DETERMINATION OF COPPER (II)

CONCENTRATION BY COLORIMETRIC METHOD

INSTITUTE OF BIOLOGY, COLLEGE OF SCIENCE
UNIVERSITY OF THE PHILIPPINES, DILIMAN, QUEZON CITY 1101, PHILIPPINES
DATE SUBMITTED: 12 MAY 2015
DATE PERFORMED: 06 MAY 2015

DISCUSSION
1. What is the significance of the addition of ammonia to Cu (II) solutions?
The addition of ammonia to the solution was done in order for the following
reaction to proceed:
Cu2+ + 4NH3 [Cu(NH3)4]

2+

The formation of the copper amine complex gives the solution a more intense
blue color than the copper solution. The higher intensity in the color of the
solution results to an increased effectiveness of the examination by the
spectrophotometer. In addition, an excess amount of ammonia was necessary
in order to prevent the hydrolysis of ammonia and the formation of copper
hydroxide precipitate which would affect the absorbance of the solution. [1]

2. Why is Beer-Lambert Law expressed in terms of absorbance instead of

transmittance?
Beer-Lambert's is an equation describing the relationship betwen absorbance
and concentration. The reason why transmittance is not used in expressing
the Beer's Law is because of its exponential relationship with concentration,
unlike the linear relationship between absorbance and concentration [1].

3. What are the limitations of the Beer's Law?

The limitations of the Beer's law include (1) real limitation, (2) chemical
limitation, and (3) instrumental limitation. (1) One condition for a
spectrophotometry procedure to adhere to Beer's Law is that the
concentrations of the solutions used must be dilute. The reason for this is
that the more concentrated a solution is, the closer proximity the molecules
are in with each other, therefore the greater the chance that the electrostatic
interactions in these solutions to affect the absorbance of the solution. (2)
Another condition for chemicals used in a spectrophotometric procedure is

that these must not undergo any chemical reactions along the procedure.
Chemical reactions such as equilibration, formation, precipitation or
equilibration cause a change in the color or the composition of the solution,
therefore changing the absorbance of the solution. (3) Instrumental
limitations include several factors which may affect the effectiveness of the
spectrophotometer. These include polychromatic light, stray light and
improper use of the cuvettes. Polychromatic light or light having multiple
wavelengths will cause a deviation from linearity while stray light always
causes a decrease in the absorbance value of the solution. Proper use of the
cuvette includes awareness of the clear and rough sides of the cuvette. The
side that must be facing the light source is the clear side; otherwise, the
rough side of the cuvette will affect the absorbance measured [1].

4. Why is it significant to scan over a wavelength range? Why is analytical

wavelength used in the determination of the absorbance of the standard and the
sample solutions?
Before measuring the respective absorbance of the samples, the lambda max
was determined through the sample with the highest concentration. The ideal
lambda max for this particular spectrophotometric analysis is 625
nanometers. This wavelength range was used for several reasons, first,
because the change in absorbance per unit concentration at this point is at
its greatest magnitude. This leads to a wider calibration curve, thus a large
working range consequentially leading to a higher probability that the
absorbance of the sample is within the calibration curve. The second reason
is that having a wavelength range wherein the sample absorbs light most
increases the sensitivity, and therefore the efficiency of the
spectrophotometer to measure the absorbance of a solution even if the
concentration of the sample is small. The last reason is that at the lambda
max, the linear relationship of the absorbance and concentration of the
sample is most observable. At any other point, a deviation from linearity
occurs[1].

5. Why do we have to measure absorbance reading against reagent blank solutions?

A reagent blank solution was necessary in order to eliminate the effects of
quantities that should not be a part of the absorbance reading. These
quantities that must be eliminated include the absorbance of the ammonia
solution, and the refraction, reflection or scattering of light that the cuvette
might have caused. This serves a similar function as the tare in a toploading
or analytical balance. Neglecting to measure the absorbance reading in a
blank solution results to an increase in the absorbance reading since it
includes all the factors mentioned above[1].

6. What is the significance of the y-intercept of your calibration curve? Discuss in

the deviation from the theoretical value.

In the construction of the calibration curve, the concentration and

absorbance values of the standard solutions are plotted and a line is taken.
The equation of the line is given by the following equation:
y= mx + b
The sample solutions concentration is given by the slope of the graph. The
variable b represents the error or the deviation from the theoretical value.
The variable y is significant in the calibration curve because it represents the
absorbance of the sample solution which is the value measured from the
spectrophotometer. Therefore, substituting the known concentration of the
sample solution, the measured absorbance from the spectrophotometer, and
the calculated error, x is then determined by isolating the variable [1].

7. Cite other analytical applications of spectrophotometry.

Aside from measuring the concentration of a given solution,
spectrophotometry can also be used for the detection of impurities. In the
graph that is given by the UV-Vis spectrophotometer, additional peaks are
observable due to impurities. Another use of the graph is the determination
of the structure of organic compounds; this may be done using the location of
peaks in the UV spectroscopic graph. UV spectroscopy can also be used to
study kinetics of reactions. Another use is the detection of functional groups
since the absence of a band at particular wavelength can be regarded as an
evidence for absence of a particular group.. Molecular weights of compounds
can also be measured spectrophotometrically by preparing the suitable
derivatives of these compounds. An example is the determination of the
molecular weight of amine through its convertion to amine picrate.

8. What are the possible sources of errors and their effect on the calculated
parameters? Rationalize.
Discrepancies in the calculated parameters include the following: (1)
ammonia was not added in excess amount, (2) the rough side of the cuvette
faces the light source, (3) the proper washing procedure for a cuvette for
different solutions was not followed, (4) failure to measure the absorbance of
the blank solution and take it into consideration, (5) failure to measure the
lambda max with the most concentrated solution among the samples, (6)
mismatched cell/cuvette, and (7) inclusion of the blank solution in the
calibration curve.
(1) Ammonia was added to the copper solution in order for the formation of
copper amine complex which results to a deeper and more intense blue
color which increases the effectiveness of the examination of the
spectrophotometer. However, a limited amount of ammonia will only result
to its hydrolysis and cause the formation of copper hydroxide based on
the following chemical reactions:

NH3 + H2O NH4+ + OHCu2+ + OH- Cu(OH)2

The formation of a precipitate in the sample solution would cause the
scattering of the light that passes through the solution and result to
increased absorbance since not all the light that is transmitted by the
solution reached the detector, therefore a lower transmittance. This is the
reason why ammonia must be added in excess.
(2) The cuvette has two faces, the clear side and the rough side. The purpose
of the rough side is for easier handling of the cuvette while the clear side
is what should face the light source. In the event that the rough side faces
the light source, the transmittance decreases, therefore increasing the
measured absorbance of the sample solution.
(3) After every measurement of the absorbance of a solution, the cuvette
used must be washed three times with distilled water and another three
times with the sample solution to be measured next. Failure to do so
might affect the concentration of the sample solution. Since there is a
linear relationship between the concentration and absorbance of the
solution, any change in concentration would also affect the measured
absorbance.
(4) The measurement of the absorbance of the blank solution which contains
pure ammonia serves the similar function as the tare in a top loading or
analytical balance. It is performed to eliminate the effects of quantities
that must not be included in the absorbance reading such as the
absorbance of the ammonia solution and the reflection, refraction, and
scattering of light by the cuvette.
(5) The lambda max must be measured using the most concentrated among
the solutions. Failure to do so might cause error in the measured
absorbance. This is because using a solution other than the most
concentrated solution reduces the probability that all the samples to be
measured have absorbances within the calibration curve.
(6) Using a different cuvette for the blank solution and the samples would
negate the necessity of having to measure the absorbance of a blank. The
cuvette used for the blank must also be used for the analysis of the
samples in order for the eliminated reflection, refraction and scattering
effect of the cuvette would not be part of the measured absorbance of the
sample solution.
(7) In the construction of the calibration curve, the blank solution should not
be included in the graph since it does not contain the substance that is
being analyzed which is the copper (II).

APPENDIX
A- REPORTED TABULATED VALUES
Table 1. Data for the construction of the calibration curve
Volume of Working
Concentration of
Standard Solution,
Absorbance
Standard Cu(II), ppm
ml
2.00
100
0.110
4.00
200
0.219
6.00
300
0.323
8.00
400
0.432
10.00
500
0.542
Table 2. Data for Sample Analysis
Concentration of
Trial
Absorbance
unknown sample
Cu(II), ppm
1
0.297
273.816156
2
0.302
278.4586815
3
0.294
271.0306407

Concentration of
stock solution
Cu(II), ppm
2489.237782
2531.442559
2463.914915

B- CALCULATIONS
Linear equation of the calibration curve: y=0.001077x + 0.0021
Trial 1:
0.297=0.001077x + 0.0021
x= 273.816156 ppm
(50 ml)(273.816156 ppm)=(5.5 ml)(M of stock Cu(II) solution)
M= 2489.237782 ppm
Trial 2:
0.302=0.001077x + 0.0021
x= 278.4586815 ppm

(50 ml)(278.4586815 ppm)=(5.5 ml)(M of stock Cu(II) solution)

M= 2531.442559 ppm
Trial 3:
0.294=0.001077x + 0.0021
x= 271.0306407 ppm
(50 ml)(271.0306407 ppm)=(5.5 ml)(M of stock Cu(II) solution)
M= 2463.9194915 ppm
REFERENCES
N.p, Chemistry 26.1 2nd Written Laboratory Examination Reviewer AY 20102011, chemistry-26-1-2nd-laboratory-exam-reviewer.pdf, p. 5-10