Determination of Calcium Content in Prime Eastern
Pharmaceutical’s Calcium Supplement (Calcinate)
Hunter H. Reagan,† Owais Sarwar†
†
Undergraduate, Department of Chemistry, Texas A&M University, College Station, TX, 77840
__________________________________________________________________
ABSTRACT: A sample of the Calcinate tablet was prepared by crushing five of the calcium
supplement tablets. A complexometric titrimetry analysis was undertaken using EDTA as the
ligand to which the calcium ion binds in a 1:1 ratio. Next, a potentiometric analysis of a solution
containing our sample using calcium solutions of known concentrations as standards was
performed. As a final technique, we used flame absorption analysis, again, using standards of
known concentration as points of comparison to the Calcinate solution. Using all three of these
methods, we determined the amount of calcium in the Calcinate tablet.
In recent years, Prime Eastern
of calcium required for the average
Pharmaceuticals has been the target of
consumer. Other ingredients (such as
multiple class-action lawsuits that allege
mineral oil, maltodextrin, etc.) are
dishonesty in the company’s labeling of
present in very small quantities. It
its products and the effectiveness of
contains no gluten, lactose, preservatives,
chemicals used in their pain-relievers.
or artificial flavors. We prepared our
Significantly, many of these lawsuits
sample by crushing five tablets into a fine
have been successful and in favor of the
powder.
consumers. Now, consumers claim that
Complexometric Titrimetry. In this
Prime Easter Pharmaceuticals has
analysis, a solution of known Ca2+
reduced the amount of calcium in its dietconcentration was prepared. It was then
supplement Calcinate tablet, again,
titrated
with
a
solution
of
without indicating this reduction on their
Ethylenediaminetetraacetic acid (EDTA)
product. Consequently, the intention of
(Figure 1) of known concentration using
this analysis was to determine the true
amount of calcium in the Calcinate tablet
and thereby validate or invalidate this
contention which, if true, is significant
Figure 1:
Anionic EDTA
not only for the ethical questions it raises
but also for the health and well-being of
Calmagite indicator. EDTA is commonly
the consumers.
used
to
determine
metal
ion
concentrations because it forms a very
stable complex with metal ions in a 1:1
ratio, regardless of the charge of the
metal ion.1 Because of these two factors,
titration with EDTA is a good method for
a quantitative analysis. The reaction for
the titration is as follows:
OVERVIEW
The Sample. The analyte used in this
analysis
was
Prime
Eastern
Pharmaceutical’s Natural Calcium Tablet
(Calcinate). It was advertised to contain
800 mg of calcium per tablet (as calcium
carbonate) with Vitamin D (as
colecalciferol). The serving size (2
tablets) is over 100% of the Daily Value
Ca2+ + H2(EDTA2-)  Ca(EDTA2-) + 2H+
1
Calmagite is used as an indicator because
it initially forms a colored complex with
the calcium ion and undergoes another
color change when the remaining calcium
is released with its reaction to EDTA.1 As
with any titration, we can determine the
amount of calcium reacted if we know
the amount of EDTA used to titrate and
the
reaction
stoichiometry.
This
knowledge can be extended to determine
the percentage of the sample which was
calcium and thereby the amount of
calcium in the tablet.
Potentiometric Analysis. Potentiometry
involves the measurement of the potential
energy of an electrode and is therefore
measured in volts (energy per unit
charge). The potential energy of the
electrode is the result of differing
activities of an ion inside and outside the
electrode’s glass membrane (i.e. the
separation of charge) and results in a
current (movement of charged molecules)
inside the glass membrane in which the
energy of the system is lowered. The
potential energy of the electrode is
measured as the amount of energy needed
to reverse the current generated through
the glass membrane.
Because the
concentration of ions inside the
membrane is constant, the amount of
potential energy of the electrode
immersed in a specific solution is a
function of the ionic concentration of the
solution. The greater the concentration of
ions, the greater the magnitude of the
potential energy of the electrode simply
because of the greater interaction
between the ions on the surface of the
electrode and the ions in solution. For
solutions in which the ionic strength is
constant, the relationship between
potential and charge can be expressed as
the Nernst equation:
29.58 is the theoretical value at twentyfive degrees Celsius and with an ion with
a charge of +2. In this analysis, we
prepared solutions of known calcium
concentration and determined their
potentials using an ion-selective electrode
(in this case, Ca2+-specific). We then
measured the potential of a solution of
the
calcium
tablet
(of
known
concentration) and used the mathematical
relationship between the logarithm of the
concentration of calcium and the
potential of the standards to determine
the amount of calcium in our sample and
thereby the amount of calcium in the
tablet.
Flame Absorption Analysis. Flame
absorption involves the vaporization of a
sample sprayed onto a flame, the
absorbance of which is then measured. It
relies on the principle that different metal
atoms absorb different and unique
electromagnetic frequencies. The amount
of radiation absorbed by a specific
sample is directly related to the amount
of metal ions in the sample. When an
atom is hit by photons from light, the
atom’s electrons jump to an excited state.
The amount of energy required to do this
is different for each atom, thereby
explaining why different elements absorb
different frequencies of light (frequency
is directly related to the energy of the
radiation). By measuring the absorbance
of a sample, we can use Beer-Lambert’s
law, which states that the absorbance is
equal to the absorption coefficient times
the path length (the distance the photons
coming into the sample have to
transverse to exit the sample) times the
concentration of the sample, to determine
the concentration of the sample. Because
we do not know the absorption
coefficient or path length, we made a set
of four standard solutions of known
E (Potential) = E (initial) + 29.58(mV*M)
log[Ca2+]
2
calcium concentration, plotted their
absorbance versus the concentration and
used the mathematical relationship to
determine the amount of calcium in a
solution of the calcium tablet.
In this experiment, we used a
Vernier calcium ion selective electrode.
The pH range of this device is between 3
and 10, it should be immersed in solution
at least 1 inch, and readings are
reproducible within 5%.2
To prepare the standards, we
weighed-out 1.000 g of CaCO3 into a 100
mL volumetric flask, dissolved it with
6M HCl dropwise, added some water,
boiled it, adjusted the pH to 6 with
NH4OH, and diluted to the mark. We
then diluted the solution further by
transferring 10.0 mL of the solution
volumetrically into another 100 mL
volumetric flask and diluted it to the
mark with deionized water. We then
prepared the standards in accordance with
the following table:
EXPERIMENTAL PROCEDURE
Preparation of the Sample. We
prepared the sample by measuring the
mass of 5 Calcinate tablets (Table 1) and
crushing them with mortar and pestle into
a fine powder.
Complexometric Titration. We first
weighed 1.0 g of EDTA salt into 250 mL
volumetric flask, dissolved it in 200 mL
of heated deionized water and diluted it
to the mark once it had reached room
temperature.
Then, we weighed 0.3 g of the
sample, dissolved it in 6M HCl dropwise
and diluted it in 100 mL of water in a 150
mL beaker. The solution was then boiled
to release CO2. Then 1M NH4OH was
added until the pH of the solution was 6.
It was then filtered and diluted with
deionized water in a 250 ml volumetric
flask.
Three 50.00 mL samples of the
calcium solution were pipetted into
separate 250 mL Erlenmeyer flasks along
with a 100 mL water blank. 15 mL of pH
buffer 10 solution was added (because
the EDTA reacts quantitatively with Ca2+
at a pH of 10) along with 6 drops
Calmagite and 4 drops methyl red. All
solutions were then titrated with EDTA
until their color turned blue.
It is important to note that 6M HCl
is corrosive and, as a preventive measure,
gloves should be worn when handling the
solution.
The amount of EDTA used to
titrate is stoichiometrically related to the
amount of calcium in the tablet solution.
Results are in Data Set 2.
Potentiometric Analysis.
Standard
mL .01M CaCO3
mL 1M KCl
1
2
3
4
1.00
2.00
5.00
10.00
10.00
10.00
10.00
10.00
To prepare the sample, we
measured .2250 g of the crushed tablet
into a beaker, dissolved it drop-wise with
6M HCl, added some water and boiled it,
adjusted the pH to 6 with ammonium
hydroxide, filtered it into a 100 mL
volumetric flask and diluted to the mark.
We then transferred 2.00 mL of the
solution and 10.00 mL of the KCl into a
100 mL volumetric flask and diluted it to
the mark.
We then transferred 60 ml of all
solutions to separate 100 mL beakers and
measured the potential of all the
solutions, rinsing the electrode with
deionized water and wiping with
Kimwipe between each trial. We
measured the potential of each solution in
two different trial runs.
The potential of the standards
verses their concentration was then
plotted for each trial and the trend line
3
generated was then used to determine the
concentration of calcium in the tablet
solution. Results are in Data Set 3.
Flame Absorption Analysis.
In this analysis, we used a
Shimadzu flame atomic absorption
spectrophotometer.
Readings
are
reproducible within 1%.3
We first prepared four standard
solutions of 5.00, 10.00, 15.00, and 20.00
ppm of calcium.
We then measured 250 mg of the
sample into a beaker, added 6M HCl drop
wise to dissolve it, added 100 mL of
deionized water to dilute it, and boiled it
to expel CO2. We then transferred it to
and diluted it in a 250 mL volumetric
flask. We then took 10 mL of this
solution and diluted it with water in a 100
mL volumetric flask. We then diluted
33.75 mL of this solution in another 100
mL volumetric flask.
To analyze, we first zeroed the
flame absorption spectrophotometer by
putting the aspiration capillary tube in
distilled water. We then put the aspiration
capillary tube into each standard and the
calcium tablet solution and recorded the
absorbance.
We then plotted a calibration curve
of the standards (absorbance verses
concentration) and extrapolated to
determine the amount of calcium in the
tablet solution. Results are in Data Set 4.
Data Set 2
RESULTS
Potential
(mV):
Standard 1
Standard 2
Standard 3
Standard 4
Sample
Table 1
Tablet
1
2
3
4
5
AVG
Mass of EDTA Sample(g)
Mass of Tablet Sample
(g)
1.0082
.2999
Tablet
Solution
Volume
EDTA
needed to
titrate
(mL)
1
2
3
48.60
46.80
46.60
Volume EDTA added
to blank (mL)
Tablet
Solution
Calcium
in
Tablet(g)
0.00
1
2
3
AVG
.629
.605
.603
.612
Sample Calculation:
Tablet Solution 1:
𝑀𝑜𝑙𝑎𝑟𝑖𝑡𝑦 𝐸𝐷𝑇𝐴: 1.0082 𝑔 𝐸𝐷𝑇𝐴
1 𝑚𝑜𝑙
1
372.24 𝑔 . 250 𝐿
= .0107 𝑀
. 0107 𝑚𝑜𝑙 1 𝑚𝑜𝑙 𝑐𝑎𝑙𝑐𝑖𝑢𝑚
40.08 𝑔
1𝐿
1 𝑚𝑜𝑙 𝐸𝐷𝑇𝐴 1 𝑚𝑜𝑙 𝑐𝑎𝑙𝑐𝑖𝑢𝑚
250 𝑚𝑙
1
= 34.74% 𝑐𝑎𝑙𝑐𝑖𝑢𝑚
50 𝑚𝑙 . 2999 𝑔 𝑠𝑎𝑚𝑝𝑙𝑒
34.74% ∗ 1.8106 𝑔 𝑇𝑎𝑏𝑙𝑒𝑡 = .629 g Calcium
. 04860 𝐿 𝐸𝐷𝑇𝐴
Data Set 3
Mass (g)
1.8169
1.8189
1.8125
1.8174
1.7875
1.8106
4
Mass CaCO3 (g)
1.0097
Mass of Sample (g)
.2319
Trial 1
Trial 2
-11.1
-2.5
8.3
17.3
6.2
-9.7
-1.6
8.5
16.5
6.0
10(6.2
Potential (mV) vs. Log[Ca2+]
20
y = 28.342x + 102
15
Potential
10
(mV) vs.
log[Ca2+]
5
-6.000
-4.000
0
-2.000 0.000
-5
-102)/28.3
= 4.17 * 10-4 M Ca2+
. 000417 𝑀 𝐶𝑎𝑙𝑐𝑖𝑢𝑚
.00208 𝑀 𝐶𝑎𝑙𝑐𝑖𝑢𝑚
Linear
(Potential
(mV) vs.
log[Ca2+])
Potential (mV) vs. Log[Ca2+]
Data Set 4
1
5.00
Standard 1
Standard 2
Standard 3
Standard 4
Tablet Trial 1
Tablet Trial 2
Tablet AVG
-6.000 -4.000 -2.000
0
0.000
-5
-10
Potential
(mV) vs.
Log[Ca2+]
Linear
(Potential
(mV) vs.
Log[Ca2+])
-15
Trial 1
Trial 2
AVG
3
15.00
0.0600
0.0400
y = 0.00214x
+ 0.0046
Absorbance
vs.
Concentrati
on Ca2+
(ppm)
0.0000
0.00 20.00 40.00
Using software
equation:
Sample Calculation:
trend
4
20.00
Absorbance vs. Concentration Ca2+ (ppm)
0.0200
Calcium in Tablet (mg)
652
630
641
Using software-generated
equation:
2
10.00
Absorbance
0.0153
0.0261
0.0365
0.0475
0.0187
0.0298
0.0243
20
15
40.08 𝑔
1 𝑚𝑜𝑙
= .08351 𝑔 𝐶𝑎𝑙𝑐𝑖𝑢𝑚
. 08351
= 36.01% ∗ 1.8106 𝑔
. 2319 𝑔 𝑇𝑎𝑏𝑙𝑒𝑡
= .652 𝑔
Concentration
of Standards
(ppm Ca2+)
Trial 1 (Top) Trial 2 (Bottom)
5
=
= .00208 𝑚𝑜𝑙 𝐶𝑎𝑙𝑐𝑖𝑢𝑚 ∗
-15
10
2 𝑚𝑙
. 00208 𝑀 𝐶𝑎𝑙𝑐𝑖𝑢𝑚 ∗ .1 𝐿
-10
y = 26.194x + 94.922
100 𝑚𝑙
generated
trend-line
Absorbance = .00214(concentration) +
.0046
line
Trial 1
.0187 (Trial 1) = .00214(concentration) +
.0046
Potential = 28.342(log[Ca2+]) + 102
(mV)
Concentration = 6.589 ppm
(6.589ppm)/(1000000)=(6.589
5
Amount of Ca2+
(mg)
1
347
2
620
AVG
484
E-6)(100L/33.75L)=(1.952E5)(100L/10L)=(1.952E-4)(250L)=
(.0488gCa2+)/(.2547gTablet)=(19.16%C
a2+)(1.81064g Tablet)= 347 mg Calcium
per tablet
theory, the analysis should be quite
precise. If the sample did not entirely
dissolve, again, this could decrease the
amount of calcium in the tablet recorded.
In the flame absorption analysis we
found the average amount of Ca2+ in the
tablet to be 484 mg. However, one of our
values was 620 mg whilst the other one
was only 347 indicating a major error.
However, using the values obtained by
all the other analyses and the Grubb’s
Test for statistical significance, we
concluded that the 347 mg reading was
undoubtedly an outlier. This error was
likely
caused
not
by
the
spectrophotometer which is highly
accurate but in the dilution process of the
sample solution, most likely in a single
step. The potential for error is somewhat
larger than in the other methods because
we filtered it multiple times and there
were three dilution steps (multiple liquid
transfers) and one of them involved
transferring a sample not volumetrically
but with a measuring pipette. Likely, the
error did not occur during the filtering
step (or else both values would have been
affected). The best explanation would be
to assume that during the transfer of
33.75 mL of the solution in the last
dilution, a significant amount of liquid
did not make it to the last volumetric
flask.
As for the relative precision of the
three methods, a paper published by the
Brazilian Society of Soil Sciences found
that within a 95% confidence interval,
there is no significant difference in the
determination of calcium and magnesium
in soil samples using these same there
analytical techniques.
Excluding the erroneous data point
from the flame absorption analysis, the
average value for the amount of calcium
in each tablet that we obtained was 623
mg indicating that the company is guilty
Trial
CONCLUSIONS
All three analyses indicated that,
indeed, the amount of calcium in the
Calcinate tablets is less than the 800 mg
advertised. The average value for the
amount of calcium in each tablet obtained
via the complexometric titration was 612
mg per tablet. For titrations in general, an
expert user should be able to replicate
results to within one percent4. Therefore,
this method is fairly accurate and precise
with only two major sources of possible
error: if the tablet sample was not
dissolved in its entirety in solution, the
EDTA needed to titrate would have been
less than stoichiometrically expected and
the value for the amount of calcium in the
tablet would have been below the actual
value. Also, because a color indicator is
used, there is always the possibility of
over-shooting the endpoint which would
lead to a higher than actual value for the
amount of calcium in the tablet.
The
potentiometric
analysis
indicated that the average value of
calcium in the tablet was 641 mg.
Readings on the electrode are
reproducible within 5%. Major sources of
errors are likely in the dissolving of the
sample and the dilution process.
However, there are only a few dilutions
that were performed and liquids were all
transferred using a volumetric pipette and
diluted in volumetric glassware so, in
6
of misinforming the consumers. In the
process they are saving roughly 22.1 %
per tablet by selling the reduced
formulation at the 800 mg formulation’s
price. The effects on the consumer can
potentially be adverse. They would
unknowingly and consistently spend
money on a product that is ineffective or
inadequate which would waste not only
their financial resources but could lead to
health complications associated with
insufficient
calcium
intake
(i.e.
osteoporosis, muscle stiffness, etc.).
ACKNOWLEDGMENT
We would like to briefly
acknowledge our T.A. Ms. Randara
Pulukkody for her consistent helpfulness
as well as Dr. Binamira-Soriaga who was
an excellent lab instructor.
REFERENCES
(1) M.W. Rowe; M. Hyman; A.E.
Miller; A.C. Javier; E. Binamira-Soriaga,
Quantitaitve Anlysis Laboratory Manual,
1983-2013.
(2)
Calcium
Ion-selective
Electrode,
www.vernier.com/products/sensors/ionselective-electrodes/ca-bta/
(3) Atomic
Absorption
Spectrophotometer AA-7000; www.tbtscitech.com/products/414-atomicabsorption-spectrophotometer--aa-7000-8e68/
(4) Volumetric Titer Determination
and
Performance
Check;
www.metrohmusa.com/support/FAQ/titra
tion/volu.html
(5) C. M. Pereira; C.A. Neiverth; S.
Maeda; M. Guiotoku; L Franciscon,
Revista Brasileria de Ciencia do Solo, 35,
4, 2011, pp. 1331-1336.
7