Calcium Analysis by EDTA titration, Ion selective electrode potentiometry and Flame Atomic Absorption Spectroscopy. Sandra K.W. Fomete, † Katie Godfrey, † Chadley Box † †
Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States
__________________________________________________________________ ABSTRACT: Consumers of calcium tablets manufactured by Prime Eastern Pharmaceuticals have recently made allegations that, the latter has reduced the amount of calcium in their tablets without relabeling their product accordingly. Thus, to verify the claim made by consumers of this tablet, the tablet was analyzed by EDTA titration, Calcium Ion selective electrode, and Flame Atomic Absorption spectroscopy. EDTA titration concluded a result of 490.6 ± 1.901mg of calcium per tablet. Potentiometric analysis with a Calcium Ion selective electrode concluded a result of 582.8 ± 62.36mg of calcium per tablet. FAAS concluded a result of 670.6 ± 10.92mg of calcium per tablet. Therefore, the consumer’s claim was validated given that all three analytical procedures concluded an amount of calcium less than 800 mg/tablet as labeled by Prime Eastern Pharmaceuticals. Prime Eastern Pharmaceuticals claims that its manufactured Calcium tablets contain ~800mg of calcium per tablet (~1.625g). However, consumers of this tablet feel that the aforementioned claim is inaccurate. EDTA titration, calcium ion selective electrode and flame atomic absorption spectroscopy was then used to analyze the calcium content of the tablet.
present in these tablets in the form of . The three analytical procedures were conducted in a three weeks’ timeframe. Ethylenediaminetetraacetic acid, commonly known as EDTA is a very good chelating agent capable of forming stable metal complexes with ions such as and . It forms very strong 1:1 complex with most metal ions. The structure of EDTA is shown below:
The various ingredients present in the tablet were: vitamin D (as cholecalciferol), maltodextin, powdered cellulose, hypromellose, mineral oil, titanium dioxide, triethyl citrate, polysorbate 80, carnauba wax, stearic acid, magnesium stearate, and crospovidone. Also, the tablet contained no gluten, no lactose, no preservatives and no additional flavors. The analytical procedures chosen were designed to determine only the amount of present and could not be affected by other ingredients in the tablet. Calcium was
In the Calcium analysis by titration with EDTA, the EDTA added combines quantitatively with the to form the complex Ca(EDTA). Calmagite and 1
methyl red indicators were used for this analysis given that EDTA and are both colorless. The color of the tablet solution changed from wine red before titration, to blue at the end point.
then brought back down to ground state, which will absorb light at a specific wavelength. To provide a wavelength that is specific to the element, a beam of light from an element specific hollowcathode lamp is passed through the burning flame. A computer program then detects the amount of absorption by the element present.
Where: In(aq) is the indicator
Ion-selective electrodes (ISE) have a sensitive membrane capable of detecting a specific type of ion. The Nernst equation is used to relate the concentration of the measured ion to the potential of the ISE. E = E’ + 2.303 (RT/nF)*log [M] Where: R=gas constant n= ion charge T= temperature in kelvin F= Faraday’s constant
The relationship between the decrease of light intensity after it passes through the flame and the concentration is described by the Lambert-Beer law:
This equation holds true only for very dilute solutions or solutions with constant ionic strength. Ionic strength is defined as: μ= ½ Σ Zi2Ci where Zi is the charge of the ion and Ci is its concentration. Because the calcium ISE can only detect free in solution, the calcium contained in the tablet in the form of has to be converted to with the help of 6M HCl.
Where: A= absorbance = initial light intensity a=absorption coefficient number of ground state atoms in the flame l= length of the way passing the light in the flame After finding the absorbance, the concentration can be obtained using Beer’s law.
Flame atomic absorption spectroscopy (FAAS) is based on the idea that an element absorbs light at a specific wavelength. The ideas that lay the groundwork for FAAS have been around for centuries, like experimenting with refracting light and measuring different wavelengths. Flame atomic absorption requires a liquid sample. The liquid sample is mixed with a variety of combustible gases and then burned at a high temperature. After the atoms of interest are combusted and excited to a higher energy state, they are
EXPERIMENTAL SECTION For these analytical procedures, all the reagents and glass wares were provided by the Texas A&M chemistry lab department. Volumetric Titration with EDTA. 2
Erlenmeyer flasks. Approximately 100 ml of DI water was added to a fourth flask as the blank. Using a graduated cylinder, 15 ml of pH 10 buffer solution was measured and added to each flask. Also, 6 drops of Calmagite indicator and 4 drops of methyl red indicator was also added to each flask. Each flask was then titrated with EDTA from a wine-red color to a blue color endpoint. The volume of EDTA standard used in each case was then recorded in the Table 1 below.
For this analytical procedure, the following reagents and glassware were used: Disodium Salt EDTA Dihydrate, 6M HCl, ammonium hydroxide, Calcium tablet, pH 10 buffer solution, Calmagite and methyl red indicators, a 250 mL volumetric flask, three 250 mL Erlenmeyer flasks, a 500 mL beaker, a 150 mL beaker, a 100 mL graduated cylinder, a mortar and pestle, pipet, a clamp and stand and a burette.
Table 1. EDTA titration
All five calcium tablet obtained from the A&M chemistry department were ground into fine powder using a mortar and pestle. Large pieces of the outer coatings were picked out by hand and discarded (the coating contains no calcium). A 0.2035 gram sample of calcium tablet was measured out on a digital scale and placed in a 150 ml beaker. Approximately 100 ml of DI water was then added to the beaker and 6 M HCl was added drop wise until the sample completely dissolved. The solution was then heated on a hot plate to expel any present. The heated calcium solution was then allowed to cool to room temperature. Once cooled, the pH of the solution was adjusted to 6 by adding NH4OH drop wise under the hood while simultaneously checking the pH with pH paper. The solution was then filtered into a 250 ml volumetric flask and diluted to the mark with DI water. A 0.01075M EDTA standard for titration was then prepared by: measuring 1.0002g of EDTA on a digital scale and diluting with heated DI water in a 250 ml volumetric flask as a standard solution for titration. The EDTA solution was then left to cool to room temperature and then 50ml were put into the burette for titration. A 50 ml volumetric pipette was used to transfer 50 ml of calcium solution into three 250 ml
EDTA used Volume of calcium solution ml Final EDTA Reading (mL) Initial EDTA Reading (mL) Volume EDTA used (mL)
Trial 1 50
Trial 2 50
Trial 3 50
28.8
28.60
28.60
0.35
0.00
0.00
28.45
28.60
28.60
Potentiometric Titration For this procedure, the following reagents and glassware were used: calcium carbonate, 0.9996g of calcium tablet powder, deionized water, potassium chloride, five 100ml beakers, five 100ml volumetric flasks 2ml, 5ml and 10mL volumetric pipettes, and a selective electrode. Four reference standards of concentrations .000102 M, .000204 M, .000510 M, and .00102 M were prepared. The standards were prepared by diluting 0.1020 gram of in a 100 mL volumetric flask to yield a .01019M solution. For standard one, 1.00 mL of the .01M solution was pipetted into a 100 mL volumetric flask. 10.00 mL of 1.0 M KCl was added, then, the
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solution was diluted to the mark with deionized water. Similarly the remaining three standards were prepared in the proportions shown below: Standard 1 2 3 4
Volume (mL) of Volume (mL) 0.01 M CaCO3 sol. of 1 M KCl 1.00 10.0 2.00 10.0 5.00 10.0 10.00 10.0
[Ca2+] 1.02E-04 2.04E-04 5.10E-04 0.00102
Potential (mV)
Potential (mV)
-22.2
-20.9
Average Potential
Log[Ca2+]
-21.6
-3.99183
-17.0
-3.69080
-3.9
-3
-3.5
-3.29280
6.1
6.6
6.4
-2.99183
33.6
31.7
32.7
Absorption
For this procedure, the following reagents and glassware were used: calcium carbonate, 6M HCl. Calcium tablet and purified deionized water, 250mL beaker, funnel, filter paper, 2 500ml volumetric flasks, a pipette/bulb combination, 100 mL volumetric flask and two plastic sample vials. Four CaCO3 standards of concentrations: 4.0ppm, 4.8ppm, 6.4ppm, and 8.0ppm were prepared. The calcium tablet solution for trial one was prepared by measuring 0.0155g of the crushed calcium tablet into a 250 mL beaker. 6 M HCl was then added to the tablet. To remove any undissolved particles, ~50 mL of DI water was added and the mixture was filtered using a funnel and filter paper. After filtering the solution, it was then transferred to a 500 mL volumetric flask. Deionized water was added to the mark, and the solution was mixed thoroughly. The previous steps were repeated exactly using a crushed tablet mass of 0.0148 grams to produce the second FAAS sample. The sample was analyzed with the flame atomic absorption spectrophotometer. Samples from trial one and trial two were poured into small cuvettes to be measured. The standards were passed through the FAAS and the absorbance were recorded on the computer. Similarly, the two calcium tablet solutions prepared above
Table 2. Ion selective electrode Trial 2
-17.1
Flame Atomic Spectroscopy
The unknown solution was prepared by adding 10.0 mL of 1M KCl to 0.9996g of calcium tablet, diluting with DI water in a 100 mL volumetric flask and mixing thoroughly. Data collection for the various potential was done using the ion selective electrode and the Visionlite software. 60 mL samples of each of the four standards and the unknown solution were placed in five different 100 mL beaker. The potential of each of the standards was then measured; starting with the least concentrated and making sure the electrode was rinsed and dried using Kim wipes before placing in the next solution. Care was taken during drying to avoid destroying the electrode’s sensitive membrane. The potential of the calcium solution was equally measured. The potential of each solution was measured twice to obtain an average and reduce the margin of error. The voltages of the standards were then plotted against the log([Ca2+]) in excel to produce a calibration curve (see Table 2 below). Then, the calibration curve equation was used to find concentration in the calcium solution. Trial 1
-16.8
4
% Ca2+ was calculated for each trial using equations (1)-(4) and results were tabulated below:
were passed through the FAAS machine and the absorbance was recorded. The absorbance of the standards were plotted against the [Ca2+] in ppm (using excel). This calibration curve was then used to find the concentration of calcium in our prepared tablet solutions. See Table 3 below
Vol. EDTA standard used
% Ca
Trial 1
28.45 ml
30.11%
Trial 2
28.60 ml
30.27%
Trial 3
28.60 ml
30.27%
Table 3. FAAS CaCO3 Standard (ppm) 4 4.8 6.4 8 Sample 1 Sample 2
Absorbance 0.0338 0.0436 0.0616 0.0767 0.1239 0.1270
After completing three trials with EDTA titration, a mean of 490.6±1.90 mg per calcium tablet was found using equation (5). Average %
St. dev. %
Average mass
St. dev.
30.19%
0.111 %
490.60 mg
1.90 mg
RESULTS & DISCUSION Analysis by Ca2+ selective electrode Analysis by EDTA titration
The calcium ion selective electrode (ISE) is used to determine concentration using a proportion between concentration of calcium and the electric potential. A linear calibration (see graph below) plot of the electric potentials for the standards was used to determine the relationship
During EDTA titration, the amount of metal ion in the solution can be determined by the volume of EDTA that is added to reach the endpoint, since EDTA and the metal ions are in a 1:1 molar ratio. (See equation (2))
Electric Potential(mV)
Electric Potential for Standard Solutions y = 28.809x + 91.672
(5)
Ca [mg]=
6
103
5
R² = 0.9808
10 0 -10
-5
-4
-3
-20 -30
Log([Ca2+])
-2
Absorban ce
Mass [g] of Ca
Mass [g] of tablet used
% Ca2+
Trial 1
.1239
6.1775*10-3
0.0148
41.74 %
Trial 2
.1270
6.3224*10-3
0.0155
40.79 %
between potential and concentration. Thus the fit of the calibration curve can be given as: (6)
2+
mV=28.809*log([Ca ])+91.672
By rearranging (6), we get an expression to solve for the concentration of in the 10 ml aliquot:
Analysis by FAAS Flame atomic absorption was used to identify the relationship between absorption and concentration, which is described by Beer’s Law:
Equation (7), was then used to determine the concentration of calcium in the unknown sample.
A linear calibration curve was plotted; using concentration of the standards and their absorbance, as seen below:
Abosrbances for Standard y = 0.0107x - 0.0083 Solutions
mV
Mass of calcium
% calcium
Trial 1
33.6 mV
0.3857 g
38.58%
Trial 2
31.6 mV
0.33139 g
31.153%
Absorbance (nm)
Equations (8)-(10) were then used to find the mass of Ca and % Ca for trials 1 and 2 as shown below:
Average mass
St. dev
35.87%
3.84%
582.83 mg
62.36 mg
0.06 0.04 0.02 0 5 7 Concentration (ppm)
Thus, the fit of the calibration curve can be given as: (11)
St. dev %
0.08
3
The average % Ca from ISE was used with equation (5) to determine there was 582.83 ± 62.36 mg per calcium tablet. Average %
R² = 0.9972
0.1
2+
Absorbance=0.0107*(ppm of Ca ) - 0.0083
Rearranging (11) we get an expression to solve for ppm of Ca2+ in diluted sample:
6
9
Equation (12) was then used to determine the concentrations in the two unknown samples.
Summary Table
Equations (13) and (9) were used to find the mass and % Ca2+
The average % Ca2+ of the two trials was used with equation (5) and yielded an average of 670.56 ± 10.92 mg per tablet. Average %
St. dev. %
Average mass
St. dev. mass
41.265%
0.6718%
670.56 mg
10.92 mg
Standard deviation
EDTA Titration
490.60 mg
1.9013 mg
Ion Selective Electrode
582.83 mg
62.3587 mg
Flame Atomic Absorption
670.56 mg
10.92 mg
Combined
581.33 mg
31.6823 mg (pooled)
% difference of analyzed calcium from labeled amount is given by: [(800-581)/800] *100 = 27.33%
One of the possible sources of errors we neglected to take into consideration was systematic error. Since, systematic errors such as improper calibrations could not have been completely removed; it was ignored in these analytical procedures. Also, some random errors such as, loss of sample during dilution due to adhesive forces between the glassware and the sample molecules might have let to small error in recording volumes.
Analysis of All Methods & Conclusion The average mass per tablet of all three methods is tabulated below (see summary table) along with the average of all three methods’ averages, or the combined average of the results. Standard deviation for three different methods is calculated using a pooled standard deviation as shown in equation (14) below: (14)
Average mass per tablet [mg]
Furthermore, In order to reduce the margin of error, each experimental procedure was repeated at least twice in order to obtain an average. Also, analyzing the Calcium content of the tablet using three different experimental procedures and taking an average of the overall calcium content reduces systematic error affiliated with a particular method (set of instruments).
Pooled standard deviation [mg]
=
Where n is the number of samples in a method and s is the standard deviation.
Conclusively, Prime Eastern Pharmaceuticals is mislabeling its calcium tablets; hence, consumer’s claim is valid. Prime Eastern claims that its 7
calcium tablets contain ~800mg of calcium per tablet. However our analytical results in Summary table show that those tablets contain an amount of calcium less than the labeled amount. This mislabeling by Prime Eastern might have repercussions on consumers in need of high calcium levels. This wrong labeling may aggravate illness such as Osteoporosis (in adults) and Rickets (in children) who consume the wrong dosage of calcium per day. AUTHOR INFORMATION S. Fomete:
[email protected] K. Godfrey:
[email protected] C. Box:
[email protected]
ACKNOWLEDGMENT Sandra, Katie and Chadley acknowledge the financial support of the Chemistry department at Texas A&M University. We equally acknowledge the support provided by Dr. Soon Mi Lim of the department of chemistry at Texas A&M. REFERENCES (1) M.W. Rowe, M. Hyman, A.E Miller, A.C Javier, E.Binamira-Soriaga, S.Lim. Quantitative analysis Laboratory Manual Fall 2013, 21-24. (2) A. Bazzi, B. Kreuz, J. Fischer; Determination of Calcium in Cereal with FAAS, Journal of Chemical education, ol 81, 2004, 1042-1044. (3) Ryan A. Verdin, Julie A. England, Myesha M. Crosby, Andrew F. Calcium tablet Analysis for percent Calcium. (4) Charles Sturt University Chemistry department. FAAS figure.
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