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
*rarnold@indiana.edu
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
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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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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
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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
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13. Jackman, D. C. J. Chem. Educ. 1985, 62, 161–162.
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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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