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Chemistry, My Research, Public Health, Undergraduate

Atomic Absorption Analysis of Three Dietary Supplements

Andrew Tolliver and S. Lee Ware


Whether called alternative, traditional, natural, integrative, or complementary medicine, this largely dietary supplement-based multi-billion dollar industry is associated with claims of extreme efficacy based more on anecdote than evidence. Meanwhile, evidence is mounting that long-term vitamin supplementation may actually be harmful (1). We wondered whether an industry known for exaggerated claims and pseudo-scientific products was similarly sloppy with its measurements and manufacture.

Ions, vitamins, and minerals are obviously critical for our basic biochemistry and normal physiology, and while a balanced diet usually provides them in ample quantities, many people carefully monitor their intake of these critical elements and compounds to manage disease or improve athletic performance. An avid marathoner or endurance athlete, for example, must keep close track of iron intake or risk anemia. If a supplement’s contents were misreported, it could have a significant effect on health or performance for these individuals.

We chose to take three well known multivitamin supplement brands (One Source, Now, and Equate) to task by measuring the amount of iron, zinc, magnesium, and copper in each using atomic absorption spectrophotometry (AAS) to confirm or expose each brand’s reported values. Of the tools available, AAS was chosen because of its superior accuracy in metal analyte analysis.

Materials and Method


     6M sulfuric acid (H2SO4)

     Ferric Nitrate (Fe(NO3)3·9H2O)

     Copper Sulfate (CuSO4 · 5H2O)

     Zinc Chloride (ZnCl2)

     Magnesium Sulfate Heptahydrate (MgSO4 · 7H2O)


        Hot Plate

        2 mL, 4 mL, 5 mL, and 10 mL volumetric pipets

        100 mL, 150 mL, 200 mL and 400 mL beakers

        100 mL and 200 mL volumetric flasks

        150 mL Nalgene bottles

        Glass stir rod w/ rubber policeman

        Iron ring

        Glass funnel

        Wattman #1 filter paper

        Mortar and Pestle


1.       Four pills (replicates) from each of three brands were selected and weighed.  Their masses were recorded to 4 significant figures.

2.       Each pill was crushed to powder with a mortar and pestle.  After each pill was crushed, it was quantitatively transferred to a labeled 150 mL beaker.

Pestle and Mortar

Pestle and Mortar, used to grind pills to powder.

3.       50 mL of 6M sulfuric acid was added to each beaker.  The beakers were placed on a hotplate and boiled for approximately 45 minutes to digest the powdered pills.

4.       The content of each beaker was quantitatively transferred through Wattman #1 filter paper into 200 mL volumetric flasks. The flasks were brought to volume with deionized water and mixed thoroughly, then set aside for later use.

5.       The content of each 200 mL volumetric flask (containing the digested contents of one pill) was diluted to allow proper readings for the atomic absorption spectrophotometer. Four different dilutions were made from each flask, one for each element to be analyzed.

6.       To create the iron (10-fold) dilution, 10 mL of solution were taken from each 200 mL flask using a 10 mL volumetric pipette and placed into separate 100 mL volumetric flasks, which were then brought to the mark with deionized water.

Four dilutions were prepared from each pill, one for each metal analyte

7.       To create the (50-fold) dilution of zinc, a 5 mL volumetric pipette was used to carry out the same procedure as before – 5 mL of solution were taken from each 200 mL flask and placed into separate 100 mL volumetric flasks before being brought to volume with dH2O.

8.       Two dilution steps were necessary to create the magnesium (1000-fold) dilution. First, 5 mL of each solution was brought to volume in separate 100 mL volumetric flasks, from which 2 mL of 50-fold diluted solution was taken and again diluted in separate 100 mL volumetric flasks.

9.       The copper required no dilution and was poured directly into a 100 mL volumetric flask.

10.   Once these dilution steps were completed, a total of 48 samples were ready for atomic absorption spectrophotometric analysis.


To make the calibration standards, four compounds, each containing an element of interest, were dissolved and subsequently diluted into a set of 4 standards containing appropriate amounts of each element of interest. These 4 standards were dilutions of a master standard made as follows.

Standardized Dilutions

1.       For iron, 0.727 g of ferric nitrate was dissolved in 15 mL of 6M sulfuric acid, as was 1.015 g of magnesium sulfate for magnesium, 0.393 g of copper sulfate for copper, and 0.208 g of zinc chloride for zinc. All of the solutions were poured into one 100 mL volumetric flask, which was brought to volume with deionized water.  Three of the four standards were graded dilutions of this master solution.

2.       2, 4, and 5 mL samples were taken from the master solution, each diluted to 100 mL.  A blank standard containing deionized water was also used.

3.       All equipment and glassware was cleaned and work areas were reset.


Atomic Absorption Spectrophotometer, ready for analysis of samples

1.       Each standard and sample was run through the atomic absorption spectrophotometer and the absorption of each element was recorded for each pill.

2.       The collected data was recorded and analyzed.


Table 1. New Brand Descriptive Statistics

The Now brand [table 1] reported 9.00 mg of iron per pill, but our results were nearly 2 mg per pill higher [m = 10.72 mg, sd = 0.67], a significant difference [t = 5.09, p < 0.05]. Its reported per pill copper content (1.00 mg) was also significantly less than measured [m = 1.40 mg, sd = 0.06, t = 12.86, p < 0.05], as was its zinc content (15.00 mg per pill reported) [m = 17.47 mg, sd = 1.03, t = 4.79, p < 0.05]. However, Now’s reported magnesium content (50.00 mg per pill) was not significantly different from our measurements [m = 67.82 mg, sd = 12.77, t = 2.79, p > 0.05].

Table 2. One Source Brand Descriptive Statistics

The One Source brand [table 2] had a reported iron content of 18.00 mg per pill, which was very close to the measured value [m = 18.19 mg, sd = 1.02, t = 0.36, p = > 0.05].  Its zinc content was found to be 15.26 mg per pill [sd = 0.18, t = 2.83] – not significantly different from the reported value of 15.00 mg.  Magnesium content per pill, measured at 109.56 mg [sd = 15.73, t = 1.22, p > 0.05], was accurately reported as 100.00 mg. But One Source’s copper content, alone among the brands and elements we tested, was over-reported at 3.50 mg per pill, which was 0.55 mg more than measured [m = 2.95 mg, sd = 0.07, t = 15.3, p < 0.05] and produced the largest t value tested.

Table 3. Equate Brand Descriptive Statistics

The Equate brand [table 3] claimed 18.00 mg of iron per pill, but our measurements found it to be nearly 3 mg per pill higher [m = 20.86 mg, sd = 0.79, t = 7.24, p < 0.05]. Its copper content (0.50 mg per pill) was significantly understated [m = 0.58 mg, sd = 0.02, t = 9.22, p < 0.05], as was its zinc content (11.00 mg per pill) [m = 12.55 mg, sd = 0.41, t = 7.54, p < 0.05], and by more than 1 mg per pill. Finally, Equate’s reported magnesium content was 50.00 mg per pill, which differed from our measured value by almost 10 mg [m = 59.88 mg, sd = 3.77, t = 5.25, p < 0.05].


Two of the three brands studied, Now and Equate, under-reported their supplement contents, while the One Source brand’s stated values were mostly accurate, with one exception: its copper content was significantly less than advertised. And while all three brands misreported their product’s copper content, the difference between One Source’s reported and measured values was the largest of the three.

In mass production, it makes sense to report minimum values, but we were surprised by the degree to which Now and Equate under-reported their contents. Clearly, more accurate reporting is possible, as One Source demonstrates, but the cost of that smaller margin may be the occasional over-reporting of certain elements. We identified two different reporting strategies: for two companies, the plan consisted of large positive margins while reporting minimum values; the other used much more accurate reporting, but the smaller margins resulted in at least one species being over-reported.


1.            Mursu, J., Robien, K., Harnack, L. J., Park, K., and Jacobs, D. R. (2011) Dietary Supplements and Mortality Rate in Older Women: The Iowa Women’s Health Study, Arch Intern Med 171, 1625-1633

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