Experiment 1. Spectrophotometeric Measurements and Beer-Lambert Law
(original prepared by Kelemu Woldegiorgis)
With added video link and virtual data.
Objectives: (1) To gain hands-on experience using a spectrophotometer.
(2) To understand the importance of the Beer-Lambert law and a cali
ation curve
Background: Spectroscopy is a simple and powerful method for performing both qualitative and quantitative analyses. It is a technique that involves the study of the interaction of light with matter. Different regions of the electromagnetic spectrum such as ultraviolet, infrared, visible, or X-ray radiation can be used to interact with matter. Ultraviolet and infrared spectrophotometeric methods are suitable for many colorless substances that abso
strongly in the UV and IR spectral regions, respectively. Visible spectrophotometeric methods can be used to determine compounds that are colored or that react to form a colored product.
The amount of light abso
ed or emitted by a sample depends on both the nature and quantity of the sample. Every substance abso
s or transmits light of certain wavelengths. For this reason, the absorption or transmission of specific wavelengths is characteristic for a substance, and a spectral analysis serves as a “fingerprint” of the substance. When light falls on a substance, one of the three things listed below could happen:
• the light can be totally reflected by the substance
• it can be totally abso
ed by the substance
• certain wavelengths can be abso
ed and the remainder transmitted or reflected.
A colored solution selectively abso
s certain wavelengths of visible light and transmits the remaining wavelengths. The color of the solution is the result of a combination of the transmitted wavelengths. A solution appears black if it abso
s all wavelengths in the visible region and appears white or colorless if it abso
s none of them. We see the various colors when particular wavelengths of radiant energy reach our eyes. Suppose we shine a beam of white light at a substance that abso
s blue light. Since the blue component of the white light gets abso
ed by the substance, the light that is transmitted is mostly yellow, the complementary color of blue. This yellow light reaches our eyes, and we “see” the substance as a yellow colored substance. The table below gives pairs of complementary colors and the co
esponding wavelength ranges.
XXXXXXXXXXTable 1. Approximate complementary color relationship between the wavelengths of
XXXXXXXXXXlight abso
ed and the wavelengths of light transmitted.
Wavelength (nm)
Color Abso
ed
Color Observed
400
Violet
Yellow-green
435
Blue
Yellow
495
Green
Purple
560
Yellow
Blue
650
Orange
Greenish blue
800
Red
Bluish green
If light of a particular wavelength is abso
ed by a sample, the intensity of the beam transmitted by the sample (It) will be less than the intensity of the incoming light beam (Io), Fig. 1.
Figure 1. Schematic Drawing for a Spectrophotometer
The ratio of It and Io can be used to indicate the percentage of incoming light abso
ed by the solution. This is called the percent transmittance, Eqn. 1.
(1)
A more useful quantity is the abso
ance (A) defined as
A = –log(T) (2)
Eqn. (2) predicts that lower percent transmittance means higher abso
ance of light by a solution.
The relationship between abso
ance and concentration of a substance in solution is given by the Beer-Lambert law, Eqn. 3.
A = ε • l • c (3)
where, A is abso
ance (dimensionless number); ε the proportionality constant called the molar extinction coefficient or molar absorptivity; l is the light path length (measured in cm); and c is the concentration of solution in mol/liter. The Beer-Lambert Law is strictly applicable only for dilute solutions. It becomes less and less accurate as the concentration of the solution increases. A picture of the spectrophotometer you will be using in this lab is shown in Fig. 2.
Figure 2. GENESYS 20 Visible Spectrophotomete
In this experiment you will construct a cali
ation curve which consists of a plot of abso
ance versus concentration using KMnO4 standard solutions whose concentrations
are known accurately. The quality of a cali
ation curve is very important since concentrations of unknown solutions are determined using the cali
ation curve. The slope of the cali
ation curve equals to the molar absorptivity, . Ideally, there should be no y-intercept on the cali
ation plot since the abso
ance should be zero when the concentration of the light abso
ing substance is zero. Once you have the cali
ation curve prepared, you may measure the abso
ance of any unknown KMnO4 solution at the same wavelength, and same experimental conditions, and read off its concentration from the graph or calculate from the slope.
Materials and Apparatus
GENESYS 20 Visible Spectrophotometer, 1 cm plastic cuvettes, 50-mL burets, 100-mL volumetric flasks, 250-mL beaker, hotplate with magnetic sti
er, oxalic acid dihydrate (H2C2O4.2H2O), stock solution of KMnO4 (~0.02 M), and unknown solutions of KMnO4.
A. Standardization of the KMnO4 Stock Solution (~ 0.02 M)
1. Accurately weigh a 0.100 g sample of H2C2O4.2H2O (MM = XXXXXXXXXXg/mol) and carefully transfer the solid to a 250-mL beaker. Dissolve the solid in about 150 mL of 1.0 M H2SO4.
2. Rinse a 50-mL buret thoroughly with tap water followed by rinsing it with distilled water a couple of times. Rinse the clean buret with about 5 mL of the KMnO4 stock solution. Dispose of the rinsing KMnO4 solution in the designated waste container.
3. Heat the oxalic acid solution to 60 – 70 oC using a hot plate/magnetic sti
er.
4. Fill the 50-mL buret with the KMnO4 standard solution. Record the initial reading on the buret to the nearest 0.01 mL. Start adding the KMnO4 stock solution directly into the oxalic acid solution while sti
ing with the magnetic sti
er. Promptly wash down any KMnO4 that spatters on the walls of the beaker into the bulk of the solution using a water wash bottle. The purple color imparted by addition of a small amount of KMnO4 should be permitted to disappear before further KMnO4 is added. The end point is marked by the appearance of a faint purple color that persists at least 30 seconds.
5. Record the final reading of KMnO4 solution to the nearest 0.01 mL. Record in your lab sheet the volume of KMnO4 required for the titration.
6. Repeat the titration using another 0.100 g sample of oxalic acid.
7. Calculate the concentration of KMnO4 in each trial and use the average for further KMnO4 concentration calculations. Under acid conditions (from the addition of the sulfuric acid), here is the balanced chemical equation for the reaction of Potassium permanganate with oxalic acid:
2KMnO4 + 3H2SO4 + 5H2C2O4 → K2SO4 + 2MnSO4 + 8H2O + 10CO2
B. Preparing Dilute KMnO4 Standard Solutions
1. Calculate the concentrations of the KMnO4 standard solutions XXXXXXXXXXusing the relation:
Vstock . Cstock = Vdilute . Cdilute.
2. The volumes of the stock solution that need to be diluted to 100-mL are given in Column 2 of the table below. Write the concentrations of the dilute solutions you calculated in Column 4.
Solution #
Volume (mL) of stock KMnO4 solution (~0.02 M)*
Final volume of dilute KMnO4 solution (mL)
Concentration of dilute KMnO4 solution (mol/L)
1
0.50
100
= (0.50 x Cstock)/100
2
1.00
100
3
2.00
100
4
2.50
100
5
3.00
100
*Use the concentration of KMnO4 stock solution determined in Part A.
3. Prepare the dilute solutions as follows. To make solution #1, use a 5-mL pipet to measure out 0.50 mL of KMnO4 stock solution. Transfer the measured stock solution to a 100-mL volumetric flask. Fill the flask with distilled water up to the 100-mL mark. Replace the stopper and mix the contents carefully.
4. Prepare solutions # 2-5 in a similar fashion. You may use the same flask to prepare the remaining solutions. Remember! The flask has to be rinsed with tap water followed by distilled water between solution preparations.
C. Measurements of Abso
ance of KMnO4 Standard Solutions
1. Turn the spectrophotometer on using the power button in the back and allow 15 min for it to warm up.
2. Set the wavelength to 555 nm by pressing-and-holding the up or down wavelength button.
3. Fill a 1-cm cuvette with distilled water about three-fourth of its volume. Wipe the outside surfaces of the cuvette with Kim-wipe to remove fingerprints and/or moisture. Insert the cuvette in the sample holder and close the cover. Press the “0 ABS” button to obtain a zero abso
ance reading for the blank – water is the blank in this experiment.
4. Use the same cuvette to measure abso
ance of the standard solutions beginning from the most dilute solution, viz. Solution #1. Between measurements, the cuvette must be rinsed with the measuring solutions at least three times to avoid slight dilution of the solutions.
5. Measure the abso
ance of your unknown solution provided to you, again using the same cuvette.
6. Prepare a plot of abso
ance (y-axis) versus concentration of the standard solutions using the Excel software. Determine the slope of the cali
ation curve by displaying the curve fitting equation on the cali
ation plot. Attach the graph to your lab report.
7. Read the concentration of the unknown solution off the cali
ation curve or calculate it using the slope of the curve.
8. Dispose of the KMnO4 solutions in the designated container.
After you have read the procedure, view a video of:
Robert Ayton, Beer’s Law Laboratory Vodcast, 12/3/201, which is similar to this procedure, except that the unknown concentration solution is that of copper(II) sulfate instead of potassium permanganate. Copy the following video link into your web
owser and play the video:
https:
www.youtube.com/watch?v=rdY41FPI9iE
Experiment 1. Spectrophotometeric Measurements and Beer-Lambert Law
Name: _________________________________ Section: _______________
Lab Partner: _____________n/a___________________ Date: _________________
Data and Calculations
I. Standardization of KMnO4 solution
Trial 1 Trial 2 Trial 3_____
1. Mass of H2C2O4.2H2O (g) ___ 0.103 ___ XXXXXXXXXX___ 0.110 ____ ____ 0.106 ___
2. Moles of H2C2O4.2H2O ___________ __________ XXXXXXXXXX___________
3. Initial buret reading (mL) ____0.0 ______ ___15