3.

Results and Discussion

When the cuvette with a solution was
placed
inside
the
UV-Vis
spectrophotometer, its absorbance reading
was determined. This is due to the light
source in the equipment, which travels
through a monochromator and a slit to
allow only the desired wavelengths,
suitable for the solution, to pass through
the sample. The sample absorbs some of
the light / energy depending on its
absorptivity, width of the cuvette used and
its concentration. These are all related as
shown in Equation _. Through measuring
the absorbance of Cu(II) solutions of known
concentrations, a graph of absorbance vs
concentration can be made. This can be
used to determine the concentration of
Cu(II) from an unknown solution through
using the best fit line of the graph to
determine which concentration it aligns to
given an absorbance value.
After measuring the absorbance of the
solutions of standard Cu(II) with known
concentrations,
the
absorbance
data
obtained are shown in the table below.
Table

Using the data from the table above, a

calibration curve was prepared. The graph
of absorbance vs concentration of Cu(II)
was made. The graph is shown below.
Graph 1. Absorbance vs Concentration of
Cu(II)
The equation of the best fit line was
found to be y = 0.0001x with an R 2 value of
0.99853. From the graph, In the equation of
the best fit line, y = absorbance of the
solution,
and
x
represents
the
concentration of the solution. The equation
of the best fit line was then used as the
equation for determining the Cu(II)
concentration of the unknown solution
given its absorbance. The absorbance
readings for all three trials of the unknown
solution were 0.055, and plugging into the
equation gives a value of 550ppm as the

concentration of Cu(II) of the unknown

solution per trial, and thus the average is
also 550ppm Cu(II).
During the preparation of the solutions,
ammonia was added to Cu(II) solutions in
order to allow the following reaction to take
place or move towards the products side.
Figure _.

The formation of the copper ammonia

complex has a more intense color so it can
be
better
examined
by
the
spectrophotometer.
Beer-Lambert Law is expressed in terms
of absorbance instead of transmittance
because absorbance gives us a more
simplified linear relationship between the
variables. On the other hand, expressing it
in transmittance would involve using
logarithms, which is more difficult to
perform.
However, there are a couple of
limitations to Beers Law. First, the
concentration of solutions used should be
dilute, approximately less than 0.01M. A
high concentration would mean molecules
are in close proximity with each other and
the electrostatic interactions in these
solutions cause a change in absorptivity of
the solution. Another is that solutions cant
undergo any kind of chemical reactions
such as dissociation and precipitation
during the experiment. This is because the
color and the absorbance will change.
In conducting the experiment, it was
important to scan over a wavelength range
first since incident light absorbed varies
with wavelength. Thus scanning over a
wavelength range will help determine the
wavelength best used for analysis. This is
the analytical wavelength, wherein the
light absorbance per unit concentration is
at its peak and the wavelength-dependent
molar absorptivity coefficient is constant.
Therefore, at max, concentration is the
only parameter affecting absorption. Where
absorbance is highest, sensitivity is
highest, and deviations are at a minimum.
Trace analytes are thereby more accurately
measured by the spectrophotometer,
resulting in more reliable values obtained
and more accurate calibration curve
generated.
In obtaining the copper(II) concentration
of the unknown solution, the equation of
the best fit line is used, and the y-intercept
is significant because it shows whether the
data obtained is consistent with Beers Law.

If
the

data obtained is consistent with Beers

Law, then there should be a direct linear
relationship between absorbance and
concentration, and the plot should pass
through the origin or near the origin. A yintercept of zero means that the calculated
concentration of copper(II) should be the
theoretical value of copper(II)
concentration, and the larger the yintercept the larger the deviation is of the
calculated copper(II) concentration. Having
a y-intercept means that there is
something more that absorbs the light from
the source apart from the solution being
analyzed, which in theory only the copper
particles should absorb the light. Thus, the
size of the y-intercept reflects the quality of
the data. The calculated concentration of
stock sample copper (II) is 550ppm and the
y-intercept of the graph is zero. This means
that the quality of the data should be
consistent with Beers Law and the value
obtained from the experiment should be
the theoretical value of copper
concentration.
In theory, the result of the concentration
of copper (II) from the experiment would be
exactly like the theoretical value. This
means that the graph should show a direct
linear relationship between the points, and
the equation of the best fit line proves this.
However, the value of R2shows that not all
points lie on a straight line without scatter.
There is a point that doesnt fit the best fit
line, which is the fourth one. This means
that errors probably occurred in the
experiment. One possible source of error is
in the methods of the experiment. The
cuvette had to be handled with bare hands
thus the cuvette could have some stains of
fingerprints on it. This will lead to an
increase in the absorbance reading of the
spectrophotometer. A way to avoid this
error is to wear gloves that wont leave
fingerprints on the cuvette. possible source
of error for this type of experiment is using
a different cuvette from the cuvette used in
standardization (absorbance reading

against reagent blank solutions). As

mentioned a while ago, if the cuvette used
was not read against a reagent black
solution, the absorbance increases due to
the longer path length that needs to be
traversed by the light, and the width of the
cuvette affects absorbance as well. Lastly,
Another possible source of error is the
presence of polychromatic light.
Polychromatic light means light with
multiple wavelengths. This will cause the
graph to deviate from linearity and become
more logarithmic. Also this takes away the
use of determining the analytical
wavelength.
In this experiment, spectrophotometry
was used to determine copper (II)
concentration, but apart from determining
substance concentration in a solution,
other analytical applications of
spectrophotometry can be found in other
areas of science such as biology, physics,
materials engineering, etc. For example it
is used to determine enzyme-catalyzed
reactions in biochemistry. It is used to
examine blood or tissues for clinical
diagnosis in clinical applications. Lastly, in
Biology, it helps with scientific research
projects and bioassays such as
differentiating between the various
pigments found in a plant cell.
4. Conclusion and Recommendations
The experiment showed how
spectrophotometry can be used to
determine unknown concentration of a
solution. Determining the absorption values
of solutions with known concentration
enables the experimenters to plot a graph
with a best fit line that will be used to solve
the unknown concentration. From the
graph it is seen that there is a direct linear
relationship between concentration of the
solution and absorption. The use of
spectrophotometry to determine the the
absorption values of copper(II) solutions
with known concentration enabled the
experimenters to obtain a graph with a
best fit line of y=0.0001x with an R2 value
of 0.99853. Using the best fit line and the
absorption value of the copper(II) solution
with unknown concentration, the calculated
concentration of stock sample copper (II) is
550ppm. Since the best fit line had no yintercept, the value of the calculated
copper(II) concentration from the
experiment should be the theoretical
copper(II) concentration. However, the R2
value is not 1, which means that there is a
point that doesnt really fit the best fit line

as seen in the graph so there might be a

slight deviation from the theoretical
copper(II) concentration, and this is due to
the errors that occurred in the experiment.
Such errors would be having fingerprints on
the cuvette and the presence of
polychromatic light.

Appendix
Calculations:
1. Sample Analysis
Best Fit Line : y = 0.0001x, where
absorbance is y and concentration is x
Trial 1:
Absorbance = 0.055
0.055 = 0.0001x
x = 550
Concentration = 550ppm
Trial 2:
Absorbance = 0.055
0.055 = 0.0001x
x = 550
concentration = 550ppm
Trial 3:
Absorbance = 0.055
0.055 = 0.0001x
x = 550
concentration = 550ppm