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Appendix J
Harris, Section 2.5, Chapter 18, and Section 19.1
INTRODUCTION
Spectrophotometric determinations based on the Beer-Lambert Law are among the most widely
used analytical procedures. These methods involve the measurement of the fraction of incident
electromagnetic radiation that is absorbed by a sample. To determine the concentration of a colored
species in solution, a cuvet containing the solution is placed in the spectrometer, which consists of a
spectrograph and a photometer. The spectrograph produces light of selected wavelengths and is
calibrated to indicate the color of the light it produces in terms of wavelength, usually expressed in
nanometers. The photometer measures the intensity of the light beam at a given wavelength. The amount
of light that is absorbed by the sample can be used to determine the amount of the colored species in
solution according to the Beer-Lambert Law.
The absorbance, A, of the solution containing a given metal ion is equal to the molar absorptivity
(in liters per mole-cm) times the path length b (in cm) times the concentration c (in moles per liter). This
law is known as the Beer-Lambert Law, A = bc. Special sample cuvets are used which have a path length
of 1.000 cm. The molar absorptivity depends on the wavelength of the light and is determined by
measuring the absorbance of a series of standard solutions of known concentration. Once and b are
known, the concentration of an unknown solution can be determined by measuring the absorbance (see
Appendix J) and using the Beer-Lambert Law given above.
You will determine the concentrations of both dyesthe Cr3+ and Co2+ simultaneously by a
spectrophotometric determination. Since the total absorbance of the solution at a given wavelength is
equal to the sum of the absorbances of the individual components, it is possible to analyze the individual
components of a mixture even when their spectra overlap. Consider the spectra of M and N given in
Figure 2.
There is obviously no wavelength at which the absorbance of this mixture is due simply to one of the
components; thus an analysis for either M or N by a single measurement is not possible. However, the
absorbances of the mixture at the wavelengths 1 and 2 may be expressed as follows:
At 1 :
A1 1M bcM 1N bcN
At 2 :
2
A2 M
bcM N2 bcN
2
The four molar absorptivities 1M , M
, 1N , and N2 can be evaluated from standard solutions containing
only M or N. Then, if the absorbances of the mixture are measured at 1 and 2, the concentration of the
individual components can be calculated by solving the two equations given above simultaneously. The
best accuracy in an analysis of this sort is obtained by choosing wavelengths at which the differences in
molar absorptivities between the two ions is large. For your unknown, you will use the absorption
spectra of yellow and red dyes 0.03 M Cr3+ and 0.1 M Co2+ to choose two wavelengths.
In this lab you will also gain experience in writing a formal laboratory report.
EXPERIMENTAL
1.
Obtain solutions of Yellow 5 (50 mg/L) and Red 40 (50 mg/L). You will need to create dilutions in
order to measure the extinction coefficientPrepare 50 mL each of solutions containing 0.01, 0.02, 0.03,
and 0.04 M Cr(III), and 0.02, 0.04, 0.06, and 0.1 M Co(II) by careful dilution of the master solutions
provided [0.05 M Cr(III) and 0.2 M Co(II)]. You do not need a separate volumetric flask for each
solution. Store solutions in Erlenmeyer flasks after they are prepared.
2.
Obtain your unknowns from the Stockroomthe unknown orange solution in the requested
glassware. It should be clean, dry, and labeled with your name and locker number.
3.
Measure the absorbance vs. wavelength for the 0.1 M Co(II)yellow dye and red dye0.03 M Cr(III)
solutions using the diode array spectrophotometer in Spectrum/Peaks mode. From these data, select
the two wavelengths that will produce the greatest accuracy in this determination. Consider the
influence of concentration. Check these values with your TA before you proceed.
4.
Measure the absorbance spectra of each known solution and your unknown mixtures in Fixed
Wavelengths mode at both wavelengths chosen. AYou should realize that an accurate absorbance
measurement is best made when the A is between 0.1 and 1.0. If your unknown has an A above 1 you
should quantitatively dilute. If your A is below 0.1 you have diluted too much.
ANALYSIS
From the absorbance data on the known solutions prepare four standard curves, one for each ion
at each of your chosen wavelengths. Using the techniques of linear regression described in Harris,
determine the slopes (b) of these curves and their 95 % confidence limits. Using these slopes and the
absorbance measurements made at the two wavelengths, determine the concentrations of both dyes
Co(II) and Cr(III) in your unknown. Determine the 95 % confidence limits, assuming that all of the
uncertainty comes from the slope measurements.
If time permits you can also use the known spectra over the entire wavelength span to perform a
component decomposition of your unknown data. This is done by importing your full standard and
unknown spectra into an Excel spreadsheet for analysis. Give it a try.
You will be given a glass blowing demonstration during this lab. You will test your skills today.
Remember: Hot glass looks just like cold glass! Retrieve one 15-inch and one 7.5-inch piece of flint (soft)
glass tubing from the Stockroom. Mark the 15-inch piece at 9 to 10 inches. Make a 90 degree bend at the
mark. The 7.5-inch piece will be pulled into a capillary with a maximum 2 mm diameter. Refer to
Appendix C & D for more details.