Enriquez and Garcia / Chemistry 26.

1/ (2014-2015) Page |1

Quantitative Determination of Copper (II) Concentration by Spectrophotometry

Giancarlo Pocholo L. Enriqueza,*
Juan Miguel Garciab,*
a
Institute of Biology, University of the Philippines – Diliman, Quezon City, PHL
b
Institute of Biology, University of the Philippines – Diliman, Quezon City, PHL

ABSTRACT
Spectrophotometry deals with the interaction of radiation and matter. It is technique that is utilized to measure the transmittance or
absorbance properties of materials as a function of wavelength. Through spectrophotometry, the experiment aims to identify the
concentration of an unknown solution since absorbance is directly proportional to concentration. The experiment was composed of
two parts. The first part aims to create a calibration curve using five trials with solutions containing 2.00, 4.00, 6.00, 8.00, and
10.00mL of Cu(II) stock solution. Meanwhile, three trials of a solution with an unknown concentration was held under
spectrophotometry for the second part. The equation obtained from the first part of the experiment which is y = 0.001x was used to
acquire the unknown concentration of the solution. For the three trials, the acquired concentration is 360ppm. These values indicate
that the data acquired is precise. Faulty handling of the cuvette, instrumental errors, and improper solution preparation are the
possible sources of error in the experiment.

1. Introduction negative logarithm of transmittance defined by (Equation

Light is a form of energy transmitted through space. It
is an electromagnetic radiation in the visible region of
the electromagnetic spectrum from 400nm to 700 nm.
On the other hand, color is perceived light based on
emittance or reflection of light rather than absorption
(Skoog et al., 2010; Blum P., 1997). For example, if a
sample absorbs all wavelengths in the visible spectrum,
it appears black. If it absorbs none, then it is white.
Moreover, as seen on (Table 1) (please refer to Appendix
A) if a beam of white light is directed at a substance that
absorbs blue light, the color perceived will then be yellow
since it has absorbed all the blue light given that yellow
is the complementary color of blue (The State University
of New York, 2005).
Meanwhile, spectrophotometry deals with the
interaction of radiation and matter. It is an analytical
technique which measures the transmission or
absorption properties of materials as a function of
wavelength. Since light is a form of electromagnetic
radiation, it can reflect, be absorbed, or be absorbed
then reflected and transmitted as it falls on a substance.
However, since spectrophotometry does not primarily
deal with reflection, only absorbance and transmittance
of light is considered (The State University of New York,
2005).
Unlike colorimetry that only uses white light that
passes through colored filters, spectrophotometry utilizes
uv-visible light that is filtered by the wavelength selector
to morph into monochromatic light that passes through
the solution that causes it to increase or decrease its
intensity. The transmitted radiation is then interpreted
as the absorbance of the solution (Skoog et al., 2010).
The absorbing characteristics of a substance or a solution
can be expressed in terms of absorbance or
transmittance. Transmittance is the ratio of the amount
of light transmitted to the amount of light radiated given
by (Equation 1). Meanwhile, absorbance is defined as the
2).
I
T=
I0
(1)
I0
A=log
I
(2)
Due to this, it can be inferred that when given two
solutions where one has a darker tone than the other, the
darker one absorbs more light. Given a higher
absorbance, it can be deduced that it also has a higher
concentration (Skoog et al., 2010; The State University of
New York, 2005; Sinvasankar, 2008). This phenomenon,
the relationship between absorption and a solution’s
concentration, is further explained by the Beer’s Law or
Beer-Lambert-Bouguer Law given by (Equation 3).
A=abc
(3)
The Beer’s Law states that absorbance is directly
proportional to the concentration of the absorbing
species, c, and to the path length, b, of the absorbing
medium (Skoog et al., 2010). However, there are
limitations to the Beer’s Law. Causes of non-linearity
include scattering of light due to particulates in the
sample or smudges and scratches in the cuvette,
fluorescence or phosphorescence in the sample, shifts in
chemical equilibria, outside light, and non-
monochromatic radiation (Kazekevich Y., 2010, David
2001).
Spectrophotometry is applied in various fields. In
microbiology. It is used in order to identify the number of
bacteria in a sample detect impurities. For example,
benzene, an impurity found in cyclohexane, can be

*Corresponding author. Mobile: 099982743524

E-mail address: pochi.enriquez@gmail.com
 Enriquez and Garcia / Chemistry 26.1/ (2014-2015)
detected at 255nm. The dissociation constants of acids
and bases can also be determined
spectrophotometrically from graphing absorbance and
wavelength at different pH values. Drugs can be
quantitatively analyzed by making a solution of the drug
and measuring the absorbance at a specific wavelength.
Chemical kinetics can also be studied through
spectrophotometry. This is done by measuring
absorbance changes based on UV radiation passing a
reaction cell. It is also applied in modern atomic theory
for the determination of certain molecular structures. It is
also used for the quantitative and qualitative
determination of both organic and inorganic compounds
(Skoog et al., 2010; pharmatutor.org n.d.).
2. Materials and Methods
Six solutions containing 0.00, 2.00, 4.00, 6.00, 8.00,
and 10.00 mL of Cu(II) stock solution were prepared for
spectrophotometry. The sample with 0.00mL of Cu(II)
stock solution will be considered as reagent blank in
order to keep the measurements precise setting a base
from which other measurements can be performed.
Ammonia is also added to the Cu(II) solutions. This is
done in order to forward the reaction between copper
and ammonia to form a copper-ammonia complex: Cu 2+
+ 4NH3 -> [Cu(NH3)4]2+ This complex gives a more
intense color than a standard copper solution which
allows the spectrophotometer to examine it more
effectively.
To determine the analytical wavelength, a UV-Vis
spectrophotometer was utilized. A cuvette was filled with
the standard solution containing the highest
concentration or the solution containing 10mL of Cu(II)
stock solution. The cuvette was only held at the tip to
prevent smudges that may give false light absorbance
readings. The cuvette must also be rinsed properly with
distilled water and the solution before measurement to
avoid discrepancies in data brought by dirt. After rinsing,
the cuvette must be wiped gently using a tissue since
water droplets may also cause a deviation in the data.
Once the cuvette is clean and dry, it is inseted gently
into the cuvette holder. The machine was then closed to
prevent outside light from entering. The
spectrophotometer was then set to Spectrum Mode
where the wavelength range was set from 300nm to
700nm and then scanned. The peak displayed by the
spectrophotometer was then recorded as it will function
as the analytical wavelength.
To prepare the calibration curve, the absorbance of
the solutions containing 2.00, 4.00, 6.00, 8.00, and
10.00mL Cu(II) stock solution were measured using the
UV-vis spectrophotometer. A reagent blank was again
used to precisely measure the absorbance of the working
standards. Only a single cuvette was used for both the
reagent blank and working standard to hold it constant
since difference in material used and thickness of the
material may affect the data (New Mexico State
University, 2006). The spectrophotometer was set to Go
to WL where the obtained analytical wavelength was
*Corresponding author. Mobile: 099982743524
E-mail address: pochi.enriquez@gmail.com
Page |2
inputted. Starting from the standard solution with the
lowest concentration, the cuvette was filled with the
solution to be measured. The cuvette was then again
handled on the edge to prevent smudges and water
droplets from rinsing were wiped to prevent
discrepancies in the data. After assuring that the cuvette
is clean and dry, the solution was scanned and the
absorbance reading was recorded. The procedure was
then repeated until the standard with the highest
concentration was scanned.
To determine the Cu(II) concentration of the unknown
solution, the cuvette was filled with the unknown
solution and scanned under the UV-Vis
spectrophotometer. The absorbance reading was then
recorded.
3. Results and Discussion
The values used for the data treatment are shown in
(Table 2). The values were used to create a calibration
curve that will find the equation of the best fit line. Using
this line, the concentration of the unknown solution can
be determined by measuring its absorbance.
Table 2: Calibration Curve Values
Volume of
Concentration of
Working
Standard Cu(II) Absorbance
Standard ppm
Solution
2.00 100 .014
4.00 200 .028
6.00 300 .041
8.00 400 .054
10.00 500 .070
Using the equation y = mx + b, absorbance could be
determined by equating it to y. This is determined by the
spectrophotometer. Meanwhile, x is equivalent to the
concentration of the solution, m is equal to the
absorptivity, and b would be the y-intercept. Using
Microsoft Excel, the calibration curve is shown as seen in
(Figure 1) (refer to Appendix 2). In the equation, y =
0.0001x thus, the absorbance is equal to 0.0001.
Moreover, since there is no y-intercept in the data
acquired, it signifies that there is no deviation from the
theoretical value. In the y-intercept, there is no
concentration of Cu(II) because x = zero. Theoretically,
the absorbance at this point would be zero because there
is no colored solution due to the lack of copper which
means that no light should be absorbed due to the
solution being colorless. The presence of a y-intercept
even after negating the effecting of the cell and the
ammonia solution on the values of absorbance signals
that something else is absorbing light due to the
presence of an absorbance value even at zero ppm
concentration. In this case, a y-intercept would deviate
from the theoretical value by increasing the values of
absorbance. The data acquired were done under the
wavelength of maximum absorption of 541nm.
Table 3: Absorbance of the unknown concentration
 Enriquez and Garcia / Chemistry 26.1/ (2014-2015)
Concentration of
Trial Absorbance Stock Sample Cu (II)
ppm
1 0.036 360
2 0.036 360
3 0.036 360
Average 0.036 360
All of the three trials for the solution containing the
unknown concentration had an absorbance of 0.036.
Replacing y with 0.036 in the equation y=0.0001x, the
concentration of the unknown sample would be 360ppm.
Since all of the trials had the same absorbance values, it
can be inferred that the data acquired is precise.
Possibles sources of errors for this experiment would
be random, systematic and gross. Random errors are
often unavoidable. They result from the usage of
glassware and may affect the precision of the results.
Systematic errors could occur from method or
instrument. The absorbance values obtained from the
spectrophotometer may increase if the reaction cell was
held improperly. This is because of the fingerprints may
be left in the cell. These fingerprints may absorb light
from the spectrophotometer which increases absorbance
value. Since the same cell was used for all tests in this
experiment, it is important that it is washed with the
solution about to be tested throughly. This is done to
ensure that the correct concentration of the Cu(II) is
being analyzed otherwise the absorbance value may
increase or decrease depending on the contaminated
concentration. The reagent blank test must be performed
in order to negate the effects of the cell and the
ammonia solution on absorbance otherwise the
absorbance values will increase. Instrumental errors may
occur in this experiment from the spectrophotometer.
Any stray light in the spectrophotometer will decrease
the absorbance value. This is due to the additional
presence of unabsorbed light. The cell used for analysis
must be standardized because a wider cell results in a
longer path length. The longer path length will increase
absorbance values, and the graph may deviate from its
linear form. The spectrophotometer must also be
inspected before use to ensure it is still effective for
analysis. Gross errors can result from contaminated
glassware especially during solution preparation.
Unwanted reagents may enter the prepared stock
solutions and affect the concentration of copper which
will affect the absorbance value. This will greatly affect
the construction of the calibration curve which will affect
the calculations for the concentration of the unknown
sample. All concentrations must be uncontaminated
before testing to prevent such errors from occurring.
4. Conclusion and Recommendations
The quantitative analysis of the copper solution via
spectrophotometry was successful. An accurate
calibration curve was constructed with the wavelength at
maximum absorption at 541 nm. The graph was nearly
completely linear which allows for an accurate equation
of the line which was found at y = 0.0001x. Based on the
absorbance value of 0.036, the calculated concentration
*Corresponding author. Mobile: 099982743524
E-mail address: pochi.enriquez@gmail.com
Page |3
of the copper solution was found at 360 ppm which is
true for all trials. This also ensures all values are precise
to one another for the sample analysis. However, the
calculated concentration of the copper solution may
slightly deviate from the theoretical value due to the
calibration curve not being completely linear which may
have resulted in some minor errors.
To increase the accuracy of the results, it is
recommended to address the systematic errors. During
the testing cell must be handled properly and with
utmost care to prevent unwanted errors. The cell must
also be throughly washed to prevent contamination and
dilution from affecting the concentration of the sample.
The curve must be constructed as linear as possible.
When testing standardized solutions for the construction
of the calibration curve, some tests may be repeated to
find the most accurate result to reduce any deviations in
the linear graph. Any instrument error may be addressed
through proper inspection of the spectrophotometer
before the performance of the experiment. In addition, it
is important that all solutions used for the construction of
the calibration curve are properly prepared and
uncontaminated during solution preparation to ensure
that the obtained absorbance values are accurate.
5. References
“Applications of Absorption Spectroscopy.”
Pharmatutor.org. n.d. Web. 7 May 2015.
Blum P., 1997. Reflectance Spectrophotometry and
Colorimetry. PP Handbook.
Brown T.. 2012. Chemistry, The Central Science. Pearson
Education, Inc.
Crouch S,. Holler F. J., Skoog D., West D., 2014.
Fundamentals of Analytical Chemistry. Cengage Learning.
9th edition.
Crouch, S, Holler, F. Fundamentals of Analytical
Chemistry. n.p: Cengage Learning, 2013. Print.
David, G. Analytical Chemistry. India: Universities Press
(India) Private Limited, 2001. Print.
Kazekevich Y., 2010. Analytical Chemistry. Seton Hall
University.
Petrucci R., 2010. General Chemistry – Principles and
Modern Applications. 10th Edition. Pearson Canada, Inc.
Scott A., 2007. Chemistry: The Practical Science.
Cengage Learning, 10th Edition.
Sinvasankar, B. Engineering Chemistry. New Delhi, Tata
McGraw-Hill Publishing Company Limited, 2008. Print.
Slowinski E., Wolsey W., and Rossi R,, 2011. Chemical
Principles In The Laboratory. Brooks/Cole. 10th edition.
 Enriquez and Garcia / Chemistry 26.1/ (2014-2015) Page |4

Silberberg M., Principles of General Chemistry. 2007. New

York: McGraw-Hill.

Spectrophotometry. State University of New York, 2006.

New York.

*Corresponding author. Mobile: 099982743524

E-mail address: pochi.enriquez@gmail.com
Appendix A: List of Tables

Table 1: Pairs of complimentary colors at their corresponding wavelength ranges

Wavelength Color Absorbed Color Observed
400-435 Violet Yellow-green
435-480 Blue Yellow
480-490 Blue-green Orange
490-500 Green-blue Red
500-560 Green Purple
560-580 Yellow-green Violet
580-595 Yellow Blue
595-650 Orange Blue-green
650-750 Red Green-blue

Table 2: Calibration Curve Values

Volume of Concentration of Absorbance
Working Standard Cu(II)
Standard ppm
Solution
2.00 100 .014
4.00 200 .028
6.00 300 .041
8.00 400 .054
10.00 500 .070

Table 3: Absorbance for the Unknown Analysis

Concentration of
Trial Absorbance Stock Sample Cu (II)
ppm
1 0.036 360
2 0.036 360
3 0.036 360
Average 0.036 360

Appendix B: List of Figures

Figure 1: Calibration Curve

Absorbance
0.08
0.07
f(x) = 0x - 0
0.06
R² = 1
0.05
0.04
0.03
0.02
0.01
0
50 100 150 200 250 300 350 400 450 500 550

Appendix C: Calculations

Concentration of Stock Sample Cu(II):

Equation of the Line (From Data Treatment):

y = 0.0001x
y = A = absorbance of the solution
x = c = concentration of the solution

1st Trial:
y = 0.036
0.036 = 0.0001x
x = 0.036/0.0001
x = 360 ppm

2nd Trial:
y = 0.036
0.036 = 0.0001x
x = 0.036/0.0001
x = 360 ppm

3rd Trial:
y = 0.036
0.036 = 0.0001x
x = 0.036/0.0001
x = 360 ppm

Average Concentration:
(360 + 360 + 360)/3 = 360 ppm