Calcium Analysis of Prime Eastern
Pharmaceuticals Calcium Tablets via EDTA
Titration, ISE, and AAS
Kelsi Brown, Jon Heibel, Robert Fuller, and Thomas Zernell
ABSTRACT: The calcium content of calcium carbonate tablets was analyzed in three
different ways. First, 500 mL of an unknown solution prepared from 0.1878 g of calcium
tablet was titrated with a 0.01336 M EDTA solution prepared from 1.2436 g of disodium
EDTA hydrate. Then, four standard CaCO3 solutions 0.0001-0.001 M were prepared and
analyzed using calcium ion selective electrode (I.S.E.) potentiometry to produce a
calibration curve of potential versus the logarithm of standard concentration. Unknown
solutions were then analyzed using the I.S.E. Lastly, four standard CaCO3 solutions were
prepared between 5 and 20 ppm and analyzed using flame atomic absorption to create
another calibration curve, which is used to analyze two unknowns. The EDTA titration,
potentiometric analysis, and atomic absorbance data yielded 34.25±0.20, 32.67±0.24, and
33.16±1.28 percent respectively. The advertised mass percentage of calcium was
approximately 44.44%, which lead to the conclusion that the tablets were considerably
mislabeled.
For many years Prime Eastern
Pharmaceuticals, Ltd has produced a
popular and very successful natural
calcium supplement. Each two-tablet
serving was labeled to have 1.6 g of
calcium in the form of calcium carbonate
(CaCO3) per every two tablet serving
size. Assuming an average tablet mass
of 1.8 grams the mass percent calcium as
advertised was 44.44 percent.
Despite
its
long-standing
tradition and popularity the calcium
dietary supplements made by Prime
Eastern Pharmaceuticals, Ltd have
recently come under legal fire for
allegedly reducing the amount of the
mineral in its tablets without altering the
label. Class action lawsuits have been
filed and consequently testing was
required to determine the legitimacy of
the claims against the manufacturer.
Three analytical techniques were
used in the determination of calcium, the
first of which was complex formation
titrimetry.
The hexadentate ligand
ethylenediamenetetraacetic acid (EDTA)
is used as a titrant
because it complexes
with most metal ions.
Figure 1 shows the
fully protonated form
and Figure 2 shows
the way in which the
fully
deprotonated
Figure 1
form of the EDTA ligand
bonds to a metal. The
bond
is
coordinate
covalent, meaning all the
electrons in each bond
come from the ligand. Other
comments about this
Figure 2
type of bonding can be
made about the structure of the complex.
For example it is clear from figure 2 that
the bonding is in a one-to-one ratio,
1
figure 3[5] typically this value is around
10-6 - 10-8M. Similarly the range of
linear response refers to the region in
which the EMF varies linearly because
at activities above 0.1 M and below 10-5
M ionic interferences cause deviation
from linearity[5].
Both of these
phenomena
are
illustrated in figure
3[5], which is a plot
of EMF versus
activity for an ion
having
a
+1
charge.
The final
analytical
test
performed on the
Figure 3
calcium
tablets
was
calcium
determination
by
flame
atomic
absorption. Also referred to as atomic
absorption spectroscopy (AAS), this
technique measures absorbance based on
the excitation of electrons in the
atomizer.
The term flame atomic
absorption is used to note that the
excitation of electrons in this specific
method is accomplished using a flame.
The atomized sample is then
exposed to electromagnetic radiation
(light). This radiation can come from
several sources but most commonly is
introduced by a hollow cathode lamp
(HCL) source. After passing through the
atomized sample the light, some of
which has been absorbed, is passed
through a monochromator, which
selectively reduces the light to a single
wavelength. A detector then measures
the strength of this light and gives an
absorbance readout. Figure 4[4] shows a
schematic of a general atomic absorption
spectrophotometer.
which makes most calculations simple.
Also, it can be noted that, because there
are six coordinate bonds per one metal
atom, the resulting complex is highly
stable. This stability combined with an
incredibly fast reaction rate explains
why EDTA titrations are reliable and
quantitative.
The
second
analytical
technique
used
for
calcium
determination took advantage of
potentiometry by using a calcium ion
selective electrode. These electrodes
measure the potential difference or
electromotive force (EMF) created
under equilibrium conditions by a
specific ion. For the analysis of the
calcium tablets a Vernier calcium
I.S.E. was used to measure the potential
difference across its PVC membrane.
Generally these electrodes are
able to measure the potential difference
because the membrane that separates the
solution from the half-cell contained in
the electrode only permits certain ions
(in this case Ca2+) to be exchanged
across it. Because the concentration of
the target ion is kept constant inside of
the electrode, the potential difference
will vary linearly with respect to the
base-10 logarithm of the ions activity in
solution. The slope of this curve is give
by the Nernst equation and only depends
on the charge of the ion (n) being
analyzed. The slope for the calcium
calibration curve is 29.58 mV.
E= E0+(59.16/n)*Log(ACa2+)
Nernst Equation
One last consideration regarding
the use of I.S.E. is the detection limit
and range of linear response. The
detection
limit
is
the
lowest
concentration that the electrode can
accurately measure. The value of the
detection
limit
is
defined
by
extrapolating the two linear sections of
the EMF versus log(A) plot as shown in
2
Figure 4
AAS results are only relevant if
comparable to the analysis of standard
solutions. This includes the calibration
of the machine to readout zero
absorbance in the pure solvent. From
this data a calibration curve can be made
to determine the concentrations of
unknown samples with a trend line
passing through the origin of the
absorbance versus logarithm of activity
plot.
All three methods were used to
obtain the mass percent of calcium in the
calcium tablets in question. The samples
in question must be proven definitively
to have a calcium content less than 44.44
mass percent to validate the claims made
in the aforementioned class action
lawsuits.
was used for titration of the unknown
solution.
A Calcium tablet was crushed
and 1.2436g of the tablet was dissolved
in a 150-ml beaker using deionized
water. 6 M Hydrochloric acid (36.4611
g/mol) was added drop wise until the
solution dissolved and the solution was
boiled to expel Carbon Dioxide.
Ammonium hydroxide (35.04588 g/mol)
was added to the solution to reach a pH
of 6. The pH was measured by
moistening a piece of pH paper. The
solution was then transferred to a 250-ml
volumetric flask and diluted to the mark
using deionized water. Using a pipette
50-ml of the unknown solution was then
transferred into each of the three 250-ml
Erlenmeyer flasks. An additional 100 ml
of deionzed water was added to a fourth
flask and this was labeled as “Blank”. 15
ml of pH 10 buffer solution along with 6
drops of Calmagite and 4 drops of
methyl red indicators were added to both
the unknown solutions and the Blank in
order to identify when the endpoint of
the titration was reached.
The
blank
and
unknown
solutions were then titrated with the
EDTA solution until they reached a
distinct blue color. The unused
ammonia, buffer, and hydrochloric acid
reagents were disposed of in the
appropriate waste bottles. All other
solutions were discarded in the sink and
flushed with water.
EXPERIMENTAL SECTION:
MATERIALS
The specifications for the Calcium
tablet in question are as follows:





800 mg/tablet with Vitamin D
1 serving of = 2 tablets
Calcium as calcium carbonate: 1.6g
Vitamin D as cholecalciferol: 800 IU
Other ingredients include
Maltodextrin, Powdered cellulose,
Hypromellose, Mineral oil, Titanium
dioxide, Triethyl citrate, Polysorbate 80,
Carnauba wax, Stearic acid, Magnesium
stearate, and Crospovidone. No gluten,
lactose, preservatives, or artificial
flavors were included in the tablet.
Ion Selective Electrode. 0.4731 g of the
crushed Calcium tablet was used and
was dissolved with 6 M HCl (36.4611
g/mol). 100 ml of deionized water was
added to the solution and then the
solution was boiled for about 2 minutes.
1 M NaOH (35.04588 g/mol) was added
to the solution in order to neutralize it.
After the solution was neutralized it was
EDTA Titrimetry.
1.2436 g of
disodium EDTA hydrate (372.24 g/mol;
volume of .250 L; molarity of 0.01336)
was used and was dissolved in a 250-ml
volumetric flask using 200 mL of boiled
deionized water. The solution was then
cooled to room temperature and diluted
to the mark. This preparation of EDTA
3
then transferred into a 500-ml
volumetric flask and diluted to the mark
using deionized water. 5 mL of the
diluted solution was transferred into a
100-ml volumetric flask and diluted to
the mark with deionzed water and this
was used for the analysis of the
unknown solution.
0.5035 g of standard Calcium
Carbonate (100.0869 g/mol) was
dissolved with 6 M HCl. 100 mL of
deionized water was added to the
solution and then the solution was boiled
for about 2 minutes and neutralized with
1 M NaOH solution. The neutralized
solution was then transferred to a 500-ml
volumetric flask and diluted to the mark
with deionized water. The following
table was then used to generate the 4
calibration standards:
mL of 0.01 M CaCO3
solution added to 100 mL
volumetric flask:
1
2
5
10
Diluted Concentrations
CaCO3
solutions
Moles/Liter
0.0001
0.0002
0.0005
0.001
The specifications for the Vernier
calcium ISE are as follows[2]:













Range: 0.20 to 40,000 mg/L or ppm (5 × 106
M to 1.0 M)
Resolution
% of reading: 1.4%
Low scale reading: 0.20 ±0.0028
High scale reading: 40,000 ±560 mg/L
pH Range: 3 to 10
Interfering Ions: Pb2+, Hg2+, Sr2+, Cu2+, Ni2+
Electrode Resistance: 1 to 4 MΩ
Electrode Slope (log voltage vs.
concentration): +28 mV/decade
Reproducibility: ± 5% of reading
Temperature range (can be placed in): 0 to
50°C (no temperature compensation)
Minimum immersion: 1 inch
Electrode Length: 155 mm
Body Diameter: 12 mm
Cap Diameter: 16 mm
Cable Length: 100 cm
Flame Atomic Absorption. For the
flame atomic absorption experiment, a
Texas A&M chemistry lab technician
used a Shimadzu flame atomic
absorption spectrophotometer. The 4
standardized solutions of CaCO3 were
created using 0.25 grams of standardized
CaCO3. The CaCO3 was dissolved in 6 M
HCl solution and diluted with
approximately 100 mL deionized water
and boiled to dispel CO2. The solution
was then diluted in deionized water in a
500 mL volumetric flask. Volumetric
pipettes were used to quantitatively
transfer 2.5, 5, 7.5, and 10 mL of the
standardized solution to separate 100 mL
volumetric flasks and diluted with
deionized water to create 5, 10, 15, and
20 ppm CaCO3 solutions respectively.
The standardized solutions were
analyzed with the flame atomic
absorption spectrophotometer to create a
calibration curve.
The unknown solution was
prepared from 0.2334 grams of Prime
Eastern
Pharmaceuticals
Natural
Calcium tablet that was ground to a fine
of
in
Table 1
The Vernier calcium ionselective electrode was used to measure
the potential for all of the samples. The
Calcium ISE has a combination-style,
non-refillable, gel-filled electrode and
the membrane has a limited life
expectancy. However, its replaceable
module allows the used membrane
module to be simply discarded and
replaced with a new one. For this
experiment two trials were run for each
of the 4 calibration samples and the
unknown sample. A calibration curve
was generated by plotting the potential
of the standards versus the log of their
respective concentrations. A trend line
was used to determine the concentration
of the unknown sample and then the
concentration was used to determine the
weight % of Calcium.
4
powder using a mortar and pestle. The
crushed tablet was dissolved with a few
drops of 6 M HCl then diluted with
approximately 100 mL of deionized
water. The solution was then filtered
with filter paper, boiled to dispel CO2,
and diluted with deionized water in a
500 mL volumetric flask. 7 mL of the
diluted unknown solution were then
transferred quantitatively using a
volumetric pipette into a 100 mL
volumetric flask and diluted using
deionized water. This solution was
analyzed in the flame atomic absorption
spectrophotometer and plotted on the
calibration curve formed from the
standardized solutions to find the
absorbance.
The specifications for the Shimadzu
atomic absorption spectrophotometer
used are as follows[1]:










EDTA
ISE
AAS
Trial 1
Trial 2
Trial 3
Average
34.326%
32.501%
34.060%
34.397%
32.835%
32.255%
34.012%
-
34.245%
32.668 %
33.158%
Table 2
Average
Standard Deviation
33.484%
0.91755%
Table 3
EDTA Titrimetry.
500 mL of a
0.01336 M EDTA solution was prepared
for titration of the unknown sample
solution from 1.2436 g of the available
EDTA hydrate. Now 500 mL of an
unknown solution of calcium tablets was
prepared from 0.1878 g of the crushed
tablet. From this sample solution a 50
mL volumetric pipette was used to
transfer three 0.03756 g (in 50 mL)
aliquots of the unknown into three
Erlenmeyer flasks for titration. Three
titrations were performed to a
phenolphthalein endpoint.
Trials 1, 2, and 3 reached the
endpoint after 24.07, 24.12, and 23.85
mL of the titrant respectively. From here
the calculations shown in “Calculations”
section of this report were used to find
the mass percent calcium in each of the
three trials. The number of moles of
EDTA react in a one-to-one ratio with
all metal ions in solution as was
illustrated by figure 2 that explains the
complex formation that EDTA is
involved in with metal ions. This fact
leads to the amount of calcium in each
titration, which can be compared to the
mass of the tablet per each 50 mL
aliquot to find the mass percent calcium.
The resulting calcium content
calculated for the trials 1-3 were 34.326,
34.397, and 34.012 mass percent
calcium respectively with an average of
34.245±0.205 mass percent.
One issue that can be presented
for this result is whether or not the
Optics: Double Beam (chopper mirror)
Monochromator: aberration corrected
Czerny-Turner monochromator
Wavelength range: 190-900 nm
Slit: 0.2 nm, 0.7 nm Manual setting
Frequency: 100 Hz
Chamber: Polypropylene
Gas control: manual setting of flow
rate; automatic Air/ N2O switching
system
Dimension and weight: W690 x D425 x
H370 mm, 38 kg
Ambient temperature and humidity
range:
Temperature:
10~35°C
Humidity: 45~80% (but less than 70%
if temperature is over 30°C
Power requirements: AC220V, 230V,
50/60Hz, 300 VA
Results and Discussion.
Through EDTA titration of metal ions,
calcium
ion
selective
electrode
potentiometry, and atomic absorption
spectroscopy the following results
(shown in table 1) were obtained for the
mass percent of calcium in the tablets.
5
EDTA titration reaction was exclusive to
calcium. From the label it is known that
magnesium stearate and titanium dioxide
are both present in the tablet in unknown
amounts. Of these two metal containing
compounds TiO2 is insoluble in water
but magnesium stearate is slightly
soluble which could contribute to a small
systematic error in the calculations
results.
in both trials are shown figure 5a and 5b
and table 5 shown below. From this data
the mass percent of calcium in the
tablets was calculated using the trend
line approximation. These calculations
are shown in the “Calculations” section
of the report.
From this data the logarithm of
molarity and therefore molarity were
calculated using the trend line for each
trial. From molarity the calcium content
of the unknown sample was then
calculated to be 32.50 and 32.84 mass
percent in trials 1 and 2 respectively
with an average value of 32.668±0.2365
mass percent calcium.
Though at first glance these
results appear precise there is some room
for discrepancy and further questioning.
The slope of the potential graph in trial 1
is within the normal allowable range of
25-30 but in trial 2 the trendline deviates
significantly from the theoretical value.
Potentiometric Analysis. 500 mL of a
0.01006 M CaCO3 standard solution was
prepared for generation of a potential
calibration curve. This stock solution
was then diluted into four 100 mL
standards by dilution factors of 100, 50,
20, and 10 respectively to each be
Table 4
analyzed using a calcium I.S.E. The
specifications for each of these solutions
are given in table 4 and were used for
the independent axis on the calibration
curve.
The logarithm of the solutions
molarity was then used to plot a
potential difference versus logarithm of
molarity plot. The plot and its trend line
as well as the readings for the unknown
Figure 5b
Table 5
Flame Atomic Absorption. When the
absorbance of the standardized
Figure 5a
6
solutions was plotted vs. their
respective concentrations in ppm, the
data yielded a linear line with a slope
of 0.0139. Taking absorbance as the
dependent variable and concentration
as your independent variable, this
graph represents Beer’s Law: A = bc
( is the molar absorptivity, b is the
path length of the light through the
solution, c is the concetration of the
species, and A is the absorbance). The
slope accounts for the two constants
in the equation ( and b). The plot of
absorbance
versus
the
ppm
concentration of calcium and the
corresponding trend line are shown in
figure 6 while the raw data for the
standards and unknowns are shown
in table 6.
certainty to say that from the
absorbencies of the two trials of our
unknown solution, trial #1 and trial
#2 yielded concentrations of 11.1295
ppm and 10.5396 ppm respectively.
This data yielded mass %
Calcium of 34.0602 % in trial 1, and
32.2548 % in trial 2. The unknown
trials
had
an
average
of
33.1575±1.2776
mass
percent
calcium.
Conclusions
Following the recent filing of
class action lawsuits against Prime
Eastern Pharmaceuticals, conformation
testing was performed to verify the
calcium content of the tablets in
question. The three methods used for
analysis were EDTA titrations, ion
selective
electrode
potentiometric
analysis,
and
atomic
absorption
spectroscopy. Through seven trials total
the resulting calcium concentrations was
determined to be 33.484±0.918 mass
percent calcium.
There were some significant
discrepancies in the data including the
systematic error in the titrimetry and the
slope in trial 2 of the potentiometric
analysis that varies from the theoretical
value. The former is likely a result of
other metal ions forming a complex with
EDTA. The magnesium stearate is
likely the culprit because of its nonnegligible solubility. The latter on the
other hand may be due to random error
resulting from the consistency with
which the data is taken.
From the label the calcium
content of the tablets should be 44.44
mass percent.
The results of this
analysis show with strong repeatability
that the claims made on the label of this
popular supplement brand are in fact a
false statement. The calcium content of
Table 6
Figure 6
The correlation coefficient
shows a minimal amount of variation
in the relationships between the
calcium
concentrations
and
absorbencies in the standardized
solutions. Therefore, we could use the
trend line equation with reasonable
7
the tablets is actually 25 percent less
than the advertised amount.
8
Calculations
9
10
References
[1]"Atomic Spectroscopy." Shimadzu
Excellence in Science. Shimadzu.
Web. 2 Dec 2012.
<http://www.ssi.shimadzu.com/p
roducts/product.cfm?product=aa6
200>.
[2]"Calcium Ion-Selective Electrode."
Vernier. Vernier . Web. 2 Dec 2012.
<http://www.vernier.com/produc
ts/sensors/ion-selectiveelectrodes/ca-bta/>.
[3]Rowe, M.W; Hyman, M.; Miller, A.E.;
Javier, A.C.; Binamira-Soriaga, E ..
Quantitative analysis Laboratory
Manual. 2012.
[4]Tissue, Brian M.. "Atomic-Absorption
Spectroscopy (AA)" The Chemistry
Hypermedia Project. 02 Dec. 2012.
The Chemistry Hypermedia
Project. 02 Dec. 2012
<http://www.files.chem.vt.edu/ch
em-ed/spec/atomic/aa.html>.
[5]Wroblewski, Wojciech . "Ion-Selective
Electrodes" Chemical Sensors
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2012
<http://csrg.ch.pw.edu.pl/tutorial
s/ise/>.
11