock Solution

1. Calculate the number of grams of copper (II) sulfate pentahydrate, CuSO 4 • 5H2O, required to prepare 100.0
mL of a 0.250 M solution. Once your calculations have been approved by your instructor, weigh out the
required amount of copper sulfate on a balance in a clean, dry weighing paper.
2. Transfer the solid to a 100-mL volumetric flask. Add distilled or deionized water and mix the solution to
give a homogeneous solution to the mark line on flask. Lable that “S” for stock solution.

Part B. Preparing Diluted Solutions

1. Select five clean and dry test tubes in a test tube rack and label them 1 through 5. Using a 10mL graduated
cylinder, measure and pour 10mL of the 0.250M stock solution into test tube #1. Record the necessary data
for this solution in the data table.

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CHEM 1412 Lab – 4 Dr. Pahlavan

3. Fill the 10-mL graduated cylinder exactly to the 2.00-mL mark with the stock solution and fill the graduated
cylinder to the 10.0-mL mark with distilled or deionized water.
4. Repeat same dilution with 3mL, 4 mL, and 5 mL for test tubes 3, 4, and 5.

5. Proceed your calculations for preparation of 10.0mL cupric sulfate solution 1, 2, 3, 4, and 5.
Check with your instructor before proceeding. record the necessary data in the data table.
6. Compare the color of the stock solution and each of the dilutions in test tubes #1-5. Record these
observations in the data table.

Part C. Colorimetry Measurements

Using Spectrum- 20 (Spectrophotometer)

1. Turn on the spectrophotometer and allow it to warm up for 5-10 minutes.

2. Turn the wavelength knob and adjust the wavelength to 635nm.
3. Fill a cuvet about ¾ full will distilled water. Then place the cuvet in the colorimeter compartment.
and adjust to read 0%T and 0 absorbance with a “blank” cuvet containing only distilled water.
4. Fill the cuvets with each solution. Wipe the cuvets with paper.
5. Record the absorbance data for solutions #1-5 in the Data Table.
6. Prepare a graph of absorbance on the y-axis versus the concentration of each solution on the x-axis.
7. Obtain an absorbance reading for the unknown sample.
8. Make a graph of concentration, c, (x-axis) vs. absorbance, a, (y-axis).
9. From the standard curve, determine the concentration of copper in unknown sample solution..

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CHEM 1412 Lab – 4 Dr. Pahlavan

REPORT FORM
Solutions Concentration
Preparing, Diluting, and Absorbance
Test Tube 1 2 3 4 5
Volume of Stock Solution
(V1) 1.0mL 2.0mL 3.0mL 4.0 mL 5.0 mL
Concentration of Stock Solution
(M1) 0.250M 0.250M 0.250M 0.250M 0.250M
Final Volume of Diluted Solution
(V2) 10.0mL 10.0mL 10.0mL 10.0mL 10.0mL
Concentration of Diluted
Solution (M2) (show Calculation)
M2 = (M1V1)/V2

Absorbance at 635 nm (A)

Percent Transmittance (%T)

Unknown Identification: _______

Absorbance (A) = ____________ Concentration (c) : ____________

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CHEM 1412 Lab – 4 Dr. Pahlavan

Solutions Concentration
Preparing, Diluting, and Absorbance
Review Questions and Exercises Name __________________________
Due before lab begins. Answer on a separate sheet of paper.
Date ___________________________
1. Convert the accompanying transmittance data to absorbance.

a) 53.86%

b) 0.567%

2. Express the following absorbances in terms of percent transmittance.

a) 0.918

b) 0.072

3. What is the relationship of percentage transmittance and absorbance? If the percentage transmittance for a
sample is 100 at a given wavelength, what is the value of absorbance?

4. Calculate the molar concentration of a solution if the absorbance (A) is 0.244, the molar absorptivity (A)
is 3.39 x 104 L/mole cm and the path length of the cell (b) is 2.00 cm.

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CHEM 1412 Lab – 4 Dr. Pahlavan

Solutions Concentration
Preparing, Diluting, and Absorbance

Post- laboratory Questions and Exercises Name _____________________

Due after completing lab. Answer in the space provided.
Date ______________________

1. Calculate the number of grams of copper (II) sulfate pentahydrate, CuSO4 • 5H2O, required to prepare
100.0mL of the following solution by dilution process.

a) 0.200 M copper (II) sulfate solution.

b) Calculate the number of milliliters of 0.250M copper (II) sulfate solution that must be diluted to prepare
10.0mL of a 0.150M copper (II) sulfate solution.

2. For a solution of 0.200 M Cu (NO3)2 in a 1-cm cell, the following data has been collected. Calculate the
percentage transmittance, %T, for each absorbance. plot A vs. C and T% vs. C on the same graph.

C(mol/L) A %T
0.010 0.112
0.030 0.336
0.060 0.672
0.090 1.008
0.120 1.120

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