1.0 INTRODUCTION

Calcium is a mineral abundantly found in the human body. It is

the main constituent of the skeleton and helps in the formation

and maintenance of our bones and teeth. Calcium plays a

significant role in the contraction of muscles and secretion of

certain hormones and enzymes. The expansion and contraction of

blood vessels depends on calcium, it also plays an important role

in the clotting of blood. This mineral is also required for

muscle contractions and relaxation, nerve and hormone function,

and blood pressure regulation. When calcium levels fall too low,

the calcium is transferred from the bones to the blood in order

to maintain the blood calcium level. Thus, nerve and muscle

impairments can result. In contrast, receiving higher amounts of

calcium over a long period of time raises the risk of kidney

stones in some people. Therefore, calcium plays a very crucial

role in the functioning of the body and it should be consumed in

sufficient amounts regularly.

In this experiment, the calcium content in a commercial

tablet is determined by using Atomic-absorption spectroscopy

(AAS) method. AAS uses the absorption of light to measure the

concentration of gas-phase atoms. Since samples are usually

liquids or solids, the analyte atoms or ions must be vaporized in

a flame or graphite furnace. AAS is an instrument that is used to

measure the amount of radiation energy absorbed by a ground-state

atom in a flame for these energy transitions. The light source

for the atomic absorption apparatus is a hollow cathode lamp in

which the cathode is made from the same metal that is being

analyzed, in this case calcium. When heated, the calcium atoms

are excited and their electrons go to higher energy levels. When

the electrons fall back to lower levels, visible radiation is

given off. The energy of the emitted photons corresponds to the

energy difference of the Ca atom electron levels. We will be

measuring the intensity of 423-nm radiation, which corresponds to

a prominent Ca atom electron transition. The 423-nm radiation

emitted by the Ca atoms in the hollow cathode lamp will be

selectively absorbed by the Ca atoms in the solutions fed into

the AAS flame chamber. The analyte concentration is determined

from the amount of absorption. Concentration measurements are

usually determined from a working curve after calibrating the

instrument with standards of known concentration.

2.0 OBJECTIVES

The objective of this experiment is to determine the amount

of calcium content in the commercial tablets by using AAS

instrumentation. The validity of the calcium content on the

commercial tablets box will be evaluated. In addition, the effect

of pH on the solubility of the calcium tablets and the efficiency

of digestion in the human body is discussed.

3.0 THEORY

Atomic Absorption Spectroscopy is an analytical technique used

for the qualitative and quantitative determination of the

elements present in different samples like food, nonmaterial,

biomaterials, forensics, and industrial wastes. Atomic absorption

is so sensitive that it can measure down to parts per billion of

a gram (μg dm–3) in a sample.

Atomic absorption spectroscopy (AAS) uses the absorption of

light to measure the concentration of gas-phase atoms. The

analyte atoms or ions must be vaporized in a flame or graphite

furnace since the samples used are usually liquids or solids. The

atoms absorb ultraviolet or visible light and energy excites the

atoms to make transitions to higher electronic energy levels.

When the homogeneous solution of a representative sample is

sprayed onto a flame (Air-Acetylene or Acetylene-Nitrous oxide),

free atoms formed in the flame absorb characteristic radiation of

the same element emitted by a hollow cathode lamp and undergo

transitions to excited energy levels. This energy absorption by

free atoms of the sample formed in the flame is proportional to

the concentration of atoms formed in the flame and is governed by

Lambert-Beer Law. Applying the Beer-Lambert law directly in AAS

spectroscopy is difficult due to variations in the atomization

efficiency from the sample matrix, and no uniformity of

concentration and path length of analyte atoms (in graphite

furnace AAS).

Atoms of different elements absorb characteristic

wavelengths of light. Analyzing a sample to see if it contains a

particular element simply means using light from that element.

For example with calcium, a lamp containing lead emits light from

excited calcium atoms that produce the right mix of wavelengths

to be absorbed by any calcium atoms from the sample. In AAS, the

sample is atomized – converted into ground state free atoms in

the vapor state – and a beam of electromagnetic radiation emitted

from excited lead atoms is passed through the vaporized sample.

Some of the radiation is absorbed by the lead atoms in the

sample. The greater the number of atoms there is in the vapor,

the more radiation is absorbed. The amount of light absorbed is

proportional to the number of calcium atoms. A calibration curve

is constructed by running several samples of known calcium

concentration under the same conditions as the unknown. The

amount the standard absorbs is compared with the calibration

curve and this enables the calculation of the calcium

concentration in the unknown sample. Consequently an atomic

absorption spectrometer needs the following three components: a

light source; a sample cell to produce gaseous atoms; and a means

of measuring the specific light absorbed.

The common source of light is a ‘hollow cathode lamp’

(Figure 3.1). This contains a tungsten anode and a cylindrical

hollow cathode made of the element to be determined. These are

sealed in a glass tube filled with an inert gas – neon or argon –

at a pressure of between 1 Nm–2 and 5 Nm–2. The shape of the

cathode concentrates the radiation into a beam which passes

through a quartz window, and the shape of the lamp is such that

most of the sputtered atoms are redeposit on the cathode.

Figure 3.1: ‘Hollow cathode lamp’

Modern spectrometers incorporate a beam splitter so that one

part of the beam passes through the sample cell and the other is

the reference (Figure 3.2). The intensity of the light source may

not stay constant during an analysis. If only a single beam is

used to pass through the atom cell, a blank reading containing no

analyte (substance to be analyzed) would have to be taken first,

setting the absorbance at zero. If the intensity of the source

changes by the time the sample is put in place, the measurement

will be inaccurate. In the double beam instrument there is a

constant monitoring between the reference beam and the light

source. To ensure that the spectrum does not suffer from loss of

sensitivity, the beam splitter is designed so that as high a

proportion as possible of the energy of the lamp beam passes

through the sample.

Figure 3.2: Double beam spectrometer

The instrument is calibrated using several solutions of

known concentrations. A calibration curve is produced which is

continually rescaled as more concentrated solutions are used –

the more concentrated solutions absorb more radiation up to a

certain absorbance. The calibration curve shows the concentration

against the amount of radiation absorbed. The sample solution is

fed into the instrument and the unknown concentration of the

element is then displayed on the calibration curve.

Figure 3.3: AAS Flame Chamber and Description of AAS used

4.0 APPARATUS/MATERIALS

AAS Hitachi Z-2000

Commercial vitamins tablet box

Calcium Chloride, CaCl

Volumetric flask

Beaker

Pipette

Weighing balance

5.0 EXPERIMENTAL PROTOCOL

1. 0.037g of calcium chloride 2-hydrate (CaCl2∙2H2O¿ powder is

weighed by using electronic balance.

2. 100ml stock solution of 100ppm calcium concentration is

prepared by diluting the weigh CaCl2∙2H2O powder with

distilled water in a 100ml volumetric flask.

3. The stock solution is then undergoing a serial dilution in

order to prepare a series of standard solution of 20ppm,

15ppm, 10ppm and 5ppm. The standard solution is prepared in

50ml of volumetric flask.

4. Each of the diluted standard solutions and four stock

solutions of commercial tablets with pH ranging 1 to 5 were

poured third quarter in the test tube provided and place on

a test tube holder.

5. The AAS Hitachi Z-2000 instrument supplied by Chemopharm

Company is turned on and the instrument software is

selected.

6. The flame and hollow cathode lamp that had been set up by

lab assistant is turned on.

7. The sucking tube which connected to AAS is placed into the

blank solution which is distilled water and soaked

approximately 30 seconds to zero the detector.

8. Each of the diluted sample solution from lower to higher

concentration of CaCl2∙2H2O is introduced with the sucking

tube. The intensity for each sample is observed and

recorded.

9. A calibration graph based on the intensity readings and the

concentrations of CaCl2∙2H2O sample is constructed.

10. Each of the commercial tablets sample solutions with

different pH range is next introduced to the sucking tube.

The intensity for each sample is observed and recorded.

11. The sucking tube is took off and inserted back to the

blank sample.

All the data is printed out for justifications.

6.0 RESULTS

Table 6.1: The absorbance and concentration readings of calcium

in standard solutions

Sample Concentration (ppm) Absorbance1 0 -0.0001

2 5 0.2341

3 10 0.4023

4 15 0.6585

5 20 0.8533

Table 6.2: The absorbance and concentration readings of the known

pH

pH Concentration (ppm) Absorbance1 32.77 1.4000 C

2 32.67 1.3957 C

3 32.41 1.3850 C

4 9.09 0.3908

5 4.21 0.1827

0 5 10 15 20 25-0.20

0.20.40.60.81

f(x) = 0.042624 x + 0.00338000000000005R² = 0.997095251915831

Graph of Absorbance vs Concentration of Ca (ppm) in standard solution

Concentration (ppm)

Abso

rbance

Figure 6.1: The graph absorbance versus of concentration of Ca

(ppm) in the standard solution

1 2 3 4 505

101520253035

Graph of Concentration of Ca (ppm) vs pH

pH

Conc

entrat

ion

(ppm)

Figure 6.2: The graph of concentration of Ca (ppm) versus pH

7.0 SAMPLE OF CALCULATIONS

The molecular weight of CaCl2.2H2O, MWCaCl2.2H2O = 147.02 g/mol.

The molecular weight of Ca, MWCa = 40.08 g/mol

1) Weight of CaCl2.2H2O powder to prepare 100ppm of Ca solutions:

¿MWCaCl2.2H2O

MWCa×0.10L×100mg/L

¿ 147.02g/mol40.08g /mol

×0.1L×100mg/L

¿36.68mg× 1g1000mg

¿0.037gofCaCl2.2H2O

2) Calculations of standard solutions 20ppm, 15ppm, 10ppm and

5ppm:

M1V1=M2V2

20 ppm M1V1=M2V2

50 x V1 = 20 x 50mL

V1 = 20 mL

15 ppm M1V1=M2V2

50 x V1 = 15 x 50mL

V1 = 15 mL

10 ppm M1V1=M2V2

50 x V1 = 10 x 50mL

V1 = 10 mL

5 ppm M1V1=M2V2

50 x V1 = 5 x 50mL

V1 = 5 mL

To calculate Ca dissolved in pH solution:

At pH 1, the concentration is 32.77ppm, hence,

Mass of Ca present = 32.77ppm x 1 L = 32.77 mg

8.0 DISCUSSION

The objective of this experiment is to determine the calcium

content present in commercial supplement tablets by using atomic

absorption spectroscopy analysis (AAS). The actual content of

calcium in the supplement is determined by using AAS. The first

step done before the experiment was the preparation of a series

of stock solution. The stock solution needed for this experiment

id 50ppm, which is prepared in a 100ml flask using CaCl2.2H2O

solid salts.

From the calculation, it was showed that 37 mg of Ca

contained in CaCl2.2H2O tablets. The amount was weighed by using

the electronic mass balance, which is more accurate to weigh a

small amount of substances. The standard solution of 20ppm,

15ppm, 10ppm, and 5ppm is prepared by diluting 50ppm of stock

solution. The concentration of commercial vitamins tablets with

five difference pH is compared with the calibration curve of the

standard solution. The blank solution (distilled water) is used

in the AAS instrument to get accurate reading for the

concentration of calcium.

The atomic absorption spectroscopy analysis can only

identified a specific type of element, where in this experiment,

only Ca can absorbed the energy emitted by the hallow cathode

lamp as the Ca material cathode lamp was used. From figure 6.2,

it showed that, the higher the concentration, the higher the

absorbance reading. It was also showed that, the higher the

concentration, the greater the energy absorbed to excite the

electrons in the samples.

The linear graph plotted, theoretically, the line with R2= 1

should be obtained, but from the experiment conducted, the line

with R2 = 0.9985 was obtained. The reading was quite not far from

the theoretical value, but it may because of the Ca salt weighed

was not exactly 0.037 mg, but it was 0.038 mg. It was quite hard

to find the smallest size of salts among the other salts and

hence, only 0.038 mg was weighed. Thus, one of the points was not

exactly in the linear line.

After all of the absorbance readings with the prepared

standard solution were obtained, the absorbance reading of five

solutions with the known pH was determined. The known pH was pH

1, pH 2, pH 3, pH 4 and pH 5, and all of them were poured into

five different cuvettes to be analyzed by using AAS.

The ‘C’ in table 6.2 showed that the value of absorbance is

exceeding the calibration data and due to this, the value of the

concentration is obtained through the extrapolation. From the

extrapolation of the calibration curve was done, the

concentration obtained were 32.77ppm, 32.67ppm, 32.41ppm, 9.09ppm

and 4.21ppm for pH 1, pH 2, pH 3, pH 4 and pH 5 respectively.

Among all the five pH readings, the highest concentration was on

pH 1 with 32.77ppm (Figure 6.1). This pH value is the optimum pH

level to dissolve the Ca tablet. From the statement, it can be

proven that the stomach digestive system is in acidic.

According to Carter (1996), the human’s stomach contains

strong hydrochloric acid (HCl), which is enough to dissolve metal

properties with pH range from 1.5 to 3. It can kills bacteria and

denatured the proteins in the food taken, making them to be more

vulnerable to be attacked by pepsin. However, from the result

obtained, the highest concentration of 32.77ppm was from pH 1,

hence the amount of Ca contained in the tablet is 32.77 mg.

However, this was contradicts from the commercial tablet box,

whereby the amount of Ca content is 200 mg per tablet.

The stomach is kind-like J-shaped and has the ability to

expands, which located on the left side of the upper abdomen.

There are some muscles layers cover the stomach and enable it to

undergo the process of food churning (Carter, 1996). The stomach

has the capacity up to 2 L to hold the food by expansion. A

person may secrete up to 400 ml of gastric juice per meal and it

contains 50 to 300 μg pepsin/ml.

There are a few factors that affecting the human digestive

system beside the pH level. According to Bailey (2013), the

factors are included the nature of the food (the fats and the

proteins content) and also the degree of muscle actions. There is

also the optimum temperature as well as the pressure, which are

included as the factors affecting the human digestive system. As

the pH environment produced was not identically as the actual one

in the human stomach because in the stomach, the Ca tablet can be

dissolved completely. Thus, the claim that the tablet contains

200 mg of Ca still can be assumed as reasonable

9.0 CONCLUSION

Generally, in this experiment, the analytical technique

which Atomic Absorption Spectroscopy (AAS) is used in order to

determine the concentration of the calcium content in the unknown

solution. The concentration of the calcium content in the unknown

solution can be determined by calibration curve. From the

standard solution which was prepared, the line with R2 = 0.9985

was obtained. From the calculation the concentration of the

calcium content is related to each of the pH value. From the

extrapolation of the calibration curve was done, the

concentration values which correspond to pH 1, 2, 3, 4, and 5 are

32.77, 32.67, 32.41, 9.09 and 4.21ppm respectively. The

concentration value and the pH value have relationship. By

plotting the graph, as the concentration of calcium that was

dissolved higher, the pH value tends to lower. Maximum amount of

calcium that recommended is 600mg/L per day. In our stomach,

gastric juice appears at pH 1 to pH 3 in order to digest our

food. The concentration of unknown sample from pH 1 to pH 3 is

suitable for human body. The total concentration from pH 1 to pH

3 can be supported by stomach. Besides, this showed the

concentration for pH in the range of 1 to 3 is adequate by blood

and bone calcium levels.

10.0 REFERENCES

Bailey, R. (2013). Digestive System. What is The Digestive System.

Retrieved May 20, 2013, from

http://biology.about.com/od/organsystems/a/aa032107a.htm.

Carter, J.S (1996). Digestive System. UC-Clermont College

Biology. Retrieved May 19, 2013, from

http://biology.clc.uc.edu/courses/bio105/digestiv.htm

National Institutes Of Health (n.d.). Retrieved May 19, 2013,

from

http://dietary-supplements.info.nih.gov/factsheets/

calcium.asp

Brian. M. Tissue. (1996). Atomic-Absorption spectroscopy (AAS).

Retrieved May 19, 2013, from

http://elchem.kaist.ac.kr/vt/chem-ed/spec/atomic/aa.htm

http://www.calciuminfo.com/about/foodsources.aspx

Skoog, D. A., Holler, F. J., & Nieman, T. A. (1998). Principles of

Instrumental Analysis. Brooks Cole.

Skoog, D. A., West, D. M., Holler, F. J., & Crouch, S. R. (2004).

Fundamentals of Analytical Chemistry. Brooks Cole.

http://weather.nmsu.edu/Teaching_Material/soil698/

Student_Reports/Spectroscopy/report.htm

http://elchem.kaist.ac.kr/vt/chem-ed/spec/atomic/aa.htm

http://www.cmetindia.org/aas.html

11.0 APPENDICES

APPENDIX B

Figure a: The CaCl2∙2H2O chemical powder used to prepare the stocksolution.

Figure b: The Nutrilite Calcium Magnesium used as commercial tablets.

Figure c: The Hitachi Z-2000 Polarized Zeeman AAS instrument.

Figure d: The AAS flame.

Figure e: The hollow cathode lamp used in the AAS intrument.

Figure f: The purified acetylene gas used to light up the AAS flame.