EXPERIMENT 3: AAS Determination of Calcium in Commercial

Supplement Tablets

3.1 Abstract 1

3.2 Introduction 1

3.3 Objectives 2

3.4 Theory 2

3.5 Apparatus and materials 4

3.6 Procedure 4

3.7 Result 5

3.8 Calculations 6

3.9 Discussion 7

3.10 Conclusion 9

3.11 Recommendations 9

3.12 References 10

3.13 Appendices 10

0
3.1 Abstract
The objective of this experiment is to determine calcium content in commercial supplement
tablets. The quantity of calcium per tablet that is stated at the supplement bottle was to be
200mg. To investigate this statement, we need to determine experimentally the actual content of
calcium in the supplement tablets. The experiment was conducted by using atomic absorption
spectroscopy analysis. Before running the analysis using AAS, first a series of standard solutions
based on calcium concentration was established. A 50ppm stock solution of calcium is prepared
in 100mL flask using CaCl2.2H2O salts. Thus we need exactly 5mg of calcium, Ca to be
dissolved in the 100mL flask to get 50ppm. From the stock solution, a series of standard solution
of 25ppm, 12.5ppm and 6.25ppm was prepared by diluting a specific volume from the stock
solution in three individual flasks to the respected concentration. The volume of 50 ppm needed
to be dissolved in 50 mL flask to get 25 ppm, 12.5 ppm and 6.25 ppm are 25 mL, 12.5 mL and
6.25 mL respectively. Further aspect of this experiment is the evaluation of the effect of pH on
the solubility of the calcium tablets and the efficiency of digestion in the human body. In the
second sets of measurement, five solutions of the dissolved tablets with known pH level from 0
to 5 are analyzed using the AAS. From the AAS analysis, the absorbance value for the solutions
with pH 0, 1, 2, 3, 4 and 5 are 0.1441, 0.1676, 0.3650, 1.4077C, 1.4018C and 1.3923C
respectively. From the absorbance reading obtained, using the interpolation and extrapolation of
the calibration curve, we get the concentration of Ca in the solutions with pH 0, 1, 2, 3, 4 and 5
to be (4.62, 5.64, 14.28, 59.91, 59.65 and 59.23) ppm respectively. The highest concentration of
Ca between the five solutions is in pH 2 with 59.91 ppm. It shows that pH 2 is the optimum pH
level in dissolving the Ca tablet. From 59.91 ppm, we get the amount of Ca in the tablet to be
59.91 mg. This result disagree with the claim from the commercial tablet box where the amount
of Ca to be 200mg per tablet.

3.2 Introduction
In 1802. The phenomenon of atomic absorption was being observed with the discovery of the
Fraunhofer lines in the sun’s spectrum. While on 1953, Sir Alan Walsh who is an Australian
physicist demonstrate the concept of atomic absorption spectroscopy where he was being
inspired during gardening at his home. This atomic absorption spectroscopy concept led to an
invention that gives high achievement in chemical analysis.

Atomic Absorption Spectroscopy (AAS) is use in order to measure the concentration of gas-
phase atom by absorbing of light which is ultraviolet light or visible light that will excite an
electrons from lower energy levels to higher energy levels. All particular elements that being
involves in experiment will be analyzed using an instrument called atomic absorption
spectrophotometer. The process of atomic absorption spectroscopy involves two steps which are
atomized the sample and absorbed a radiation from the light source by free atoms.

1
At the first stage of the mechanism, the analyte atoms or ions must be vaporized in a flame or
graphic furnace. The flame is lined up in a beam of light of the appropriate wavelength. Atom
will undergo a transition from the ground state to the first excited state because of flame. When
atoms make their transition, they absorb some of the light from the beam. The basic structure of
atomic absorption spectrophotometer are hollow cathode lamp, an atomizer section for atomizing
the sample, a monochrometer for selecting the analysis wavelength of the target element and also
a detector to convert light into an electrical signal. Hollow cathode lamp is consists of hollow
cathode, anode, neon or argon gas.

The hollow cathode lamp is use to emits the light at correct wavelength which is equal to that
absorbed by the atoms of the sample. The hollow cathode lamp needs to align so that the beam
will across to the hottest part of the flame and travels to the detector. Detector is use to measure
the intensity of the beam of light. When metals absorbs some light, the intensity will
automatically reduced and its propotional. So, the detector will record the reduction as
absorption.

3.3 Objective
1. To determine the commercial calcium supplement by Atomic Absorption
Spectrophotometry.
2. To compare the concentration of unknown sample (commercial calcium supplement)
with the amount of calcium that needed by human.
3.4 Theory
Before experiment was conducted, we need to prepared calcium stock solution to make up a
100ml 50ppm and dilute the solution to give a series of standard solution (25, 12.5 and 6.25ppm)
to be used in the calibration of the spectrometer.

Atomic spectroscopy is the study of absorption or emission of light by lone atoms (the free atoms
or ions NOT a compound). Generally, AAS is only used for inorganic compounds. Absorption
and emission of light correspond to absorption and emission of energy by electrons of the atom.
The range of those energetic transitions is very wide, so that the absorption and emission and be
observed in any part electromagnetic spectrum.

The most common source for atomic absorption measurements is HCL (Hollow Cathode Lamp).
In this experiment the cathode of the HCL is constructed of lead. The most important
atomization device for atomic spectroscopy consists of a nebulizer and a burner. The nebulizer
converts a liquid sample into a fine spray or aerosol, which is then fed into the flame. The fuel-
oxidizer combination is air-acetylene, which produces a flame temperature of 2400-2700K.

2
AAS schematic/ flow diagram.

Figure 3.1 Atomic-absorption spectroscopy (AA) (by Brian M. on http://www.files.chem.vt.edu/chem-

ed/spec/atomic/aa.html)

As implied previously, only a very small number of the atoms in the flame are actually present in
an excited state at any given instant. Thus there is a large percentage of atoms that are in the
ground state and available to be excited. AAS takes advantage of this fact and uses a light beam
to excite these ground state atoms in the flame. Thus AA is very much like molecular absorption
spectrophotometry in that light absorption (by these ground state atoms) is measured and related
to concentration. The light source, called a hollow cathode tube, is a lamp that emits exactly the
wavelength required for the analysis (without the use of a monochromator).

The light from this lamp is exactly the light required for the analysis, even though no
monochromator is used. The reason for this is that atoms of the metal to be tested are present
within the lamp, and when the lamp is on, these atoms are supplied with energy, which causes
them to elevate to the excited states. Upon returning to the ground state, exactly the same
wavelengths that are useful in the analysis are emitted, since it is the analyzed metal with exactly
the same energy levels that undergoes excitation. The light is directed at the flame containing the
sample. The flame is typically wide about 4-6 inches, giving a reasonably long path length for
detecting small concentrations of atoms in the flame.

The light beam then enters the monochromator, which is tuned to a wavelength that is absorbed
by the sample. The detector measures the light intensity, which after adjusting for the blank, is
output to the readout, much like in a single beam molecular instrument. The absorption behavior
follows Beer's Law and concentrations of unknowns are determined in the same way. All atomic
species have an absorptivity, ε, and the width of the flame is the pathlength, l. Thus, absorbances
(Abs) of standards and samples are measured and concentrations determined as with previously
presented procedures, with the use of Beer's Law (Abs = ε l c ).

Double beam instruments are also in use in AAS. In this case, however, the second beam does
not pass through a second sample container. It's difficult to obtain two closely matched flames.

3
The second beam simply bypasses the flame and is relayed to the detector directly. This design
eliminates variations due to fluctuations in source intensity (the major objective), but does not
eliminate effects due to the flame (cuvette) or other components in the sample (blank
components). These must still be adjusted for by reading the blank at a separate time.

3.5 Materials and Apparatus

i. CaCl2.2H2O solids
ii. Distilled water
iii. AAS
iv. Ca supplement tablet

3.6 Procedure
1. The sample was prepared prior to the experiment.
2. The computer and apparatus was turned on, and the Varian software was selected.
3. Place the bulb (accurate to the sample we are going to analyse) to the sample
4. The sucking tube was place inside a beaker filled with distilled water. The ‘analyse’
button was clicked in the computer.
5. The flame was turned on before the samples were put to the test.
6. The sucking tube was then inserted into the first sample. The ‘Analyse’ button was
clicked on the computer. The sucking tube was left in the beaker, until the sample was
done analysed.
7. The sucking tube was then taken off, and inserted into the beaker filled with distilled
water.
8. Steps 6 and 7 was repeated for every sample, and between every sample’s analysis, the
sucking tube was put into a beaker filled with dilute water.
9. The data was recorded and hard copied.

4
3.7 Results
Sample Concentration (ppm) Absorbance
STD 1 0 0.0001
STD 2 6.25 0.1772
STD 3 12.5 0.3442
STD 4 25 0.6645
STD 5 50 1.1496

1.4
1.2
f(x) = 0.02 x + 0.04
1 R² = 0.99
0.8

0.6
0.4
0.2
0
0 10 20 30 40 50 60

Sample pH Concentration (ppm) Absorbance

5 4.62 0.1441
4 5.64 0.1676
3 14.28 0.365
2 59.91 1.4077C
1 59.65 1.4018C
0 59.23 1.3923C

5
3.8 Sample calculation
We need to dilute 5 mg of CaCl2.2H2O solid in 100 mL solution to obtained 100 ppm of Ca stock
solution. From the solids of CaCl2.2H2O, we know that Ca = 40.08 g/mol, Cl = 45.45 g/mol, H=
1.008 g/mol and O= 16g/mol.

Percentage of Ca in CaCl2.2H2O:

RMM CaCl2.2H2O = (40.08 + 1.008 x 4 + 16 x 2 + 45.45 x 2) g/mol = 167.01 g /mol

40.08 g /mol
Zn% = x 100% = 27.2 %
167.01 g/mol

Total mass of CaCl2.2H2O to obtain 5mg of Ca:

10 mgCa W mgof CaCl 2.2 H 2 O

27.2 % = 100 %

W = 18.38 mg Ca

Thus we need to dissolve 18.38 mg of CaCl2.H2O in 50 mL solution.

To get series of standard solution of 25 ppm, 12.5 ppm and 6.25 ppm:

M1V1 = M2V2 ; let M1 = 50 ppm V2 = 50 mL

To get 25 ppm in a 50 mL flask from 100 ppm:

M1V1 = M2V2
50 ppm (V1) = 25 ppm (50mL)
V1 = 25 mL

thus, we take 25 mL of 100 ppm Ca solution and diluted it in a 50 mL flask with distilled water.

If, M2 = 12.5 ppm V1 = 12.5 mL

M2 = 6.25 ppm V1 = 6.25 mL

To calculate Ca dissolved in pH solution:

At pH 2, the concentration is 59.91 ppm,

Mass of Ca present = 59.91 ppm x 1 L = 59.91 mg

6
3.9 Discussion
The objective of this experiment is to determine calcium content in commercial supplement
tablets. The quantity of calcium per tablet that is stated at the supplement bottle was to be
200mg. To investigate this statement, we need to determine experimentally the actual content of
calcium in the supplement tablets. The experiment was conducted by using atomic absorption
spectroscopy analysis.

Before running the analysis using AAS, first a series of standard solutions based on calcium
concentration was established. A 50ppm stock solution of calcium is prepared in 100mL flask
using CaCl2.2H2O salts. Thus we need exactly 5mg of calcium, Ca to be dissolved in the 100mL
flask to get 50ppm. Using relative atomic mass of each elements, it is found out that the element
Ca distribute 27.2% composition in CaCl2.2H2O. From this knowledge, we know that to get 5mg
of Ca, we need to dissolve about 18.4mg of CaCl2.2H2O in the flask.

Because the CaCl2.H2O salts are not fine grains and more to smaller chunks of coarse-grained
solids that are irregular in shapes, it makes it very difficult to finely tune our mass measurement
of CaCl2.H2O to be exactly 18.4mg in the electronic mass balance. The exact mass of CaCl 2.H2O
taken was 19.3mg. The weighed CaCl2.2H2O was then diluted in a small beaker before being
placed in the 100mL flask to ensure that all the solids are dissolved entirely.

From the stock solution, a series of standard solution of 25ppm, 12.5ppm and 6.25ppm was
prepared by diluting a specific volume from the stock solution in three individual flasks to the
respected concentration. The volume of 50 ppm needed to be dissolved in 50 mL flask to get 25
ppm, 12.5 ppm and 6.25 ppm are 25 mL, 12.5 mL and 6.25 mL respectively.

The AAS analysis was straight forward and direct. Basically, AAS analyzed a specific element at
one time depending on the hollow cathode lamp used. In this case, since we want to study the Ca
content, a Ca hollow cathode lamp was placed in the spectrometer. Main role of the hollow
cathode lamp was to emit cathode material (Ca) in excited state towards the sample. But first, the
sample containing Ca was atomized using flame atomizers with temperature at around 2700K.

In the flame atomization process, we can see that the flame inside the AAS changes colour to
orange. The atomization of Ca causes the excitation of electrons. The transition of electrons will
produce energy and emit in the form of specific wavelength that corresponds to the colour
orange for Ca. Since every element has their unique excitation energy, this thus play the main
key aspect in AAS where specific element can be identified using its respective hollow cathode
lamp. The Ca hollow cathode lamp emits photons where only Ca atoms can absorb giving the
absorbance value.

From the result of the calibration line using series of standard solution with known
concentration, it can be seen that higher concentration of Ca gave higher absorbance reading.
This shows that higher quantities of Ca atoms in the sample absorb greater energy released from

7
the lamp to excite electrons. Theoretically, the line obtained should be linear with r = 1. By
comparison of the r value for the line, we get r = 0.9964 which are close to one.

Further aspect of this experiment is the evaluation of the effect of pH on the solubility of the
calcium tablets and the efficiency of digestion in the human body. In the second sets of
measurement, five solutions of the dissolved tablets with known pH level from 0 to 5 are
analyzed using the AAS. From the AAS analysis, the absorbance value for the solutions with pH
0, 1, 2, 3, 4 and 5 are 0.1441, 0.1676, 0.3650, 1.4077C, 1.4018C and 1.3923C respectively.
Notice the letter C in three of the reading showing that the absorbance value exceed the
calibration data, thus the concentration values are obtained through extrapolation.

From the absorbance reading obtained, using the interpolation and extrapolation of the
calibration curve, we get the concentration of Ca in the solutions with pH 0, 1, 2, 3, 4 and 5 to be
(4.62, 5.64, 14.28, 59.91, 59.65 and 59.23) ppm respectively. The highest concentration of Ca
between the five solutions is in pH 2 with 59.91 ppm. It shows that pH 2 is the optimum pH level
in dissolving the Ca tablet. This is analogous to the human stomachs of the digestive system that
are acidic in nature.

The interior of the stomach is able to secrete about 2 to 3 litres of gastric fluid every day. Gastric
juice in the stomach keeps a pH level anywhere between 1 and 3 (Brooker, 2009). From 59.91
ppm, we get the amount of Ca in the tablet to be 59.91 mg. This result disagree with the claim
from the commercial tablet box where the amount of Ca to be 200mg per tablet. In a realistic
point of view, before questioning the statement made from the company, we must first look at
the method in which we dissolved the Ca tablet.

From Gore (2007) said that the stomach releases proteases (protein-digesting enzymes such as
pepsin) and hydrochloric acid, which kills or inhibits bacteria and provides the acidic pH for the
proteases to work. Food is churned by the stomach through muscular contractions of the wall -
reducing the volume of the fundus, before looping around the fundus and the body of stomach as
the boluses are converted into chyme (partially-digested food). In adult humans, the stomach has
a relaxed, near empty volume of about 45 ml. Because it is a distensible organ, it normally
expands to hold about 1 litre of food, but can hold as much as 2-3 litres.

We may produce a pH environment identically to a human stomach, but there are many other
factors that affect the human digestive system beside pH level. There is the optimum
temperature, mechanical induced movement of the system, pressure and many more. The
statement highlights that the Ca tablet is not in the ideal and identical environment as in the
human stomach to be dissolved completely. The “lab’s stomach” has much lower efficiency than
the human stomach. Thus, the claim that the tablet holds 200mg of Ca still can be considered as
plausible.

8
3.10 Conclusion
The volume of 50 ppm needed to be dissolved in 50 mL flask to get 25 ppm, 12.5 ppm and 6.25
ppm are 25 mL, 12.5 mL and 6.25 mL respectively. From the AAS analysis, the absorbance
value for the solutions with pH 0, 1, 2, 3, 4 and 5 are 0.1441, 0.1676, 0.3650, 1.4077C, 1.4018C
and 1.3923C respectively. From the absorbance reading obtained, using the interpolation and
extrapolation of the calibration curve, we get the concentration of Ca in the solutions with pH 0,
1, 2, 3, 4 and 5 to be (4.62, 5.64, 14.28, 59.91, 59.65 and 59.23) ppm respectively. The highest
concentration of Ca between the five solutions is in pH 2 with 59.91 ppm. From 59.91 ppm, we
get the amount of Ca in the tablet to be 59.91 mg. This result disagree with the claim from the
commercial tablet box where the amount of Ca to be 200mg per tablet.

3.11 Recommendation
Based on what we had done, we would like to recommend the following to ensure a more
accurate and smooth future experiments:

1. Always ensure that the sucking tube is in a beaker of solvent; either samples or distilled
water, at all time. This was done to ensure that the AAS apparatus experience no harm or
damage.
2. It is also recommended that the flame was lighted at least 10 minutes before the test.
3. The bulb (hollow cathode lamp) was recommended to be placed at least 20minutes before
the experiment.
4. There should be a gap of at least 1 minute between each sample. During this period of
time, the sucking tube is left in the beaker filled with distilled water to ensure that there is
no trace of the previous sample.
5. It is also recommended that we use the background solvent of our sample to rinse the
apparatus as a substitute to using the distilled water.

9
3.12 References
Atomic Absorption Series, 2011. Wikipedia [online] Available at
<http://en.wikipedia.org/wiki/File:NovAAseries.jpg> [Accessed 31 March 2011]

Atomic Absorption Spectroscopy, 2010. GMU [online] Available at

<http://www.gmu.edu/depts/SRIF/tutorial/aas/aas2.htm> [Accessed 31 March 2011]

Gore, R.M., Levine, M. S., 2007. Textbook of Gastrointestinal Radiology. Philadelphia: P.A.
Saunders

Human Stomach, The Basics, 2009. Brooker, J.J. [online] Available at <
http://EzineArticles.com/86428 Human stomach - the basics> [Accessed 31 March]

Schematic of Atomic Absorption, 2010. Brian M. [online] Available at

<http://www.files.chem.vt.edu/chem-ed/spec/atomic/aa.html> [Accessed 31 March]

3.13 Appendices

10