Atomic Absorption Analysis of Calcium and Magnesium in the Lillian Anderson

Arboretum
Alexandra L. Gothard, Agust A. Olafsson, Pornkamol Huang, Maythita Eiampikul, Dr. Jennifer
R.W. Furchak*, Dr. Kari Anderson*
June 5, 2014

Chemistry Department, Dow Science Center, Kalamazoo College, 1200 Academy Street, Kalamazoo, Michigan
49006, United States

Abstract.
Metals found in water samples are not uncommon; however when found in high
concentrations they can be dangerous. Metals can have a serious detrimental effects both on
ecological and human health. Calcium and magnesium are two metals which account for the
hardness of water, and may have adverse effects in high concentrations. In this study the levels
of calcium and magnesium were monitored for two sites in the Lillian Anderson Arboretum,
over an 8 week period, using atomic absorbance spectroscopy. The analytical method showed
that on average the concentration of calcium was both higher and significantly different in site 1
compared to site 2. The method showed that magnesium also on average had a higher
concentration in site 1 compared to site 2, and was generally statistically different. Furthermore
the weekly concentrations were observed of both magnesium and calcium was typically observed
to be statistically different over the 8 week period. According to the state wide averages the sites
are at risk for harmful effects from high calcium and magnesium.

Introduction.
Metals found in water samples are often the byproduct of geological processes or
biological cycles which result in naturally occurring low concentrations of dissolved metals in
water. These metals can be assimilated into organisms, and some are necessary for survival.
However when found in high concentrations, certain metals may be toxic to aquatic
environments, as well as affect the overall health of the ecosystem. These effects may be direct,
such as lead poisoning, or indirect, such as turnover prevention due to increased solution density.
Therefore the concentration of various metals in an aquatic system is an important aspect of
water quality.
There are many metal ions that can be found in aquatic systems, and each of them are
used to observe different aspects of environment health. Because of this the Environmental
Protection Agency regulates and sets guidelines for acceptable levels of each ion. Calcium and
magnesium are two such ions that have guidelines, and are often used as a measure of the
hardness of water. In general calcium is more abundant in natural water systems compared to
magnesium; and typically a ratio of 4:1 Ca:Mg is observed in surface waters. Thus when the
concentration of each ion increases, the hardness also increases.
Calcium and magnesium are important to human health, another reason they are
monitored by the EPA. The role of calcium in bone development and neuromuscular excitability,
as well as heart muscle contraction, is paramount.
1
Calcium deficiency can lead to osteoporosis
Magnesium is an important cofactor and activator for over 300 enzymes, and is a natural
antagonist of calcium
1
. Magnesium deficiency has been linked with pathological conditions such
as hypertension and cardiac arrhythmia
1
. Magnesium helps the body to metabolize. Furthermore,
the ratio of both metal ions is essential for plant-growth.
Because ions may exist in several charge states in aqueous environments, atomic
absorbance spectroscopy is an effective technique for analyzing metal concentrations in water
samples. The linear relationship between concentration and signal allows calculation for
concentration to be done easily, typically with low error. This technique also has a high
specificity for determining the concentrations of only the metal of interest. Atomic absorbance
also has the ability to determine the concentrations of multiple analytes in a single solution.
In this study, atomic absorption analysis was used in order to determine the
concentrations of calcium and magnesium present in the surface water composition of two
wetland sites in the Lillian Anderson Arboretum in Kalamazoo, Michigan. The calcium and
magnesium concentrations are being monitored to observe the effects of a recently developed
subdivision which is adjacent to the arboretum wetland site, in order to determine if there have
been any adverse effects.

Experimental.

Table I. Metal concentrations (ppm) used for the Site samples and QC controls standard solutions in the AAS analysis of metals
in wetland water.
3

Sample Metal Concentration (ppm)
Blank 500 ppm K and 1000 ppm La
Calibration Standard 200 ppm Ca, 100 ppm Mg, 500 ppm K,
and 1000 ppm La
Quality Control 10 ppm Ca, 10 ppm Mg, 2ppm Pb, 500
ppm K, 1000 ppm La
Blind Quality Control Unknown concentration (Ca, Mg, Pb, K,
La)
Site 1 Water Unknown concentration
Site 2 Water Unknown concentration
Matrix Spike Add 5 ppm Ca to site 1 water
Add 15 ppm Ca to site 2 water

Chemicals and materials. Reagents were purchased from Sigma Aldrich (St. Louis, MO) and
used without further purification. Water samples were collected biweekly for 8 weeks starting on
April 7
th
, 2014. Samples were collected from two different wetland sites within the Lilian
Anderson Arboretum Kalamazoo, Michigan. Site 1 samples were collected from the road side
and site 2 samples were collected from the outflow. The water samples were collected and
filtered through a 0.45 L filter before being used.

Calibration Standard Preparation. The standard solutions were prepared by pipetting the
appropriate volumes of the stock solutions of Ca, Mg, K, and La in order to yield a solution with
100 ppm Mg, 200 ppm Ca, 500 ppm K, and 1000 ppm La. The standard solution was then auto-
diluted by the SpectrAA Varian 220FS (Palo Alto, CA). with Millipore water to give
concentrations of 1, 3, 5, 7, 10, 13, 15, 17, 20, and 30 ppm Ca and 0, 1, 2, 3, 4, 5, 7, 10, 13, 15,
and 20 ppm Mg. A calibration blank was also prepared, which contained 500 ppm K and 1000
ppm La.

Site Sample Preparation. In order to prepared the site samples, 20 mL of each site water was
transferred to a 25 mL volumetric flask and had K and La added to give concentrations of 500
ppm K and 1000 ppm La. A drop of 12 M HCl was added to each solution and then they were
diluted to the mark with Millipore water. If a matrix spike was performed, 5 ppm Ca was added
to site 1 and 15 ppm calcium was added to site 2.

Atomic Absorbance. The atomic absorbance was read by a light emitted from a Ca/Mg/Al hollow
cathode lamp. The sample was nebulized and burned in an acetylene/O
2
flame, which had an air
flow of 13.5 L/min and an acetylene flow of 2.00 L/min. All samples had 500 ppm K for ion
suppression and 1000 ppm La as a releasing agent added to them. Calcium and magnesium
signals were read separately, each of them in triplicate. Between each sample the system was
rinsed with Millipore water. The calcium signal was read at 422.7 nm with a slit width of 0.5nm
and a lamp current of 10.0 mA. The magnesium signal was read at 285.2 nm with a slit width of
0.5 nm and a lamp current of 4.0 mA.

Results and Discussion: Because of the dangers of high concentrations of metals to aquatic
organisms and ecosystems, their analysis is essential for assessing the water quality and
environment health. The concentrations calcium and magnesium in two wetland sites in the
Lillian Anderson Arboretum were quantified over an 8 week period using atomic absorbance
spectroscopy. Calibration curves were first generated for each analyte by measuring the atomic
absorbance of the metal (fig. 1,2).


Figure 1. A representative standard curve of calcium concentration read by atomic absorbance spectroscopy in an O
2
/acetylene
flame, reading absorbance at 422.7 nm. The calibration curve had a linear dynamic range of 1 to 30 ppm calcium and the limit of
detection was X. y=0.0029 + 0.004, R
2
= 0.9955.
y = 0.0029x + 0.004
R = 0.9955
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.1
0 5 10 15 20 25 30 35
M
e
a
n

A
b
s
o
r
b
a
n
c
e

[Calcium] (ppm)

Figure 2. A representative standard curve of magnesium concentration read by atomic absorbance spectroscopy in an
O
2
/acetylene flame, reading absorbance at 285.2 nm. The calibration curve had a linear dynamic range of 1 to 15 ppm
magnesium and the limit of detection was X. y=0.0161x + 0.0051, R
2
= 0.9972.

The calibration curve for the concentration of calcium was linear from 1ppm to 30 ppm
calcium, which included the all of the standard solution points. Using this method, the limit of
detection was then calculated to be X ppm, and the limit of quantification was determined to be
X ppm. This signifies that all of the calibration points were measured with reasonable accuracy.
The same analysis was used on the calibration curve for the concentration of magnesium. The
magnesium calibration curve was linear from 1 ppm to 15 ppm magnesium, where the 20 ppm
calibration standard showed a 8% deviation from linearity, thus being outside of the linear range.
The magnesium calibration curve then showed that the atomic absorbance method had a limit of
detection of X and a limit of quantification of Y. Each respective calibration curve was then used
to analyze the concentrations of samples from each site as well as a 10 ppm Mg, 10 ppm Ca
quality control and a blind quality control sample.
In order to ensure a valid representation of calcium and magnesium concentrations at
each site, parameters were used to assess whether data was prime for analysis. Experimental QC
concentrations that had a % difference > 60% from the known QC concentration, that were
values outside the LDR, and/or that had values below the LOD were cause for dismissal of
calculated sample site concentrations.
After priming the data for analysis, statistical tests were performed to compare analyte
concentrations between sites within each week (fig x). A 2-mean t-test showed that the
concentration of calcium was significantly different between site 1 and site 2 for each week (t
values?). The concentration of magnesium was significantly different between site 1 and site 2,
with the exception of the 4/21 2-week period (t values).

Table 2. Table showing the calculated t values from a 2-mean t-test comparing concentrations of each analyte between site 1 and
site 2 for each week over an 8-week period at the 95% confidence level. Calculated t values about students t test values were
statistically significant. No usable magnesium concentrations were collected for 5/5.
Calcium Magnesium
Week t value Week t value
0.3011
y = 0.0161x + 0.0051
R = 0.9972
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0 5 10 15 20 25
M
e
a
n

A
b
s
o
r
b
a
n
c
e

[Magnesium] (ppm)
4/7 52.0 4/7 4.77
4/21 19.2 4/21 0.520
5/5 21.9 5/5 -
5/19 14.7 5/19 39.2

Next, a 2-mean t-test was used to compare analyte concentrations for one site, week by
week (fig). Analysis revealed the concentration of calcium was significantly different at both
sites for each week, with the exception of 4/21 and 5/5 (t values). The concentration of
magnesium at both sites was not statistically different between 4/7 and 4/21, but was
significantly different between 4/21 and 5/19 (t values).

Table 3. Table showing the calculated t values from a 2-mean t-test comparing concentrations of one site from week to week
over an 8-week period. Calcluated t values greater than the students t test values at the 95% confidence level were determined to
be significantly different. No usable concentrations were collected for 5/5.

Calcium Magnesium
Week Site 1
t value
Site 2
t value
Week Site 1
t value
Site 2
t value
4/7 to 4/21 4.25 0.754 4/7 to 4/21 0.028 0.064
4/21 to 5/5 11.23 24.21 4/21 to 5/5 - -
5/5 to 5/19 11.27 30.57 5/5 to 5/19 9.077 7.367



0
100
200
300
400
500
600
700
800
C
a
l
c
i
u
m

(
p
p
b
)

Site 1
Site 2
4/7 4/21 5/5 5/19

On average, the concentrations of calcium at site 1 and site 2 drastically exceeded the
statewide median of 50 ppm. The average concentrations of magnesium for site 1 and site 2
remained similar to the state wide median for the first four weeks, and the proceeded to spike
above the statewide average for the last 2 weeks of the 8-week period. From these results, it
would appear that the wetland sites at the Lillian Anderson Arboretum are at risk for toxic
effects. However, the data was rather limited and with high variance from week to week. Factors
such as changes in precipitation, pH and individual laboratory group techniques could have
contributed to the variance in the data.


Conclusion.
Metal composition in surface water can be an important aspect of aquatic environment
health. Calcium and magnesium contribute majorly to the hardness of water, and may have
adverse effects on human health as well as negatively affect an ecosystem. Through the use of
atomic absorbance spectroscopy the levels of calcium and magnesium were monitored over an 8
week period at the Lillian Anderson Arboretum. Based on the experimental results, the
concentration of each calcium and magnesium was typically higher in the roadside site (site 1)
compared to the outflow site (site 2). It can be hypothesized that there is a natural filtering effect
provided by the marsh area, decreasing the site 2 concentrations. Statistical analysis also
determined that the concentration was significantly different on a biweekly basis. Based on the
state wide averages the calcium concentration far exceeds the average concentration. Magnesium
concentration spiked heavily, making it also much higher than the state wide average. Based on
the EPA guidelines both sites are at risk for high calcium and magnesium concentration.


Acknowledgement
We would like to thank Dr. Jennifer R.W. Furchak and Dr. Kari Anderson for their guidance
throughout the experiment; Joe Widmer and Virginia Greenberger for assisting us with the
instruments during the wet laboratory; our fellow colleagues for sampling the water from the
arboretum and performing the data analysis; and the Kalamazoo College Department of
Chemistry for the use of their laboratory and instrumentation.


0
100
200
300
400
500
600
700
800
C
a
l
c
i
u
m

(
p
p
b
)

Site 1
Site 2
4/7 4/21 5/5 5/19
References
1. Kozisek, F. National Institute of Public Health. 2003. 1-3.
2. N.J. Miller-Ihli Trace element determinations in foods and biological samples using
inductively coupled plasma atomic emission spectrometry and flame atomic absorption
spectrometry. J. Agric. Food Chem., 1996, 44 (9), pp 26752679 DOI:
10.1021/jf950616l
3. Furchak, J. Spectrometric detection: atomic absorption analysis of metals, Laboratory
Manual: Analytical Chemistry:40-43





Sample Calculation for QC Sample from Week 4

For Calcium:
Calibration curve y=mx+b
y=0.0103x +0.0137
0.0835 = 0.0103x + 0.0137
x = [Ca+] = 6.8076

Use output of LINEST on Excel to calculate Sy
Slope Intercept
Parameter 0.01025244 0.013705
Sm 0.00021736 0.003199 Sb
R^2 0.99641707 0.005763 Sy

Standard Deviation Calculation:

||

=0.3866 0.4

The concentration of QC sample with standard deviation is 6.80.4 ppm.
Supplemental Information.
The limit of quantification was determined by where m is slope

The limit of detection was determined by

The concentrations were calculated based on the following linear calibration equations
Calcium
Magnesium

Sample Calculation:

The standard deviation in the LOQ and LOD were calculated using the equation:

The t value was calculated using the s
pooled
and t-test equation:

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