In the Laboratory
Overcoming Matrix Effects in a Complex Sample: Analysis of Multiple Elements in Multivitamins by Atomic Absorption Spectroscopy Randy J. Arnold,* Brett Arndt, Emilia Blaser, Chris Blosser, Dana Caulton, Won Sog Chung, Garrett Fiorenza, Wyatt Heath, Alex Jacobs, Eunice Kahng, Eun Koh, Thao Le, Kyle Mandla, Chelsey McCory, Laura Newman, Amit Pithadia, Anna Reckelhoff, Joseph Rheinhardt, Sonja Skljarevski, Jordyn Stuart, Cassie Taylor, Scott Thomas, Kyle Tse, Rachel Wall, and Chad Warkentien Department of Chemistry, Indiana University, Bloomington, Indiana 47405, United States *
[email protected] Atomic absorption spectroscopy (AAS) is among the most valuable tools in analytical chemistry (1, 2). AAS is capable of analyzing samples for the presence of elements, particularly metals. The examination of multivitamin samples can facilitate teaching students this important technique and the theory behind it. Because multivitamins are consumed by millions of people worldwide, they can serve as a familiar and relevant example for the students. Multivitamins often contain relatively large quantities of calcium, magnesium, and iron and smaller quantities of zinc, copper, and manganese. The variety of elements available allows students to investigate as few as one or as many as six different analytes, depending on the availability of the appropriate light source(s) and the time allotted for the experiment. The measured quantity of each element can be directly compared to the manufacturer's label, providing students with a sense of the validity of their results, as demonstrated previously for iron (3). Other analytical methods, such as colorimetric (4) and voltammetric (5) analysis of metals, may also be used for comparison. In addition to offering a variety of different elements to be analyzed, multivitamins also present the analytical chemist with a challenging matrix. Non-elemental constituents of multivitamins may include vitamins, binders, and artificial colors. These components also provide interesting samples for analysis by luminescence (6), infrared spectroscopy (7), and high-pressure liquid chromatography (8). This matrix varies depending on the type of multivitamin, for example, coated tablets, chewable tablets, children's chewable tablets, and liquid multivitamins. The protocol presented here includes two options for removing these components that can potentially interfere in AAS measurements: vacuum filtration and solvent extraction. Although others have shown that additional procedures (9) may be used to enhance extraction of metals from multivitamins, a simple protocol described here and elsewhere (10) can be accomplished with standard laboratory equipment. Demonstrated herein is an undergraduate laboratory experiment designed for an analytical chemistry course in which students learn the theory of absorption spectroscopy, the technique to prepare samples, and operation of the instrument, as well as the concept of matrix interference. If resources allow, the experiment could also be adapted for an introductory-level laboratory to demonstrate the principal of atomic transitions 484
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or to illustrate the importance of a particular element such as calcium (11, 12). In this experiment, the students prepare elemental standards, use the linear calibration method to determine the concentration of each element in the multivitamin sample, and then use standard addition to establish the extent of matrix interference, if present. By establishing the relationship between concentration of each analyte and its absorbance, students come to understand the Beer-Lambert law. Moreover, fundamental concepts of atomic orbital structure related to absorption spectroscopy as well as saturation effects that produce a deviation from Beer-Lambert law could be included in the experiment to teach additional concepts. Materials and Methods Chemicals Calcium carbonate, CaCO3 Copper sulfate pentahydrate, CuSO4 3 5H2O Iron sulfate heptahydrate, FeSO4 3 7H2O Lanthanum nitrate hexahydrate, La(NO3)3 3 6H2O Magnesium metal, Mg Manganese sulfate, MnSO4 Zinc oxide, ZnO Centrum Multivitamin/Multimineral Supplement (Wyeth Consumer Healthcare, Madison, NJ) Centrum Multivitamin/Multimineral Supplement Liquid (Wyeth Consumer Healthcare, Madison, NJ)
Equipment This experiment requires a flame atomic absorption spectrometer and hollow cathode lamps (or other equivalent light sources) for the elements found in multivitamins: calcium, magnesium, iron, zinc, copper, and manganese. A PerkinElmer (Shelton, CT) AAnalyst 200 Atomic Absorption Spectrometer is used to acquire the data. Two multielement hollow cathode lamps, PerkinElmer Lumina Lamp Ca-Mg-Zn and PerkinElmer Lumina Lamp Co-Cr-Cu-Fe-Mn-Ni, were used. Data were acquired using a delay time of 5 s followed by triplicate integration times of 3 s each. A vacuum filtration apparatus, hot plate, sonicator, separation funnel, mortar, and pestle are also recommended.
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Vol. 88 No. 4 April 2011 pubs.acs.org/jchemeduc r 2011 American Chemical Society and Division of Chemical Education, Inc. 10.1021/ed100039v Published on Web 02/04/2011
In the Laboratory
Experimental Procedure Preparation of Standards Standards for magnesium, calcium, manganese, copper, zinc, and iron were prepared. Each standard was prepared by digesting the metal or ion in 0.1 M or concentrated nitric acid, then diluting the solutions with 0.1 M nitric acid. Solutions of known analyte concentration can be prepared via serial dilution. Refer to the supporting information for the details related to each element. Preparation of Multivitamin Samples Multivitamin tablets are first ground to a fine powder using a mortar and pestle and digested in 0.1 M nitric acid. To prevent clogging of the instrument's nebulizer, a full vitamin tablet should be diluted in 100 mL of 0.1 M nitric acid and then further diluted another 10-fold before introduction in the instrument. If desired, this mixture can be extracted three times with equivalent volumes of hexanes. The aqueous solution is filtered via a vacuum filter apparatus. Though students may omit the hexane extraction step, including this step was shown to have no effect on efficiency or precision. If it is desired that the nonpolar organic components of the sample be analyzed by another technique, this extraction with hexanes may serve as a straightforward way to separate them from the metals. Much faster filtration times were achieved without loss of sensitivity or accuracy by weighing out as little as one-quarter of the ground tablet and dissolving this smaller sample in 0.1 M nitric acid. Alternatively, liquid multivitamins can be used without filtration or solvent extraction. Although some of the elements listed above, such as calcium and magnesium, are not found in these liquid samples, they require much less preprocessing. In a shorter lab period, liquid multivitamins may be a viable option. Hazards Lanthanum and other heavy metals are toxic and should be disposed accordingly with aqueous waste. Nitric acid solutions should be prepared with care, as concentrated nitric acid is highly caustic. Digestion of metals should be performed with ventilation, as some reactions can release gaseous byproducts. Results and Discussion Using the absorption data from the standards and the multivitamin sample, students can fit the absorption data from the standards to a line and use the equation of the best fit to this data to determine the metal concentrations in the multivitamin sample. Data for iron are shown in Figure 1. A Centrum sample was prepared by dissolving one-quarter of a tablet in 100 mL of 0.1 M nitric acid followed by a factor of 25 dilution. The absorbance measured for this sample was 0.053, which corresponds to 1.47 ppm and 14.7 mg of iron per tablet. After analyzing the standards and the prepared samples in the flame AAS, it is worthwhile to repeat the experiment using standard addition (2). Comparing results from the linear calibration method and the standard addition method may reveal the magnitude of any matrix interference. The standard addition plot for iron is shown in Figure 2. The resulting concentration from this plot is 1.78 ppm, which corresponds to 17.8 mg of iron per tablet. The results, as shown in Table 1, indicate that
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Figure 1. Calibration curve for iron. Least squares best fit line is displayed.
Figure 2. Standard addition plot for iron in Centrum tablet. Least squares best fit line is displayed.
standard addition yields results that are either similar to linear calibration, as for copper and manganese, or results that are greater than linear calibration, as with magnesium, iron, and zinc. These results demonstrate the utility of the standard addition technique where all absorbance measurements are made with the matrix of the sample present. This is not the case for calibration curve measurements where only the sample contains the matrix, but the standards do not. Thus, if the matrix suppresses the ability to measure the analyte (in this case reducing the absorbance), results are artificially lower when using matrix-free standards and linear calibration. These results seem to indicate that some signal suppression from the matrix occurs for magnesium, iron, and zinc. It is worth noting that the results in Table 1 are for a single sample analysis. To make conclusive and quantitative comparisons between calibration and standard addition measurements, replicate samples should be analyzed. The releasing agent lanthanum was use to eliminate matrix interference in the analysis of calcium, resulting in a linear plot closer to what is expected for the Beer-Lambert law. Calcium is relatively strongly influenced by matrix interference. One can also simulate interference effects for calcium by the addition of phosphate (13), which is found as a counterion for calcium in multivitamins. In addition to using releasing agents, others have shown that optimization of flame stoichiometry and observation height can be used to reduce solute vaporization interference by aluminum (14) and other metal salts (15) in the analysis of calcium. Because of the need to add lanthanum and known problems with standard addition analysis of calcium (16), only linear calibration data were obtained for calcium.
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In the Laboratory Table 1. Typical Results for Single Analysis of Ca, Mg, Fe, Zn, Cu, and Mn in Multivitamins Element
Wavelength/nm
Label/ (mg/serving)b
Linear calibration/ (mg/serving)
Standard addition/ (mg/serving)
Linear Range (ppm)
Centrum Multivitamin - Tablet Ca
a
422.67
200
223.5
n/a
0-10
Mg
285.2
100
85
112
0-2
Fe
248.33
18
14.7
17.9
0-6
Zn
213.9
11
10.7
12.5
0-1
Cu
324.75
0.5
0.67
0.72
0.5-5
Mn
279.8
2.3
2.2
2.0
0-5
Fe
248.33
9
7.5
8.9
0-6
Zn
213.9
3
3.3
3.9
0-1
Mn
279.8
2.0
2.1
6.3
0-5
Centrum Liquid Multivitamin
a
b
Lanthanum was added in a 10- to 20-fold excess to obtain these results. Serving size is one tablet or 15 mL (one tablespoon) for the liquid multivitamin.
Sources of error for this experiment are encountered at multiple steps in the analysis. First, error can be introduced at the level of measuring both masses for standards and samples and volumes of diluted samples. The use of commercial standards could reduce some of this source of error. Second, the improper use or functioning of the atomic absorption spectrometer, particularly in regards to inconsistent sample uptake and flame profile, is another potential source of error. Thus, the care and quality of instrument used can affect measurement accuracy. For the results presented here, replicate absorption measurements agreed within 1% relative standard deviation or within a reasonable tolerance for lower absorbance samples such as blanks. Finally, the sample itself and the complex matrix of a multivitamin sample likely provide the largest source of error. Many of the vitamins and other organic molecules present in the sample have broadband absorption in the ultraviolet, and although these should be destroyed in the flame, incomplete combustion products may have nonreproducible absorption in the experiment. Whereas the relative error associated with measurement and instrument errors is estimated to be 2% or less for calibration and 5% or less for standard addition (assuming measurements are made in the working range of the instrument), the error from the sample is potentially much larger. The results presented in Table 1 are for a single sample analysis and do not include standard deviation or confidence intervals. Replicate analysis of the same element in the same vitamin sample is an interesting application of this experiment that could be readily adopted. Finally, with proper understanding of the errors associated with the measured quantity of each element in the sample, the values can be compared to the manufacturer's labels. In addition to the vitamins described here, Centrum Chewables Multivitamin/Multimineral Supplement (Wyeth) and Flintstones Complete Children's Multivitamin/Mulitmineral Supplement (Bayer Healthcare, LLC, Morristown, NJ) were also analyzed by these methods and produced similar results. It is expected that multivitamins other than those listed here will be amenable to these procedures. Our goals in developing this experiment included determining whether a sample with a complex matrix would be suitable for accurate quantitative analysis by atomic absorption spectrometry, establishing a sample preparation protocol that would be 486
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readily achievable in the undergraduate laboratory setting, and enabling students to work independently in sample preparation and data analysis. These goals were met as reasonable results were obtained for six different elements found in multivitamins, the entire protocol for a single element can be completed in a 3-h laboratory period, and as many as six individuals or groups of students can each be assigned to a unique analyte. When students are assigned to work somewhat independently on their own analyte, each student accepts a certain level of responsibility for their work and cannot rely on their classmates to help them resolve all problems that may arise. Nevertheless, students were observed to work together and share tips with one another for general concepts such as sample preparation and the standard addition method that are introduced in this experiment. This experiment was developed during the final five weeks of an upper-level analytical chemistry course after an initial two-week experiment written by the instructor. Student coauthors were involved not only in sample preparation and analysis, but also with preparation of the manuscript. Acknowledgment We would like to thank Indiana University for support in the development of this laboratory exercise. We also acknowledge Hyuna “Esther” Lim and Niya Sa for their helpful insights and guidance in this project. Literature Cited 1. Hieftje, G. M.; Copeland, T. R.; De Olivares, D. R. Anal. Chem. 1976, 48, 142R–174R. 2. Harvey, D. T. Modern Analytical Chemistry, 1st ed.; McGraw-Hill: Dubuque, IA, 2000. 3. Pinnell, R. P.; Zanella, A. W. J. Chem. Educ. 1981, 58, 444. 4. Atkins, R. C. J. Chem. Educ. 1975, 52, 550. 5. Herrera-Melian, J. A.; Dona-Rodriquez, J. M.; Hernandez-Brito, J.; Pena, J. P. J. Chem. Educ. 1997, 74, 1444–1445. 6. Utecht, R. E. J. Chem. Educ. 1993, 70, 673. 7. Wilk, I. J. J. Chem. Educ. 1976, 53, 41–43. 8. See, for example, Goodney, D. E. J. Chem. Educ. 1987, 64, 187– 188. Bohman, O.; Engdahl, K.-A.; Johnsson, H. J. Chem. Educ. 1982, 59, 251–252. Umagat, H.; Tscherne, R. Anal. Chem. 1980,
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52, 1368–1370. Holcomb, I. J.; Fusari, S. A. Anal. Chem. 1981, 53, 607–609. Barnett, S. A.; Frick, L. W. Anal. Chem. 1979, 51, 641– 645. Soriano, S.; Netto, A. D. P.; Cassella, R. J. Anal. Bioanal. Chem. 2007, 387, 1113–1120. White, C. Atomic Absorption Determination of Zinc and Copper in a Multivitamin 2009, http://www.scribd.com/doc/10513921/ Atomic-Absorption-Determination-of-Zinc-and-Copper-in-aMultivitamin (accessed Jan 2011). Kostecka, K. S. J. Chem. Educ. 2000, 77, 1321–1323. Bazzi, A.; Kreuz, B.; Fischer, J. J. Chem. Educ. 2004, 81, 1042– 1044.
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13. Jackman, D. C. J. Chem. Educ. 1985, 62, 161–162. 14. Dockery, C. R.; Blew, M. J.; Goode, S. R. J. Chem. Educ. 2008, 85, 854–858. 15. Hosking, J. W.; Snell, N. B.; Sturman, B. T. J. Chem. Educ. 1977, 54, 128–130. 16. Hosking, J. W.; Oliver, K. R.; Sturman, B. T. Anal. Chem. 1979, 51, 307–310.
Supporting Information Available Detailed instructions for students and notes for the instructor. This material is available via the Internet at http://pubs.acs.org.
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