Joana Rita Costa Rodrigues 1220662

Mia Di Santo 1230042

2DC

4. QUANTIFICATION OF COPPER IN LABORATORY WASTE BY

ATOMIC ABSORPTION SPECTROPHOTOMETRY

Laboratory III
Professor Dr. Maria Madalena Freitas

21 de September de 2023

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Index
1. Abstract
2. Introduction
3. Experimental part
3.1 Reagents
3.2 Equipment
3.3 Material
3.4 Experimental procedure
4. Results and Discussion
4.1 Previous calculation
4.2 Obtained experimental results
4.3 Calculation of the results
4.4 Critical analysis and discussion of the results
5. Conclusion
6. References

Figures List
Figure 1: Photo of the volumetric flasks with all solutions (standards and samples).
Figure 2: Calculations for the initial volume of the standards.
Figure 3: Calculations for the initial volume of the sample.
Figure 4: Calculations for the initial volume of the standard copper solutions.
Figure 5: Graphic representation of the calibration curve, with a ‫ = ג‬324,7nm, of the
standard copper solutions with concentrations between 1,000mg/L and 5,000mg/L.
Figure 6: Graphic representation of the calibration curve, with a ‫=ג‬324,7nm, of the
standard solutions made of the sample of laboratory residue and the standard copper
solution, with a variation between 0,5mg/L and 2,5mg/L, by atomic absorption
spectrophotometry.
Figure 7: Obtained results with the method of direct calibration.

Tables List
Table 1: Absorbance media reading, with a ‫ = ג‬324,7nm, of the standard copper solutions
by atomic absorption spectrophotometry
Table 2: Absorbance media reading, with a ‫ = ג‬324,7nm, of the problem solutions by
atomic absorption spectrophotometry.
Table 3: Absorbance media reading, with a ‫=ג‬324,7nm, of the solutions made of problem
sample and standard copper solution added increasingly, by atomic absorption
spectrophotometry.

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Abstract
In this work, the aim was to measure the copper content in laboratory waste using atomic
absorption spectrophotometry. To accomplish this objective, two distinct approaches
were employed: the direct calibration method and the standard addition method.
The initial approach adopted was the Direct Calibration Method. This method
encompasses a strategy utilized in machine learning to bolster the dependability of
model forecasts. It encompasses the segmentation of forecasted probabilities into
categories, the evaluation of outcome occurrence within these categories, and the
refinement of probabilities to achieve a closer alignment with real-world observations.
This procedure enhances the precision of projected probabilities and provides more
nuanced avenues for appraising the model's performance.
On the other hand, the analyte addition method is both the simplest and the most used.
The concentration of the sample is found by comparing its absorbance to a curve of the
concentration of the standards versus the absorbances of the standards. For this method
to be applied the standards and the sample must have the same behaviour when
atomized. When the matrix is unknown the analyte addition technique uses an aliquot of
the sample itself as the matrix. The aliquots are then spiked with various amounts of the
analyte. Interference is caused by contaminants within the sample that absorb at the
same wavelength as the analyte, and thus can cause inaccurate measurements.
Corrections can be made through a variety of methods such as background correction,
addition of chemical additives, or addition of analyte.
After analysing the first method mentioned earlier, the method has a precision error of
32% and because of this it’s not possible to determine the copper concentration in the
residue. Also, due to errors in the procedure, it is inconclusive whether the method is
precise or not. As for the standard addition method, a copper concentration wasn’t
determined as well due to errors during the procedure. However, this method cannot be
deemed precise as the coefficient of variation was 0.567%.

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 1. Introduction
The Atomic absorption spectroscopy (AAS) is an instrumental analytical technique used
for the quantitative and qualitative determination of metal ions in solutions. For molecules
the absorption of electromagnetic radiation by excitation at a higher level occurs per
frequency band while for the atom at a single frequency. The source of the
electromagnetic radiation is given by a hollow cathode lamp, which emits with a very
narrow and characteristic spectrum of the element of which the cathode itself is made.
These lamps can be selective for a single chemical species when the cathode is coated
with a single metal, or they can be composite lamps suitable for the analysis of multiple
analytical species when the cathode is coated with multiple metals. Specific lamps offer
greater reliability, stability, and durability than composite lamps. The atomization system
is the system by which the sample under analysis, and therefore the metals to be
searched for, is reduced to the state of monatomic gas, a necessary condition for the
measurement as this occurs by measuring the difference in intensity of the
electromagnetic radiation before and after the passage through the atomized sample,
which absorbs energy through the electrons of the outermost shell. Atomization by flame,
uses the temperature of a much hotter air-acetylene or nitrous oxide-acetylene flame or
air-hydrogen flame for nebulizing the solution.
The relationship between absorbance (A), concentration of the analyte in the solution
(c), path length of the cuvette (typically 1 cm) (l), and the molar absorptivity or molar
extinction coefficient, which is a constant for a given substance at a specific wavelength
(ε), is described by the Beer-Lambert Law:

𝐴 = − log(𝑇) = 𝜀𝑐𝑙

Where T is the transmittance, and:

𝐼
𝑇=
𝐼0
Where “I” is the intensity of the transmitted radiation and “ 𝐼0 ” is the intensity of the
incident radiation.

2. Experimental Part

2.1 Reagents
• Nitric acid solution (𝐻𝑁𝑂3 ) 0.5 %(m/m)
• Copper stock solution 1000 mg/L
• Sample (given by the laboratory)
• Deionized water

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 2.2 Equipment
• Atomic absorption spectrophotometer GBC 904AA with copper hollow cathode
lamp

2.3 Material
• Micropipettes (100-1000 ±10) 𝜇L (1-5 mL ±10 𝜇L)
• Volumetric flasks (50 ±0.06) mL
• General laboratory material
• Goblets
• Pasteur pipette
• Deionized water sprayer
• Micropipette tips

2.4 Experimental procedure

1. Choice of wavelength and concentration range. Consult the equipment’s manual
to define the wavelength and the concentration range.
2. Quantification using the direct (external standard) calibration method.
a. Prepare 5 standard copper solutions in the concentration range according to the
chosen wavelength. Dilute the copper stock solution in 50-mL volumetric flasks
to obtain the desired concentrations (note: use the nitric acid solution to complete
the volume).
b. Place an adequate volume of the sample in a 50-mL volumetric flask and
complete the volume with the nitric acid solution.
c. Optimize the experimental conditions of the spectrometer according to the
equipment's instruction manual.
d. Measure the absorbances of the standard and sample solutions at the selected
wavelength.
e. Measure the absorbance of the blank 20 times.
f. Calculate the copper concentration (mg/L) in the sample.
3. Quantification using the standard addition method.
a. According to the copper concentration calculated in point 2.5, in 5 volumetric
flasks of 50 mL place the adequate volume of the sample solution.
b. To each volumetric flask add an adequate volume of the copper stock solution so
as not to exceed the maximum concentration of the defined concentration range.
c. Complete the volumes with the nitric acid solution.
d. Measure the absorbances of the solutions using the conditions established in
point 2.c.

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 1st Figure: Volumetric Flasks with all solutions
mentioned above.

3. Previous calculation
Following a review of the equipment manual, the decision was made to employ a
wavelength of 324.7nm within the concentration range spanning from 1mg/L to 5mg/L.
This wavelength was chosen due to its superior sensitivity when detecting copper.
Enhanced sensitivity translates to reduced potential for errors, making it the preferred
option.

3.1 Preparation of the standard solutions for the calibration curve

Five standard solutions ranging in concentration from 1mg/L to 5mg/L were
meticulously crafted. This involved a precise calculation of the necessary volume of
the initial solution to achieve these desired concentrations.

2nd Figure: Calculations for the initial volume of the standards

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 3.2 Preparation of the problem samples
Due to constraints within the laboratory, the problem samples were prepared without an
initial reading of their concentrations. Instead, a visual comparison of colour intensity was
made between the samples and the original copper solution. It was inferred that the
sample was more diluted as its colour appeared less intense than that of the copper
mother solution. Therefore, it was estimated that the sample had a concentration of
7000mg/L.
Following this estimation, two solutions were prepared with an estimated concentration
of 2.5mg/L using the calibration curve. With this information, the required volume of the
initial sample was calculated. Both solutions were prepared in the same manner and with
identical volumes.

3rd Figure: Calculations for the initial volume of the sample

3.3 Preparation of the standard copper solutions for the standard addition
method
Assuming the sample had a 7000mg/L concentration, it was prepared 5 volumetric flasks
of 50,00mL, with the adequate volume of the initial sample, pointing to a concentration
of 2.5mg/L. It was calculated the volume of the standard copper solution in order to obtain
standard solutions of 0.5mg/L, 1mg/L, 1.5mg/L, 2.0mg/L and 2.5mg/L.

4th Figure: Calculations for the initial volume of the standard copper solutions

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 4. Results

4.1 Direct Calibration Method

As mentioned earlier, following a thorough review of the equipment manual, the decision
was reached to employ a wavelength of 324.7nm, within a concentration range spanning
from 1mg/L to 5mg/L.
After the preparation of the five standard solutions using the direct calibration method,
with concentrations of 1mg/L, 2mg/L, 3mg/L, 4mg/L, and 5mg/L, absorbance
measurements were conducted.

C (Standard Cu) (mg/L) Absorbance

1 0.064
2 0.266
3 0.295
4 0.320
5 0.360
1st Table: Absorbance media reading, with a ‫ = ג‬324,7nm, of the standard copper solutions by
atomic absorption spectrophotometry.

Based on the collected data, a calibration curve was constructed, establishing a

relationship between absorbance (A) and the concentration of the standard solution
(𝐶𝐶𝑢 ). This resulted in the equation: A = 0.0646𝐶𝐶𝑢 + 0.0672, accompanied by a
correlation coefficient (r) of 0.885. Notably, the calibration curve exhibits a standard
deviation represented as 𝑆𝑦⁄ = 0.0620. The data presented have a poor correlation
𝑥
coefficient.

5th Figure: Graphic representation of the calibration curve, with a ‫ = ג‬324,7nm, of the standard
copper solutions with concentrations between 1,000mg/L and 5,000mg/L.

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 4.1.1 Quantification of Copper in the problem sample, by the direct
calibration method

Additionally, two test solutions were formulated, both featuring identical

concentrations. These solutions were created with the objective of determining the
copper concentration within them. To achieve this, 18µL of the sample solution were
added to each 50.00mL volumetric flask, and the remaining volume was adjusted by
the addition of nitric acid.

Problem Solution (mg/L) Absorbance

1 0,015
2 0,016
2nd Table: Absorbance media reading, with a ‫ = ג‬324,7nm, of the problem solutions by atomic
absorption spectrophotometry.

Since the obtained calibration curve does not exhibit good linearity, it is not possible to
accurately determine the copper concentration in the laboratory residue sample.

4.2 Standard addition method

After that, 5 standard solutions were made, within the concentration range, by the
method of standard solution addition, adding up in the 50,00mL volumetric flasks the
same volume of the sample, 18µL, and increasing volumes of the standard copper
solution, 25µL, 50µL, 75µL, 100µL and 125µL. The solutions were analysed with a
wavelength of 324,5nm.

Csample+standard (mg/L) Absorbance

0,5 0,023
1 0,011
1,5 0,067
2 0,059
2,5 0,056
3rd Table: Absorbance media reading, with a ‫=ג‬324,7nm, of the solutions made of problem
sample and standard copper solution added increasingly, by atomic absorption
spectrophotometry.

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Using the collected data, a calibration curve was constructed to illustrate the relationship
between absorbance (A) and solution concentration (C). This resulted in the following
equation: A = 0.0114C + 0.009, accompanied by a correlation coefficient (r) of 0.7321.
The calibration curve displays a standard deviation of Sy/x = 0.01937. With the help of
this equation, it became feasible to identify the point of intersection with the abscissa
axis, thereby enabling the determination of the copper concentration within the sample.
Notably, the data presented exhibits a correlation coefficient that falls short of
expectations.

6th Figure: Graphic representation of the calibration curve, with a ‫=ג‬324,7nm, of the
standard solutions made of the sample of laboratory residue and the standard copper
solution, with a variation between 0,5mg/L and 2,5mg/L, by atomic absorption
spectrophotometry.

4.2.1 Quantification of copper in the sample, by the standard addition

method
As the obtained calibration curve lacks good linearity, it is not possible to accurately
determine the copper concentration in the laboratory residue sample through curve
extrapolation.

4.3 Detection Limit (LOD) and the Quantification Limit (LOQ)

The Detection Limit (LOD) of the method can be defined as the analyte concentration
that can be detected with a certain level of confidence, resulting in a distinguishable
signal compared to the blank assay or background noise. The Quantification Limit (LOQ)
is the lowest quantity or analyte concentration that can be quantitatively determined with
precision and accuracy.
4.3.1 Direct calibration method
𝑦
𝑆 ⁄𝑥 3 ×0,0620
LOD = 3 × = = 2,88 𝐿/𝑚𝑔
𝑚 0,0646
𝑦
𝑆 ⁄𝑥 10 ×0,0620
LOQ = 10 × 𝑚
= 0,0646
= 9,60 𝐿/𝑚𝑔

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 4.3.2 Standard addition method
𝑦
𝑆 ⁄𝑥 3 ×0,0194
LOD = 3 × 𝑚
= 0,0228
= 2,55 𝐿/𝑚𝑔
𝑦
𝑆 ⁄𝑥 10 ×0,0194
LOQ = 10 × 𝑚
= 0,0228
= 8,51 𝐿/𝑚𝑔

4.3.3 White

To calculate the LOD and LOQ based on the blank signals, it was necessary to perform
20 absorbance readings of this assay. However, this requirement was not met, which
means that an absorbance average cannot be obtained. Consequently, it is not possible
to calculate the absolute standard deviation, and as a result, the LOD and LOQ cannot
be determined.

4.4 Precision
Precision can be defined as the level of agreement between successive measurements
performed in the same manner during an experiment. It can be influenced by random
errors such as the instrument's lack of sensitivity and observer variability, incorrect
readings, and noise, all of which contribute to variations in the standard deviation.
Therefore, the results were derived from the method's standard deviation and,
consequently, the coefficient of variation of the method.

4.4.1 Direct Calibration Method

- Method Precision
𝑆𝑦/𝑥 0,0620
𝑆𝑥0 = = = 0,960 𝐿/(𝑚𝑔 ∙ 𝑐𝑚)
𝑚 0,0646

𝑆𝑥0 0,960
𝑉𝑥0 = × 100 = × 100 = 32%
𝑥̅ 3

4.4.2 Standard addition method

- Method Precision
𝑆𝑦/𝑥 0,0194
𝑆𝑥0 = = = 0,851
𝑚 0,0228

𝑆𝑥0 0,851
𝑉𝑥0 = = = 0,567%
𝑥̅ 1,5

4.5 Accuracy
In this work, it is not possible to assess the accuracy of the method since we are not
provided with a true value for the copper concentration in the laboratory residue.
Therefore, it is not possible to determine the value of the relative error.

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 5. Critical analysis and discussion of the results
For the quantification of copper in laboratory waste it was used the direct calibration
method and the standard addition method. Nitric acid was used to atomize the solutions
more easily. The calculations did in the previous parts say that the method’s values are
different from the blank. The LOD is always lower that the LOQ in this case. Our results
show that something went wrong in the making of the standards in both the methods and
that in both methods there are some standards that are too far from the others. The error
could have been caused by the wrong use of the micropipette or some wrong
calculations. Unfortunately, because of this we can’t calculate the concentration and the
laboratory waste.

6. Conclusion
In summary, it was not possible to achieve the outlined objective for this work due to
failures during the procedure. Therefore, it is not possible to characterize and assess the
methods used, nor is it possible to choose the most recommended method for the
intended purpose.

7. References

I. Manual de Laboratório de Métodos Instrumentais de Análise I, 2000/2001,

Departamento de Engenharia Química, Instituto Superior de Engenharia do
Porto.
II. L. Ebon, A. Fisher and S. J. Hill, An Introduction to Analytical Atomic
Spectrometry, Ed. E. H. Evans, Wiley, New York (1998).
III. B. Welz and M. Sperling, Atomic Absorption Spectrometry, 3rd Ed, Wiley-VCH,
New York (1999).
IV. J. W. Robinson, Atomic Spectroscopy, 2nd Ed. Marcel Dekker, Inc., New York
(1996).
V. K. S. Subramanian, Water Res., 1995, 29, 1827.
VI. M. Sakata and O. Shimoda, Water Res., 1982, 16, 231.
VII. J. C. Van Loon, Analytical Atomic Absorption Spectroscopy Selected Methods,
Academic Press, New York (1980)
VIII. Douglas A. Skoog et al., Chimica analitica strumentale, Edides, 2009.
IX. Harvey, D. T. J. Anal. Bioanal. Chem. 2011

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 Attatchments

7th figure: Obtained results with the method of direct calibration.

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