S. Diaz / Chemistry 26.

1 (2019) Page | 1

Quantitative Determination of Copper(II) Concentration by

Spectrophotometry
S. Diaz, D. Galela
Institute of Biology, College of Science, University of the Philippines, Diliman, Quezon City 1101
Performed 4 July 2019; Submitted 8 July 2019

_________________________________________________________________________________________________________________________

ABSTRACT

The purpose of the experiment was to determine the copper(II) concentration of an unknown solution by
applying concepts involving spectrophotometry. Specifically, UV-Vis spectrophotometry was employed in
the quantitative analysis, and the Shimadzu UVmini-1240 single-beam spectrophotometer was the device
used to measure absorbance. A deep blue colored complex was formed from the addition of concentrated
ammonia to the standard copper(II) solutions, and a calibration curve was drawn from the obtained
absorbance values. The linear equation of the calibration curve was y = 0.0004x - 0.0049, showing that
Beer’s law was obeyed. In the sample analysis, the absorbance values of the unknown fell within the
range reported by the standards, and the average concentration of the stock sample copper(II) was
224.75 ppm. Overall, the experiment was successful in applying spectrophotometry in determining the
concentration of an unknown copper(II) solution.
_________________________________________________________________________________________________________________________

1. Introduction fact that the species absorbs in the red region of

the visible spectrum (Petrucci, Herring, Madura, &
Generally, solutions are colored because they Bissonnette, 2017). The addition of concentrated
contain species that can absorb visible light. The ammonia, NH3, converted the copper(II) solutions
species responsible for light absorption is known to a tetraamminecopper(II) complex, as shown in
as a chromophore (McMurry, 2016). The light (1). The [Cu(NH3)4]2+ species absorbs in the
energy absorbed is used to promote electrons to yellow region of the visible spectrum, hence the
higher energy levels; therefore, by emitting transmitted light is deep blue.
radiation, electrons go back to their ground state
(Silberberg & Amateis, 2015). The absorption and
Cu2+¿ 2+¿¿
emission spectra are the primary basis of (aq )+ 4 NH 3(aq) ⇌[Cu(NH ¿¿ 3)¿ ¿ 4 ](aq) ¿ ¿ ¿
spectrophotometry. (1)

UV-Vis spectrophotometry involves measuring the The objective of the experiment was to determine
transmission properties of substances across the the copper(II) concentration of an unknown
ultraviolet-visible spectral region (Harris, 2010), solution using Beer’s Law (2), which shows that
and thus, it has broad applications in both the absorbance, A, is directly related to the
qualitative and quantitative analysis. UV-Vis concentration of the light-absorbing species in the
spectrophotometry may be used to determine the solution (Harris, 2010).
identity of an unknown substance. Analyte
concentration may also be determined using Beer’s A=ε bc (2)
Law, which describes the linear relationship
between absorbance and concentration. Molar absorptivity, ε, describes how well a
substance absorbs light at a particular wavelength,
Applications of UV-Vis spectrophotometry include and it has units of ppm−1 ∙ cm−1. The width of the
heavy metal tracing and the analysis of proteins in cuvette that contains the sample is the pathlength.
clinical samples. In the experiment, however, UV- The pathlength, b, is expressed in cm, while the
Vis spectrophotometry was utilized in the concentration, c, is in units of ppm. Hence, the
quantitative analysis of copper(II) solutions, and a absorbance value is unitless (Harris, 2010).
single-beam spectrophotometer was the device
used to measure the transmission properties of the
sample.

The color of a species in a solution is the

complement of the color of the light that it absorbs.
Aqueous copper(II) solutions usually exhibit a
characteristic pale blue color which is due to the
 S. Diaz / Chemistry 26.1 (2019)
2. Methodology
2.1 Solution Preparation
From the 250.0 mL standard 2500 ppm copper(II)
stock solution, the working standard solutions
were prepared. 0.00, 2.00, 4.00, 6.00, 8.00, and
10.00 mL of the copper(II) stock solution was
pipetted into six 50-mL volumetric flasks. Then, 10
mL of concentrated NH3 solution was added to
each of the flasks and diluted to mark with
distilled water.
2.2 Determination of the Analytical Wavelength
The absorbance of the working standard
copper(II) solution with the highest concentration
was measured against the reagent blank from 300
nm to 700 nm using a UV-Vis spectrophotometer.
The analytical wavelength was then determined.
2.3 Preparation of the Calibration Curve
The absorbance of the remaining working
standard solutions was measured at the analytical
wavelength obtained previously.
2.4 Determination of the Copper(II) Concentration
of the Unknown Solution
To a solution of unknown copper(II)
concentration, 10.0 mL of concentrated NH 3
solution was added and diluted to mark with
distilled water. Then, the absorbance readings for
the unknown solution were measured thrice.
3. Results and Discussion
3.1 Purpose of Reagents
In aqueous solution, transition metals form
colored ions (Skoog, West, Holler, & Crouch, 2013),
and in the experiment, the copper(II) stock
solution exhibited a pale blue color. Concentrated
ammonia was added to the copper(II) stock
solution to produce a deep blue copper-ammonia
complex. The resulting complex had a more
intense blue color than the original copper(II)
stock solution, thus allowing the
spectrophotometer to measure the absorbance of
the solution more effectively.
The solvent used to prepare the sample was
distilled water since it is colorless and will not
interfere with the absorbance of light measured by
the spectrophotometer (Christian, Dasgupta, &
Schug, 2014). Also, the copper-ammonia complex
Page | 2
is sufficiently soluble in distilled water, thus
resulting in a homogeneous solution.
A reagent blank was prepared, which contained
only the concentrated NH3 solution and distilled
water. The preparation of a reagent blank was
essential to measure the absorbance of the
uncomplexed NH3 and the cuvette. Furthermore,
the prepared blank was used to calibrate the
spectrophotometer and to account for the
absorbance of the solvent as well as the sample
cuvette (Harris, 2010). The Autozero function
primarily serves as background correction, so that
in the sample analysis, the spectrophotometer will
only measure the absorbance of the analyte.
3.2 UV-Vis Spectrophotometer
The UV-Vis spectrophotometer used in the
experiment was the Shimadzu UVmini-1240
single-beam spectrophotometer. The components
are shown in figure 1. The UV-Vis
spectrophotometer is used to identify various
organic, inorganic, and biochemical species given
that they absorb ultraviolet and visible radiation
(Skoog, West, Holler, & Crouch, 2013).
Figure 1. Block diagram illustrating the
components of a single-beam UV-Vis
Spectrophotometer.
As the name suggests, a single-beam
spectrophotometer has only one beam of light
(Harris, 2010). The four basic components of a
single-beam spectrophotometer include a light
source, a monochromator, a sample cuvette, and a
detector. The monochromator separates light into
narrow bands of wavelength to be passed through
the sample and measured by the detector. The use
of a single-beam UV-Vis spectrophotometer is
inconvenient since the absorbance of the sample,
and the blank must be measured separately
(Harris, 2010). Consequently, the use of a double-
beam UV-Vis spectrophotometer is more
advantageous because it splits the light alternately
to pass between the sample and blank cuvettes, as
shown in figure 2.
 S. Diaz / Chemistry 26.1 (2019) Page | 3

Figure 2. Block diagram a double-beam UV-Vis products with different absorbance values from
Spectrophotometer. the analyte. In terms of instrumental limitations,
3.3 Interpretation of Results when stray light from outside the instrument is
introduced, a decrease in absorbance value is
Measurements were made at the wavelength of observed. If the cells holding the sample and the
maximum absorbance, λmax, where the sensitivity reagent blank are of unequal pathlengths, a
of the analysis is highest, and the curve is flat, so deviation in Beer's law would also be observed.
there is only little variation in absorbance (Harris, Mismatched cells cause an intercept in the
2010). The absorbance of the standard copper(II) calibration curve, and the new equation is shown
solutions was measured at the previously obtained in (3), where k is the intercept.
analytical wavelength, as shown in table 1.
A=ε bc +k (3)
Table 1. Calibration Curve Data
Volume of Concentration Absorbance The deviation of the y-intercept from the
Working of Standard theoretical value is the difference of the intercept
Standard Cu(II), ppm from zero. In the experiment, the y-intercept of the
Solution, mL calibration curve is y = -0.0049, which shows the
2.00 100 ppm 0.034 occurrence of mismatched cells. This also indicates
4.00 200 ppm 0.069 that the reagent blank solution did not account for
6.00 300 ppm 0.109 all interferences.
8.00 400 ppm 0.144
10.00 500 ppm 0.185 In the sample analysis, the absorbance of the
unknown falls within the region covered by the
The calibration curve is shown in figure 2. The standards, as shown in table 2. The average
equation of the line of plot concentration of Cu(II) concentration of the stock sample copper(II) was
vs. absorbance was y = 0.0004x - 0.0049. The 224.75 ppm
positive slope indicates that the concentration is
directly proportional to the absorbance, and Beer’s Table 2. Sample Analysis
law is obeyed. However, the measure of linearity is Trial Absorbance Concentration
R2=0.9993. Though the value is still close to one, of stock sample
the relationship between the concentration and Cu(II), ppm
the absorbance is still not entirely linear. 1 0.089 234.75 ppm
2 0.083 219.75 ppm
3 0.084 222.25 ppm
0.2
Average 0.085 224.75 ppm
0.18
0.16
0.14 3.4 Sources of Errors
Absorbance

0.12
0.1 The presence of fingerprints or dirt on the non-
0.08 frosted side of the cuvette may cause false
0.06
0.04 absorbance readings. Mismatched cell placement
0.02 may also be a possible source of error. The failure
0 of the reagent blank to compensate for
0 100 200 300 400 500 600 interferences results in high absorbance readings.
Concentration of Standard Cu(II), ppm Thus the cuvette should be cleaned and
appropriately positioned in the cuvette holder to
Figure 2. Calibration curve: Concentration of Cu minimize errors. In operating the UV-Vis
(II) vs. Absorbance. spectrophotometer, the compartments must also
be tightly closed to avoid stray light, which results
The linearity of Beer’s law is challenged by certain in low absorbance readings. The multiple errors
real, chemical, and instrumental limitations. In mentioned could be the cause of the occurrence of
terms of real limitations, Beer’s law can only be a y-intercept and the non-linearity of the
observed in dilute solutions with less than 0.01M calibration curve.
because high concentrations cause deviations in
absorptivity due to the electrostatic interactions
between molecules in close proximity. In terms of
chemical limitations, the solutions must not
experience shifts in equilibria that may result in
 S. Diaz / Chemistry 26.1 (2019)
4. Conclusion and Recommendations
The measured absorbance values correlate with
the concentration of the standard copper(II)
solutions. Though the calibration curve obtained
was not entirely linear and produced a y-intercept,
the results still agree with Beer's Law. The non-
linearity may only be attributed to human errors
such as failure to clean the cuvettes properly and
failure of the prepared reagent blank to account for
impurities. Overall, the experiment was successful
in applying spectrophotometry in determining the
concentration of an unknown copper(II) solution.
The extensive applicability of UV-Vis
spectrophotometry to identify various inorganic,
organic, and biochemical species (Christian,
Dasgupta, & Schug, 2014), with much accuracy and
ease due to modern instrumentation, are what
makes this technique advantageous in quantitative
chemical analysis.
Page | 4
References
[1] Christian, G.D., Dasgupta, P.K., & Schug, K.A.
(2014). Analytical chemistry (7th ed.). Hoboken, NJ:
John Wiley & Sons.
[2] Harris, D.C. (2010). Quantitative chemical
analysis (8th ed.). New York, NY: W. H. Freeman.
[3] McMurry, J. (2016). Organic chemistry (9th ed.).
Boston, MA: Cengage Learning.
[4] Petrucci, R.H., Herring, F.G., Madura, J.D., &
Bissonnette, C. (2017). General chemistry:
Principles and modern applications (11th ed.).
Toronto, CA: Pearson.
[5] Silberberg, M., & Amateis, P. (2015). Chemistry:
The molecular nature of matter and change (7th
ed.). New York, NY: McGraw-Hill Education.
[6] Skoog, D.A., West, D.M., Holler, F.J., & Crouch,
S.R. (2013). Fundamentals of analytical chemistry
(9th ed.). Boston, MA: Cengage Learning.
 S. Diaz / Chemistry 26.1 (2019) Page | 5

Appendix

A. UV-Vis Spectrophotometer

Operating Procedures

1. Turn on the spectrophotometer.

2. Determine analytical wavelength.
a) Fill up the cuvette with the standard solution with highest concentration.
b) Place the cuvette at the cuvette holder. Make sure the clear side faces the light source.
c) Go to Spectrum Mode.
d) Set the wavelength range.
e) Scan.
f) Obtain peak. This corresponds to the analytical wavelength.
3. Autozero.
a) Fill up the cuvette with the blank solution.
b) Place the cuvette at the cuvette holder. Make sure the clear side faces the light source.
c) Press Autozero for background correction.
4. Read
a) Press Go to WL and input the obtained analytical wavelength.
b) Starting from the standard solution with lowest concentration to highest concentration, fill up
the cuvette with the solution to be measured.
c) Place the cuvette at the cuvette holder. Make sure the clear side faces the light source.
d) Scan each solution.
e) Record the absorbance readings for each solution.
5. After reading all the standard and sample solutions, rinse all cuvettes with distilled water and allow it
to drain. Return the cuvettes in their storage container.

Cleaning and Handling of Cuvettes

1. The cuvette must be rinsed properly with distilled water. In the analysis of the sample, rinse the
cuvette at least three times with a small amount of the sample solution.

2. In most spectrophotometers, radiation passes through the clear (non-frosted) side of the cell.
Therefore, you should handle only the frosted portion of the cell when filling and rinsing with solution
and when placing it into the cuvette holder.

3. Be sure to wipe off any liquid on the outside of the cuvette as much as possible, including the entire
bottom half.
 S. Diaz / Chemistry 26.1 (2019) Page | 6

B. Calculations

Concentration of Standard Cu(II), ppm

M wstd V wstd
M=
V sol' n

2500 ppm ×2.00 mL

M= =100 ppm
50 mL

2500 ppm ×4.00 mL

M= =200 ppm
50 mL

2500 ppm ×6.00 mL

M= =300 ppm
50 mL

2500 ppm ×8.00 mL

M= =400 ppm
50 mL

2500 ppm ×10.00 mL

M= =500 ppm
50 mL

Concentration of Stock Sample Cu(II), ppm

y = 0.0004x - 0.0049

y +0.0049
x=
0.0004

Trial 1
0.089+0.0049
x= =234.75 ppm
0.0004

Trial 2
0.083+0.0049
x= =219.75 ppm
0.0004

Trial 3
0.084+ 0.0049
x= =222.25 ppm
0.0004

Average
0.085+0.0049
x= =224.75 ppm
0.0004
 S. Diaz / Chemistry 26.1 (2019) Page | 7

C. Raw Data

Absorption Spectrum
Wavelength at maximum absorption (λmax): 621 nm

Calibration Curve
Concentration of working standard Cu(II) solution: 2500 ppm

Volume of Working Concentration of Absorbance

Standard Solution, mL Standard Cu(II), ppm
2.00 100 ppm 0.034
4.00 200 ppm 0.069
6.00 300 ppm 0.109
8.00 400 ppm 0.144
10.00 500 ppm 0.185

Linear equation of calibration curve: y = 0.0004x - 0.0049

Sample Analysis
Volume of stock sample solution: 50 mL

Trial Absorbance Concentration of stock

sample Cu(II), ppm
1 0.089 234.75 ppm
2 0.083 219.75 ppm
3 0.084 222.25 ppm
Average 0.085 224.75 ppm