Chem126 Lab – Instrumental Analysis
EXPERIMENT 5
Chemical Interferences in Atomic Absorption Spectrophotometric
Measurements
Vanessa Olga J. Dagondon and Ken M. Menez
Department of Chemistry, College of Arts and Sciences,
University of the Philippines – Visayas, Miag-ao Iloilo
ABSTRACT
Calcium content in prepared samples containing other species such as
K, P, La, EDTA and Al was determined using AAS or Atomic Absorption
Spectroscopy. Four schemes were adapted in the experiment: (1) absorbance
of Ca standards determined using air/acetylene flame; (2) absorbance of Ca +
K standards determined using air/acetylene flame; (3) absorbance of Ca
standards determined using N2O/acetylene flame; and (4) absorbance of Ca +
K standards determined using N2O/acetylene flame. Addition of K and P
contributes to the chemical interference due to the incomplete dissociation
of compounds. This can be minimized by using a high temperature flame
N2O/acetylene flame instead of air/acetylene and by adding a releasing
agent, La. Addition of Al can cause another type of chemical interference
using interference due to effects of ionization. This can be minimized by using
a low temperature flame, air/ acetylene and by adding a protective agent
such as EDTA.
INTRODUCTION
Atomic spectroscopy is a series of different qualitative analyses regarding the
concentration of a specific substance in an analyte, with each analysis applied based on the
characteristics of the substance.1 As with any atomic spectroscopic method, the identity and
concentration of a substance in a sample solution could be determined by exciting the
molecules of the solution using a source, such as heat or strong light. This excitation
produces neutral atoms in the gas phase, which emit a specific wavelength and intensity of
light that gets captured and analyzed by detectors.1 These processes are considered very
successful in a wide array of applications in data analysis, with most processes already built
into special automated machines.10
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Chem126 Lab – Instrumental Analysis
Figure 2. A schematic diagram for the process of atomic
absorption spectroscopy.1
Figure 1. Varian SpectrAA 55B atomic
absortion spectrometer. The user interface is
located top right, the light source and
detector at the lower right, the nebulizer and
aspirator (clear tube) at the lower left, and
the covered burner at the top left which
This experiment uses the flame atomic
spectroscopy, where an analyte is taken up by an
atomic absorption spectrometer to be excited using includes an exhaust chimney.1
different mixtures of acetylene flame. The mixtures
depend on the substance being analyzed, as not all substances can be atomized with the
same type of flame. Each substance has a certain temperature needed to atomize them in
gas phase, as well as a maximum speed for the flame to attain. In the case of this
experiment using metallic substances, too high a temperature can ionize the metal while
too low will have less metal atoms excited; both decrease sensitivity.9 As only air –
acetylene and nitrous oxide – acetylene torches were used in this experiment, the maximum
flame speed of air – acetylene is 160 cm s-1 and maximum temperature at 2300 ⁰C while
nitrous oxide – acetylene attains a max flame speed of 180 cm s-1 and a max temperature of
2955 ⁰C. 7 An example of an atomic absorption spectrometer using the flame is shown in
Figure 1, however a SHIMADZU model was instead used for the experiment. This model had
very similar parts as the example, including an easy switch between two cathode lamps.
The process of flame atomic spectroscopy needs the analyte to be dissolved in a
solvent; this analyte is then nebulized into the flame. Nebulization is the application of an
oxidant gas to force the dissolved substance to spray evenly over the flame. 1 The flame will
atomize, or turn the sprayed analyte into neutral atoms in gas form by increasing the
temperature until the gas forms a plasma; in this plasma the ions stabilize by bonding with
free electrons and radicals to form neutral atoms.1 A finer aerosol will easily vaporize the
analyte, and a hotter flame will easily vaporize stable compounds; both reduce
interferences at this step.9 After excitation, the released energy is converted as light, which
travels through a monochromator. The monochromator is a device that helps select one
certain wavelength to reach the detector. However, the light coming from the neutral
atomic gas is so narrow that it causes gaps in the monochromator slit which greatly deviates
the amount of light obtained by the detector; this can be solved by activating a hollow –
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Chem126 Lab – Instrumental Analysis
cathode ray tube that delivers radiation patterns similar to the substance being analyzed.1
After the light reaches the detector, it is then analyzed. A simplified process is shown as
Figure 2.
Due to different substances having different properties, the metals featured in this
experiment have to be dissolved in certain concentrations and have specific flames to
atomize.
Aluminum was present in the form of aluminum chloride hexahydrate. Being a more
easily dissolvable form of aluminum, it was added directly to the standard solution. The
optimal wavelength for aluminum is 396.1 nanometers while using a nitrous oxide –
acetylene flame; due to it being partially ionized in the flame, a small amount of potassium
chloride was added to the solution as a suppressor.2 Calcium was used as a standard for all
the other solutions in this experiment. Used in the form of calcium carbonate, a small
amount of nitric acid was added before dilution of water to help it dissolve completely. The
optimal wavelength for aluminum is 422.7 nanometers, while using a nitrous oxide –
acetylene flame; in air – acetylene flames the interferences of calcium can be reduced by
adding lanthanum, while in nitrous – oxide acetylene flames potassium chloride is added to
suppress ionization.3 Potassium was present as potassium chloride. The optimal wavelength
is at 766.5 nanometers with an air – acetylene flame; this same flame helps eliminate
interferences in the sample.4 Lanthanum was present as lanthanum chloride in the
experiment. The optimal wavelength is at 441.7 nanometers, with a nitrous oxide –
acetylene flame; the interferences caused by partial ionization were suppressed by the
potassium present in the solution.5 Phosphorus was present in the experiment as sodium
phosphate. The optimal wavelength is at 213.6 nanometers at a nitrous oxide – acetylene
flame; due to being an uncommon substance determined in AAS and having a lack of
sensitivity in the process only a few studies contain interference information about
phosphorus.6
The standard solutions are prepared to obtain a common constant between all other
solutions. This standard was based around calcium. If the concentration of the solution is
plotted against the absorption of each standard, the slope of the resulting line equals the
common constant or molar absorptivity ε. This is used in the Beer – Lambert’s Law, which
relates absorbance (A) to the molar absorptivity, path length of light (b), and concentration
of the sample solution (c):9
𝐴 = 𝜀𝑏𝑐
(1)
The obtained absorbance from the different solutions is then converted to
transmittance:9
𝑇 = 10−𝐴
(2)
In vice versa, absorbance could also equal the logarithmic function of transmittance.
To decrease interferences in the samples the same suggestions that were mentioned earlier
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Chem126 Lab – Instrumental Analysis
were used as well as adjusting several aspects of the flame and samples to obtain the
maximum sensitivity in high precision.9
METHODOLOGY
Two series of standard solutions were prepared. The first series contained only
calcium carbonate. Five 100 milliliter volumetric flasks were used to contain 0, 1, 2, 3, and 5
ppm of calcium carbonate, respectively. These flasks were labeled from 1A to 5A in the
manner of increasing calcium concentration, and then diluted to mark with distilled water.
The second series was prepared similar to the first, however an added approximate of
0.0381 grams of potassium chloride was added to each flask. The second series flasks were
labeled from 1B to 5B, same in order as the first series.
A set of sample solutions were prepared in 100 mL volumetric flasks, with different
mixtures of substances for each. A stock solution of 250 mL calcium was prepared by
dissolving 2.4976 grams calcium chloride into 250 mL water in a 250 mL flask. This stock
calcium solution was used for each of the following sample solutions:
The first flask, labeled 1S, and all other flasks were each added with 3 mL of stock
calcium solution. The second flask, labeled 2S, was added 0.0053 grams of sodium
phosphate. The third flask, labeled 3S was added 0.0053 grams of sodium phosphate, and
0.3818 grams of potassium chloride. The fourth flask, labeled 4S, was added with 0.0053
grams of sodium phosphate, 0.3818 grams of potassium chloride, and 0.0017 grams of
lanthanum chloride. The fifth flask, labeled 5S, was added with 0.0908 grams of aluminum
chloride hexahydrate. The sixth flask, labeled 6S, was added with 0.0908 grams of aluminum
chloride hexahydrate and 2.3146 grams of EDTA.
The spectral interference of the each solution was recorded using atomic absorption
spectroscopy by air/acetylene and N2O/acetylene flames. Each set of solutions were
analyzed by air/acetylene and N20/acetylene flames, with the standards being tested first
before the samples.
RESULTS AND DISCUSSION
Two sets of standards were prepared in the experiment: one is composed of calcium
in different concentrations and the other one is composed of calcium and potassium in
different concentrations. The absorbances of each set of standards were analyzed in the
AAS using two flames: air/acetylene and N2O/ Acetylene flame. This resulted to four
schemes: Scheme 1 used air/acetylene flame to analyze the calcium standards; scheme 2
used the same flame to analyze the calcium plus potassium standards; scheme 3 used
N2O/acetylene flame to analyze the calcium standards; and, scheme 4 used the same flame
to analyze the calcium plus potassium standards. These four schemes will result to five
calibration curves used to obtain the concentration of calcium in the prepared samples. This
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Chem126 Lab – Instrumental Analysis
variation of conditions is done to identify the effect of the added interference in the
prepared samples and also the effect of the type of flame used in the analyses.
Table 1 summarizes the result of absorbances obtained from each schemes. Notice
that in scheme 1 at the concentration of 1 mg/L, there was no recorded absorbance. This is
because the absorbance read was negative and therefore would be erroneous. The data was
discarded instead. Figure 3 shows the calibration curves obtained from each schemes.
Linearity of the calibration curves was not satisfactory. The calibration curve is somehow
curved up or has an “upward curvature”. While this may be due to the inaccurate
preparation of the standards, it is a fact that it is rare for atomic absorption calibration
curves to show ideality (i.e. linear plot). The “upward curvature” in the calibration curves
generated is usually observed on the standards of small concentration range.11
Table 2 shows the absorbances recorded in each samples prepared. As shown, there
are six samples with the same concentration of calcium, each of which contains different
interferences. Table 3 shows the concentration of calcium of the samples obtained from
each scheme. It can be observed the fluctuation of the values for the concentration of
calcium in each sample despite the fact that they are of the same concentration of calcium
when prepared. This shows how much an addition of interference affects the analysis done
in AAS.
In the first sample which theoretically contains only 3mg/L calcium, the obtained
concentrations from the four calibration curves were much lesser than the theoretical value
(Table 3). This indicates that there are indeed errors in the preparation of the calibration
curves (i.e. preparation of standards). All the other samples contain the same amount of
calcium as that of the first sample. However, the remaining samples contains other
components such as P, K, La, Al, and EDTA.
Interferences in atomic absorption fall into six categories: chemical interferences,
ionization interferences, matrix interferences, emission interferences, spectral
interferences, and background absorption. The most common interferences are chemical
interferences. A chemical interference emerges when the sample being analyzed contains a
thermally stable compound with the analyte that is not totally decomposed by the energy of
the flame and thus, the number of atoms in the flame capable of absorbing light is reduced.
12 There are to general forms of chemical interferences: ionization and incomplete
dissociation of compounds.13
The effect of phosphorous and potassium in calcium, as in the second and third
sample, is an example of a chemical interference due to incomplete dissociation of
compounds. These interferents form compounds which are not completely dissociated at
the temperature of the flame and hence prevent the formation of neutral ground state
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Chem126 Lab – Instrumental Analysis
atoms.13To overcome this interference, a higher temperature flame can be used, as in in
scheme 2 which uses N2O/Acetylene flame, or La can be added as a releasing agent, as in
sample 4. A releasing agent, which can be referred also as a competing cation, reacts with
the interferent releasing the analyte.12The presence of Al, as in samples 5, are another
example of a chemical interference. This time, the interference is due to ionization. To
overcome this inference, a lower temperature flame such as air/ acetylene flame must be
used, as in in scheme 1, because high temperature flames such as nitrous N 2O/acetylene
may cause appreciable ionization of the analyte element. The alkali and alkaline-earth
metals such as Al are more susceptible to ionization. To control this interference, a suitable
cation with an ionization potential lower than that of the analyte is added. A protective
agent such as EDTA can also be added to reduce this effect, as in sample 6. A protective
agent is a ligand reacts with the analyte forming a relatively volatile complex.
It can be observed that the results of the experiment did not coincide with the
theory of the experiment. This can be accounted with the inaccurate preparation of the
standards and also of the samples.
Table 1. Absorbance Readings of the Standards
Air/Acetylene Flame
Scheme 1
Scheme 2
Ca Standard
0 mg/L
0.0005
1 mg/L
0.0020
2 mg/L
0.0778
3 mg/L
0.0824
5 mg/L
0.1963
Ca Standard + K
0 mg/L
1 mg/L
2 mg/L
3 mg/L
5 mg/L
0.0030
0.0068
0.0779
0.0824
0.1818
Table 2. Absorbance readings of the samples
Sample
Air/ Acetylene Flame
3 mg/L Ca
0.0824
3 mg/L Ca + P
0.0413
3 mg/L Ca +P + K
0.0519
3 mg/L Ca + P + K + La
0.0508
3 mg/L Ca + Al
0.0292
3 mg/L Ca + Al + EDTA
0.0002
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N2O/Acetylene Flame
Scheme 3
Scheme 4
0.0002
0.0613
0.0661
0.1780
0.0029
0.0070
0.0897
0.0908
0.1367
N2O/ Acetylene Flame
0.0661
0.0140
0.0516
0.0693
0.0452
0.0200
Chem126 Lab – Instrumental Analysis
Calibration Curve:
Scheme 1
Calibration Curve:
Scheme 2
0,2500
0,2000
y = 0,0403x - 0,0169
R² = 0,939
0,1500
0,1000
0,0500
0,1000
0,0500
0,0000
0,0000
-0,0500 0
2
4
6
Concentration of Ca Standards (mg/L)
0
2
4
6
-0,0500
Concentration of Ca + K Standards (mg/L)
(a)
(b)
Calibration Curve:
Scheme 3
Calibration Curve:
Scheme 4
0,2000
0,2000
Absorbance
y = 0,0344x - 0,0095
R² = 0,9332
0,1500
Absorbance
y = 0,0368x - 0,0106
R² = 0,9454
0,1500
Absorbance
Absorbance
0,2000
0,1000
0,0500
0,0000
0
2
4
6
-0,0500
Concentration of Ca Standards (mg/L)
(c)
y = 0,0286x + 0,0019
R² = 0,8969
0,1500
0,1000
0,0500
0,0000
0
2
4
Concentration of Ca + K Standards (mg/L)
(d)
Figure 3. Calibration curves generated from each scheme: (a) plot of the absorbance
obtained using air/acetylene flame against the concentration of Ca standards, scheme 1; (b)
plot of the absorbance obtained using air/acetylene flame against the concentration of Ca +
K standards, scheme 2; (c) plot of the absorbance obtained using N2O/acetylene flame
against the concentration of Ca standards, scheme 3; (4) plot of the absorbance obtained
using N2O/acetylene flame against the concentration of Ca + K standards, scheme 4.
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6
Chem126 Lab – Instrumental Analysis
Table 3. Concentration of Ca in the samples in mg/L
Air/ Acetylene Flame
Sample
Scheme 1
Scheme 2
3 mg/L Ca
2.4630
2.5266
3 mg/L Ca + P
1.4433
1.4098
3 mg/L Ca +P + K
1.7063
1.6978
3 mg/L Ca + P + K + La
1.6790
1.6679
3 mg/L Ca + Al
1.1430
1.0809
3 mg/L Ca + Al + EDTA
0.4235
0.2929
N2O/ Acetylene Flame
Scheme 3
Scheme 4
2.2004
2.2448
0.6848
0.4231
1.7786
1.7378
2.2935
2.3566
1.5924
1.5140
0.8594
0.6329
CONCLUSION
Atomic Absorption Spectroscopy (AAS) is a technique for measuring quantities of
chemical elements present in a sample by measuring the absorbed radiation by the
chemical element of interest. The sample is excited by radiation making its atoms absorb
ultraviolet or visible light and make transitions to higher energy levels. In this experiment,
flame atomic spectroscopy is used to excite the analyte using different mixtures of
acetylene flame. The analyte in this experiment is calcium. Calcium content of 6 samples
containing interferences and other components were determined. To determine the
calcium content in the samples, two sets of calcium standards (Ca standards and Ca+K
standards) were prepared to create calibration curves. The absorbances of the standards
were determined using air/acetylene and N2O/acetylene flames as indicated in the four
schemes followed in the experiment. Four calibration curves were obtained in the
experiment. Effects of interference in the determination of calcium in the sample were
examined. Potassium and phosphorus caused a chemical interference due to incomplete
dissociation of compounds. This interference can be aided by using a higher temperature
flame (N2O/acetylene flame instead of air/acetylene) and also by adding Lanthanum which
is a releasing agent. Aluminum can cause another type of chemical interference due to
effects of ionization. To aid this, a lower temperature flame such as air/ acetylene flame
must be used because high temperature flames such as nitrous N2O/acetylene may cause
appreciable ionization of the analyte element and also by adding a protective agent such as
EDTA. These theories were not reflected in the results of the analysis because of the
inaccurate preparation of standards and samples.
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Chem126 Lab – Instrumental Analysis
LITERATURE CITED
1. “Introduction”. Determination of Calcium by Atomic Spectroscopy. Chem 334:
Quantitative Analysis Laboratory, Colorado State University. March 24, 2016. p. 1 –
2.
2. “Standard Conditions: Al (Aluminum)”. Flame Atomic Absorption Spectrometry:
Analytical Methods. Agilent Technologies, Australia. 13th ed. November 2015. p. 16.
3. “Standard Conditions: Ca (Calcium)”. Flame Atomic Absorption Spectrometry:
Analytical Methods. Agilent Technologies, Australia. 13th ed. November 2015. p. 24.
4. “Standard Conditions: K (Potassium)”. Flame Atomic Absorption Spectrometry:
Analytical Methods. Agilent Technologies, Australia. 13th ed. November 2015. p. 42.
5. “Standard Conditions: La (Lanthanum)”. Flame Atomic Absorption Spectrometry:
Analytical Methods. Agilent Technologies, Australia. 13th ed. November 2015. p. 43.
6. “Standard Conditions: P (Phosphorus)”. Flame Atomic Absorption Spectrometry:
Analytical Methods. Agilent Technologies, Australia. 13th ed. November 2015. p. 54.
7. Amos, M. D. & Willis, J. B. “Choice of Flame”. Spectrochimica Acta: Use of high –
temperature pre – mixed flames in atomic absorption spectroscopy. Pergamon Press
Ltd., Northern Ireland. vol. 22. 1966. p. 1327.
8. Martizano, J. “Materials & Procedures”. Chemical Interferences in Atomic Absorption
Spectrophotometric Measurements. University of the Philippines Visayas – Miagao
Campus, Philippines. March 2016. p. 1.
9. Melville, J. “Theory”. Atomic Absorption Spectroscopy of Metal Alloys. Chemistry
105: Instrumental Methods in Analytical Chemistry, Berkeley College of Chemistry,
University of California, California. March 3, 2014. p. 2.
10. Walsh, A. “Introduction”. Spectrochimica Acta: The application of atomic absorption
spectra to chemical analysis. Chemical Physics Section, Division of Industrial
Chemistry, Commonwealth Scientific and Industrial Research Organization,
Melbourne, Australia. Pergamon Press Ltd., London. vol. 7. 1955. pp. 108 – 177.
11. Harvey, D. Modern Analytical Chemistry. United State of America: The McGraw-Hill
Companies, Inc.; 2000 [cited 2016 February]. Available from: http://elibrary.bsu.az/
12. Skoog D. A., West D. M., Holler F. J., Crouch S. R. 2014. Fundamentals of Analytical
Chemistry Ninth Edition. Canada: Nelson Education, Ltd. 1026p.
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Chem126 Lab – Instrumental Analysis
APPENDICES
I. Tables
Table 4. Statistical Data for the Schemes 1, 2,3 and 4
Statistical
Parameter
M
B
sr
sm
sb
sc
r
Scheme 1
Scheme 2
Scheme 3
Scheme 4
0.040304054
-0.016868919
2.28x10-2
5.93x10-3
1.66x10-2
6.22x10-1
0.939
0.036798649
-0.01057703
1.96 x10-02
5.10 x10-03
1.43 x10-02
6.42 x10-01
0.036439189
-0.01916622
2.28 x10-02
5.92 x10-03
1.65 x10-02
6.91 x10-01
0.0286
0.0019
2.15 x10-02
5.60 x10-03
1.56 x10-02
8.34 x10-01
0.9454
0.9267
0.8969
II. Sample Calculations
Least Square Method
*For Scheme 1
x
y
x2
y2
xy
(y1-mx1+b)2
0
1
2
3
5
0.0005
0.0020
0.0778
0.0824
0.1963
0
1
4
9
25
0.00000025
0.000004
0.00605284
0.00678976
0.03853369
0
0.002
0.1556
0.2472
0.9815
0.000301679
0.000459465
0.000197706
0.00046843
0.000135691
sum
11
0.3590
39
0.05138054
1.3863
ave
2.2
0.0718
7.8
0.01027610
0.27726
Let x be the concentration of the Ca standards and y be the absorbance
𝑆𝑥𝑥 = 𝛴𝑥 2 −
(𝛴𝑥)2
( 11)2
= 39 −
= 14.8
𝑛
5
𝑆𝑦𝑦 = 𝛴𝑦 2 −
(𝛴𝑦)2
( 0.3590)2
= 0.05138054 −
= 0.02560434
𝑛
5
𝑆𝑥𝑦 = 𝛴𝑥𝑦 −
(𝛴𝑥𝛴𝑦)2
[ (11)(0.3590)]2
= 1.3863 −
= 0.5965
𝑛
5
Page 10 of 11
0.00156297
0.00031259
Chem126 Lab – Instrumental Analysis
𝑚=
𝑆𝑥𝑦
0.5965
=
= 0.040304054
𝑆𝑥𝑥
14.8
𝑏=
𝛴𝑦
𝛴𝑥
0.3590
11
− 𝑚( ) =
− ( 0.02560434) ( ) = −0.01686892
𝑛
𝑛
5
5
Determination of calcium in sample where y = 0.0824
𝑒𝑞𝑢𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑙𝑖𝑛𝑒: 𝑦 = 0.040304054x − 0.01686892
𝑥=
0.0824 + 0.01686892
= 2.4630 mg/L
0.040304054
Uncertainty of Measurement
𝑆𝑦𝑦 − 𝑚2 𝑆𝑥𝑥
0.02560434 − (0.0403040542 )(14.8)
𝑠𝑟 = √
=√
= 2.28 × 10−2
𝑁−2
5−2
𝑠𝑟 2
(2.28 × 10−2 )2
𝑠𝑚 = √
=√
= 5.93 × 10−3
𝑆𝑥𝑥
14.8
𝑠𝑏 = 𝑠𝑟 √
1
1
−2
√
=
2.28
×
10
= 1.66 × 10−2
2
5 − (11)2⁄39
𝑁 − (∑ 𝑥𝑖 ) ⁄∑ 𝑥𝑖2
𝛴𝑦 2
0.3590 2
−2
(𝑦
−
)
(0.0824
−
) |
𝑖𝑛𝑡
1
|𝑠𝑟 √ 1
𝑛 | = | 2.28 × 10 √1 + 1 +
5
𝑠𝑐 =
+ +
|𝑚 𝑀 𝑁
|
|0.02560434 1 5 (0.0403040542 )(14.8)|
𝑚2 𝑆𝑥𝑥
= 6.22 × 10−1
𝑢𝑛𝑐𝑒𝑟𝑡𝑎𝑖𝑛𝑡𝑦 = √(2.28 × 10−2 )2 + (5.93 × 10−3 )2 + (1.66 × 10−2 )2 + ( 6.22 × 10−1 )2
= 6.22 × 10−1
*All calculations for the remaining schemes are done is the same way shown.
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