Calcium Content Analysis of Supplementary Tablet

Phillip Thane and three other group members (deleted for privacy)
Department of Chemistry, Texas A&M University, College Station, Texas, United States

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ABSTRACT: The purpose of these experiments was to determine the amount of calcium in Natural Calcium tablets from Prime Eastern Pharmaceuticals. EDTA titration, Calcium ionselective electrode potentiometry, flame atomic absorption analysis methods were used to determine the amount of calcium in the tablets. The results from different methods were significantly different. While results from EDTA titration were precise they were not accurate. The results from FAAS contained a standard deviation. ISE contained the most accurate data. Calcium is the most abundant element in the human body. Calcium is tightly linked to many of the roles that vitamin D plays in the body. Calcium must be constantly consumed to build bone and maintain the blood level of calcium. Taking calcium as a supplement can have a positive impact on health, playing a key role in muscle contraction, nerve impulse transmission, heart beat regulation, blood clotting, enzyme activation, and fluid balance in cells. The calcium supplement chosen to analyze in this lab is manufactured by Prime Eastern Pharmaceuticals. The manufacturer claims the supplement contains 800mg per tablet. A serving, two tablets, exceeds the daily value of calcium of 1000mg. The supplement contains 800 micrograms of vitamin D and traces of other compounds such as maltodextrin and powdered cellulose. These can be neglected in the analysis of calcium in the tablet. The three methods chosen to analyze the calcium content are EDTA (Ethylenediaminetetraacetic acid) titration, Ion-selective electrode potentiometry, and flame atomic absorption. EDTA forms stable complexes with many metal ions (Figure 1). The formation reaction for calcium-EDTA complex has a relatively large formation constant (Kf = 4.47x10^10) and the complex is quite stable. EDTA forms 6 coordinate covalent bonds with the metal ligand where both electrons of the lone pairs on the two nitrogens and four carboxyl oxygens are donated to the metal ligand. Regardless of the identity and charge of the metal ligand, the ratio of complex formation is 1:1. The formation of calcium-EDTA complex is pH-dependent. The pH must be buffered to 10 for effective chelation.

Figure 1. Metal EDTA Complex Equation 1. EDTA calcium formation reaction. Endpoint titration can be determined with

an indicator such as Calmagite. Calmagite bound to metal ions is a deep red color and blue when uncomplexed. Calmagite does not bind as strongly to the metal as EDTA and so EDTA displaces the Calmagite metal complex to assure complete titration. Methyl red indicator can be added to ensure that the pH of the solution is within the appropriate range. Titration is a rudimentary method for quantitative analysis. Therefore, it provides a good baseline for content of calcium in the tablet while being relatively inexpensive to conduct.[1] Ion selective electrodes use potentiometry to determine potential across a reference and indicator electrode. Ion-selective electrodes use a special glass membrane which can detect more than 20 different cations and anions, including calcium. The potential is logarithmically related to the activities of the half reaction according to the Nernst equation. Relative to calcium, KCl is at a much higher concentration and so almost exclusively contributes to the ionic strength, thus the activities can be substituted with concentrations.

concentrations did not change. Thus, the potential difference between the reference and indicator electrodes was solely due to changes in indicator electrode potential. [2] Because the electrode only detects ions, it is very important to fully ionize the sample. However the Nernst equation is only applicable for very dilute solutions. This is because interferences from other ions contribute to the potential difference. Potentiometry is probably the most frequently used electroanalytical method. [2] Flame atomic absorption spectroscopy (FAAS) analysis can be used to determine the calcium content in a sample by relating absorption to concentration. This method uses heat to excite electrons to higher energy levels. E = hf Equation 4 Energy equation (f = c/, h = Plancks constant). This equation indicates that the atomic spectrum is the absorption of light as a function of wavelength, where the spectrum of an atom depends on the electronic transitions between energy states. The basic principle of FAAS is that the sample is introduced into the atom cell, where it is desolvated and then atomized. The analyte atoms so formed then quantitatively absorb light in a way that is proportional to the concentration of the atoms of the analyte in the cell. The light, which is at a specific wavelength, is then isolated from other wavelengths, amplified, and then detected. [3]

Equation 2. Nernst Equation (n = number of moles of electrons transferred in half reaction, E0 = standard reduction potential, Q = reaction quotient)

Equation 3. Ionic Strength (C = concentration of ion, Z = charge of ion) For the specific ion-selective electrode used for the experiment, the reference electrode was a Ag+/AgCl half cell whose 2

Figure 2 FAAS Method The light emitted by a calcium cathode is selectively absorbed by only calcium atoms. Using Beers Law, the amount of radiation that is absorbed by the calcium can be related to the concentration.

Equation 5 Beers Law One drawback of FAAS is that only small ranges of concentrations, usually only a few ppms of ion, can be detected. For this reason, solutions must be diluted to a great extent. [4] EXPERIMENTAL SECTION Materials. The Calcium supplementary tablet was obtained from Prime Eastern Pharmaceuticals. The calcium ionselective electrode was obtained from Vernier Software & Technology. The flame atomic absorption spectrophotometer was obtained from Shimadzu Scientific Instruments. Deionized water was used as solvent to prepare all solutions. All unknown samples were boiled to expel excess carbon dioxide and filtered prior to analysis. For the complexometric titration, the analyte solutions were buffered to pH 10 with an ammonium salt/ammonia buffer. EDTA in powder form was used as the primary standard for the titration. For the ion-selective potentiometry

(ISE) the solution was set to a pH of 6 (confirmed with litmus paper) using 1M ammonium hydroxide and 6M hydrochloric acid. Calcium carbonate powder was used for the preparation of standards in each experiment. For the standards prepared for the ISE, 1M potassium chloride was added to maintain ionic strength. Various volumetric flasks, graduated pipets, beakers, Erlenmeyer flasks, graduated burets, mortar and pestle, and a digital balance were used throughout the experiments. Calmagite and phenyl red were used as indicators for complexometric titration. Complexometric Titration with EDTA (Na2H2Y.H2O;Y=C10H12N2O84-) A ~0.01M EDTA standard was prepared using approximately 1.0g of solid EDTA dissolved in 250mL of deionized water. The unknown sample was prepared by taking ~0.24g of crushed calcium tablet dissolving with hydrochloric acid. The pH was then adjusted to 6 using ammonium hydroxide. The sample solution was diluted in a 500 mL volumetric flask. The analyte solution consisted of 50.00mL aliquots, pH 10 buffer and calmagite with methyl red indicators. A blank was prepared with deionized water and indicators. The aliquots were titrated with EDTA. Calcium Ion-Selective Electrode Potentiometry. Standards were prepared using 1.0 g of calcium carbonate dissolved with 6M hydrochloric acid. The sample was diluted to 100mL to prepare an approximately 0.1M solution. The solution was diluted further to prepare an approximately 0.01M solution. The standards for the ion selective electrode were then prepared by taking 1.00, 2.00, 5.00, and 10.00 mL of the 0.01M solution. Ten milliliters of potassium

chloride were added to each standard solution and diluted to 100 mL. The calcium tablet sample was prepared by dissolving approximately 0.25g in 10 mL of water. The sample was diluted to 100 mL to prepare a ~0.0005M solution. The electric potential of the standards were measured and used to generate a calibration curve to calculate the concentration of calcium in solution. The electric potential of the unknown solution was analyzed with a calcium ion selective electrode. Flame Atomic Absorption Analysis Two trials were performed on two separately measured tablet samples. Approximately 0.3 g of the calcium supplementary tablet was dissolved in deionized water with the aid of 6M hydrochloric acid. This sample was diluted to 100 mL (0.0133 M Ca2+). 1.00 mL aliquot of the 0.0133 M solution was taken and diluted to 100 mL. Then 2.00 mL aliquot of the 0.0133 M solution was take and diluted to 100 mL. Flame atomic absorption analysis on the sample was performed on the prepared sample. RESULTS AND DISCUSSION The unknown sample used in all methods was prepared from five tablets, ground into powder and homogenized. The average mass of a single tablet was 1.7981 g. Calcium carbonate is sparingly soluble in water and will only dissolve in acidic solutions upon conversion to bicarbonate. For the analyses that required pH to be basic, all calcium must be kept in solution by removing carbonate so that calcium carbonate cannot reform and precipitate. Boiling the solution will lower the solubility of carbon dioxide gas, and this will drive the formation of more carbon dioxide from bicarbonate and ultimately carbonate such that no carbonate remains in

solution. Complexometric Titration A 0.0111 M EDTA solution was used to titrate three 50.00 mL aliquots of the unknown solution. The mass of tablet used in the sample was 0.2453 g. The volume of EDTA required to reach the endpoint (red to blue with no purple remaining) was 19.10, 19.50, 19.10, and 19.30 mL for trials one through four, respectively. This corresponds to a mass of 627 mg of calcium per tablet. Complexometric titrations with EDTA have a high formation constant and essentially proceed to completion. The stoichiometry of EDTA to any metal ligand is 1:1. It can be assumed that the amount of EDTA added to a solution is equal to the amount of metal ions present. The formation of the EDTA-metal complex is pH dependent. In the case of calcium, pH must be kept at approximately 10. This was achieved by an ammonia/ammonium hydroxide buffer. It is possible for other metal ions to be present in the water used to prepare the samples which would be evident from the color of the blank before titration. In our case, the blank was blue, which would indicate that there was no appreciable amount of metal bound Calmagite. Calmagite bound to metal ions is a deep red color, and blue in absence. Had the solution not been blue, it would have to have been titrated with EDTA until the endpoint and this volume subtracted from the unknown solution endpoint volumes. Methyl red indicator was added to ensure that the pH of the solution was within the appropriate range, red in acidic solutions, and yellow in neutral to basic solutions

Ion-Selective Potentiometry The mass of the tablet used in the sample was 0.2599 g, which corresponds to a concentrations of 4.44*10-4 M Ca2+. This is within the range defined by the standards, 10-4 to 10-3 M. The electric potentials of the standards and the unknown sample are summarized in Table 1.
Sample (M) Standard 1 (10-4) Standard 2 (2*10-4) Standard 3 (5*10-4) Standard 4 (10-3) Unknown Table 1. solutions. Trial 1 (mV) 0.5 4.2 14.4 22.2 16.8 Trial 2 (mV) -7.6 7.5 11.4 19.5 17.2 Average (mV) -3.6 5.9 12.9 20.9 17.0

value is relatively close to the theoretical value and is indicative of an ion with a positive charge of 2. If calcium were the only ion in solution, the ionic strength of each solution would vary drastically invalidating the substitution of concentrations for activities in the Nernst equation. To mitigate this, an excess of potassium chloride was added to each solution (0.1 M KCl compared to 10-3 M Ca2+ at most). Atomic Absorption Spectroscopy The mass of the tablet used in the sample was 0.3082 g, and 0.3051 g for trials one and two, respectively. Using the calibration curve data provided (Figure 2), the amount of calcium per tablet was determined to be 650 mg and 451 mg calcium per tablet, respectively.

Electric potentials for standard and unknown

The calibration curve obtained from the standards is presented in Figure 1. Using the linear relationship from this curve, the amount of calcium in the unknown solution was 960 mg per tablet.
25.0

0.35 0.3 Absorbance 0.25 0.2 0.15 0.1 0.05 0 -2 0 10 20 [Ca2+] (ppm) 30
y = 0.0171x - 0.0469 R = 0.9587 E = 23.486*log[Ca2+] + 91.204 R = 0.9889

20.0 15.0 10.0 5.0 0.0

potential (mV) -4.5

-4

-3.5

-3

-2.5

-5.0

log(Ca2+)
Figure 1. Electric potential versus the logarithm of standard solution calcium concentration for ion selective potentiometry.

Figure 2. Absorbance versus calcium concentration of standard calcium solutions for atomic absorption spectroscopy.

The slope of the calibration curve was 23.49 compared to a theoretical value of 29.58 from the Nernst equation (2.303 , where R is the gas constant, T is the temperature, F is the Faraday constant and n in the number of moles of electrons transferred in the half-reaction (2)). Our

CONCLUSIONS The calcium tablets were found to have the claimed amount of calcium within each tablet with reasonable level of error.

AUTHOR INFORMATION (DELETED FOR PRIVACY OF OTHER GROUP MEMEBERS)

September 1998, Pages 95-102, ISSN 0039-9140, 10.1016/S00399140(98)00075-7. (http://www.sciencedirect.com/scienc e/article/pii/S0039914098000757)

ACKNOWLEDGMENT This group acknowledges the chemistry department of Texas A&M University for its resources. REFERENCES (1) Jeewoong Kim, C Vipulanandan, Effect of pH, sulfate and sodium on the EDTA titration of calcium, Cement and Concrete Research, Volume 33, Issue 5, May 2003, Pages 621-627, ISSN 0008-8846, 10.1016/S0008-8846(02)01043-8. (2) "analysis, chemical." Encyclopdia Britannica. Encyclopdia Britannica Online School Edition. Encyclopdia Britannica, Inc., 2012. Web. 30 Nov. 2012. <http://school.eb.com/eb/article80817>.

(3) Steve J. Hill, Andy S. Fisher, Atomic Absorption, Methods and Instrumentation, In: Editor-in-Chief: John C. Lindon, Editor(s)-in-Chief, Encyclopedia of Spectroscopy and Spectrometry, Elsevier, Oxford, 1999, Pages 24-32, ISBN 9780122266805, 10.1006/rwsp.2000.0006. (4) Michael S Epstein, Bradley Buehler, Margaret F Bullard, Evaluation of the precision and accuracy of an automated sample introduction accessory for the flame atomic absorption spectrometric measurement of calcium in serum, Talanta, Volume 47, Issue 1, 6