1. What is the significance of the addition of ammonia to Cu (II) solutions?

Ammonia is added to the solutions to forward the reaction:

Cu2+ + 4NH3 > [Cu(NH3)4]2+
A copper-ammonia solution will give a more intense blue color copper solution. With a more
intense color the solution will be more effectively read by the spectrophotometer. If only a limited
amount of ammonia is added then the hydrolysis of ammonia might proceed and copper hydroxide
will form demonstrated by the following reaction.
NH3 + H2O <-> NH4+ + OHCu2+ + OH- <-> Cu(OH)2

2. Why is Beer Lambert Law expressed in absorbance instead of transmittance?

Absorbance is directly proportional to concentration. A linear relationship will make the
calculations easier. If transmittance was used, the relationship would be more complicated:
log(T) = -bc or T = 10^(-bc) with T being (T = I/I0) instead of A = bc

3. What are the limitations of the Beers law?

The Beers law is limited by chemical and instrumental factors.
It is instrumentally limited because only a certain range of absorbance values is required for
obtaining an accurate data. It is only limited to monochromatic radiation as polychromatic
deviation gives negative deviation to Beers law. There are also chemical limitations which occur
when the absorbing species is caught up in a state of static equilibrium. Furthermore, the
concentration of solutions must be diluted. They must be less than 0.1M. If the concentration is the
higher it will mean that the molecules are close to one another. The close proximity will result in
electrostatic interactions and cause a change in absorptivity.
The solution should not go through any kind of chemical reaction like equilibration,
dissociation, precipitation or formation during the experimental process. If the solution goes
through any such reaction the color and absorbance will change.

4. Why is it significant to scan over a wavelength range? Why is the analytical wavelength
used in the determination of the absorbance of the standard and sample solutions?

The analytical wavelength is used to determine the absorbance, the wavelength for which
the absorbance is highest. The wavelength absorbed by the solution is the complement of the
wavelength that is actually seen by our eyes.
Spectrophotometry is performed using monochromatic light. Monochromatic light is light in
which all photons have the same wavelength. The absorbance spectrum shows the extent to which
a sample absorbs light depends strongly upon the wavelength of light. The absorbance spectrum
shows how the absorbance of light depends upon the wavelength of the light.
5. Why do we have to measure absorbance reading against reagent blank solution?
During the experiment, a series of standard solutions were prepared. The absorbances of
the standard solutions were measured and used to prepare a calibration curve, which is a graph
showing the experimental absorbances varies with the concentration. For this experiment, the
points on the calibration curve should yield a straight line (Beer's Law). The slope and the intercept
of the line describes the relationship between absorbance and concentration.
An unknown solution is also analyzed. The absorbance of this unknown solution is then
used with the slope and intercept from the calibration curve to calculate the concentration of the
unknown solution.

6. What is the significant of the y-intercept of your calibration curve? Discuss its deviation
from the theoretical value.
The blank solution is often not included in the graph.
The Y-intercept of the curve is the absorbance of the reagent blank. The only chemical
species in responsible solely for absorbance is ammonia. Ammonia was present in all the solutions
examined, 10mL to be exact. It contributed to the absorbance of the working solutions in the
calculations for this experiment. It is experimentally revealed that the absorbance of the reagent
unknown sample solution was equal to 0.240, the y-intercept of the calibration curve. Y-intercept
may deviate from its theoretical value by being affected by things such as light diffraction,
absorption of light by the cuvette or any containing apparatus used to house the analyte.

7. Cite other analytical applications of spectrophotometry.

Other analytical applications of spectrophotometry include determination of the iron content
in wastewater and transferring protein in blood.

8. What are the possible sources of errors and their effect on the calculated parameters?
Rationalise.

Personal errors such as having fingerprints on the cuvette surface prevents some of the
light being from passing through the sample to reach the detector.This gives a negative deviation
towards the theoretical value of absorbance. Careful handling of the cuvette, such as wiping off the
cuvette with tissue and not allowing tissue fibers to be left on the container can help avoid such
errors.

References:
Harvey, D., 2000. Modern Analytical Chemistry. New York City: McGraw-Hill Higher
Education. 385-389

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