Activity

Cite This: J. Chem. Educ. XXXX, XXX, XXX−XXX pubs.acs.org/jchemeduc

Activity Analysis of Iron in Water Using a Simple LED

Spectrophotometer
Benjamin J. Place*
Chemical Sciences Division, National Institute of Standards & Technology, 100 Bureau Drive, Gaithersburg, Maryland 20899,
United States
*
S Supporting Information

ABSTRACT: Access to clean water is a vitally important part of the

lives of all people, yet there is often a concern about the contamination
of water by heavy metals. The detection and measurement of heavy
Downloaded from pubs.acs.org by WEBSTER UNIV on 02/15/19. For personal use only.

metals in water can be performed using colorimetric reagents paired

with ultraviolet−visible light (UV−vis) spectrophotometry. The
presented activity includes construction of a simple, inexpensive
single-wavelength spectrophotometer using LEDs as the light source
and detector. Using a colorimetric reagent, 2,4,6-tripyridyl-s-triazine,
the concentration of iron can be measured by the LED spectropho-
tometer. Multiple natural water samples were collected and analyzed
by this technique, and the performance of the LED spectrophotometer
was compared to that of a conventional UV−vis spectrophotometer.
The LED spectrophotometer had comparable results to those of the
conventional UV−vis spectrophotometer while teaching students about simple circuit design, spectroscopy, and water quality
J. Chem. Educ.

measurements.
KEYWORDS: Analytical Chemistry, Environmental Chemistry, High School/Introductory Chemistry,
First-Year Undergraduate/General, Hands-On Learning/Manipulatives, Computer-Based Learning

■ INTRODUCTION
Water is a vital part of everyday lives, and the access to clean
water can be a concern as the result of changes to municipal
water treatment or natural disasters.1,2 According to the United
States Geological Survey, the total withdrawal of water by the
U.S. is approximately 1.3 trillion liters of water per day
(including irrigation and mining), with the total public-
supplied water use at 159 billion liters of water per day.3
With such a large dependency of the U.S. on water, it is
important that the water has low levels of toxic chemical
contaminants. Under the authority of the Safe Drinking Water
Act 40 CFR 141, the U.S. Environmental Protection Agency
(USEPA) sets regulatory standards for drinking water; the
USEPA requires maximum concentration limits on toxic metal
concentrations in drinking water, including arsenic, lead,
chromium, and mercury.4,5
The compound of interest for this activity is iron in natural
waters. There is no regulatory limit to the concentration of
iron in drinking water,4,5 but there can be nontoxic effects from
water with high concentrations of iron, such as damage to
plumbing fixtures (ref 6, pp 170−224). In addition, there is a
wide range of iron concentrations in natural waters,6 which
allows the activity to provide varying results with different
water sources without the need for spiked water samples. The
purpose of this activity is to teach students about water quality
via spectrophotometry and environmental analytical chemistry.
Through the creation of a simple and inexpensive spectropho-
This article not subject to U.S. Copyright.
Published XXXX by the American Chemical
Society
tometer and a fast, observable reaction with an iron indicator,
students can measure the concentration of iron in natural
waters, directly linking them with a natural, relevant
phenomenon.
The construction of the light-emitting diode (LED)
spectrophotometer introduces students to the concept of
analytical instrumentation, including circuit design and spec-
troscopy. Spectrophotometers with a single multiwavelength
light source require a diffraction grating to isolate single
wavelengths.7,8 Student users of these commercial spectropho-
tometers may see them as “black boxes” with complicated
circuitry, many mechanical parts, and software that returns
interpreted data. In contrast, the LED spectrophotometer is a
simple design that gives students an intimate look at the
function of a spectrophotometer and may provide a better
understanding of the interaction of light with a solution. LEDs
can be manufactured to emit light over a narrow range of
wavelengths, and LEDs are commercially available and
relatively inexpensive. A unique feature of LEDs is their
function as both a light source and detector at similar
wavelengths, as demonstrated in other published LED
spectrophotometer activities.9,10
Received: July 3, 2018
Revised: January 28, 2019
A DOI: 10.1021/acs.jchemed.8b00515
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Journal of Chemical Education
Figure 1. Graphical display (top) and electrical circuit diagram (bottom) for the LED spectrophotometer.
■ HAZARDS
The organic chemicals and nitric acid should be handled and
disposed according to their respective safety data sheets.
Goggles and gloves should be worn at all times while handling
solutions. While the current and voltage from the battery pack
with 2 AA batteries are low, care should be taken to make sure
the wires are placed into the breadboard before the battery
pack is turned on.
■ EXPERIMENTAL SECTION
LED Spectrophotometer Construction
The spectrophotometer consists of a miniature breadboard,
two LEDs (bright yellow; 585−590 nm maximum emission
wavelength), four wire connectors with alligator clips, a AA
battery pack for two AA batteries in series (with on/off
switch), one 100 Ω resistor, and a multimeter capable of
measuring 0−2 V, which were acquired from Spark Fun
Electronics (Niwot, CO). Polystyrene cuvettes were purchased
from VWR International, LLC (Radnor, PA). The con-
struction of the LED spectrophotometer is detailed in the
Supporting Information. Briefly, the cuvette is glued to the
center of the breadboard, and the LEDs are placed on opposite
sides of the cuvette and bent to 90° (parallel to the
breadboard) so that the top of the LEDs are aimed directly
at each other through the cuvette. The wires with alligator clips
are placed to create a circuit between the detector LED and the
multimeter, and between the battery pack, the resistor, and the
source LED. A graphical display and circuit diagram are shown
in Figure 1.
The typical range for the voltage response is between 0 and
200 mV, but this is dependent on the position of the LEDs and
the brightness of the source LED (which can vary by
manufacturer). With distilled water in the cuvette, the LEDs
can be carefully moved until the output voltage is as high as
possible, which indicates the optimal position (highest
transmittance of light).
B DOI: 10.1021/acs.jchemed.8b00515
Activity
The approximate cost for a single spectrophotometer was
$9.00, not including batteries or the multimeter.
Calibration Solutions
An iron(II) [Fe2+] stock solution (nominally 100 mg/L) was
made in the lab using laboratory distilled water, and nitric acid
was added for a nominal concentration of 1.5% (mass percent)
nitric acid. Solutions of 100 mg/L Fe2+ are commercially
available. Calibration solutions were prepared by volumetric
dilutions in laboratory distilled water. The procedures are
described in the Supporting Information; the calibration
solutions concentrations were 1, 0.5, 0.04, and 0 mg/L Fe2+
in water.
Collecting the Water Samples
For the present study, water was collected from five different
locations in Maryland: a river, a pond, water from a household
well (the sample was taken prior to household water
treatment), and Atlantic Ocean coastal water. The water
samples were collected in polypropylene bottles and stored in a
refrigerator until analysis. In addition to the natural water
samples, a laboratory-spiked water sample was prepared by
adding the stock solution to distilled water to create a 0.2 mg/
L solution and labeled as an unknown spiked solution.
Measuring Iron in the Solution
The iron indicator reagent (2,4,6-tripyridyl-s-triazine; TPTZ)
was purchased from the Hach Company (Loveland, CO) in
reagent powder pillows for 25 mL water samples. In addition
to the iron indicator, the reagent powder includes a reducing
agent that converts all precipitated or suspended iron to Fe2+.11
In most aquatic systems, the dominant form of ionic iron is in
the Fe2+ state, although in highly oxidizing and acidic
conditions, Fe3+ can be present (ref 6, pp 100−103). The
structure and reaction of TPTZ with Fe2+ are shown in Figure
2. The maximum applicable Fe2+ concentration for the reagent
is 1.8 mg/L.11 The absorption spectrum for the iron-TPTZ
coordination compound is shown in Figure 3, with the
maximum absorption between 580 and 600 nm.
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Journal of Chemical Education
Figure 2. Observed reaction between Fe2+ and the TPTZ iron
indicator reagent. The resulting coordination compound is an
observable violet color.
To prepare samples, one packet of the iron indicator reagent
was added to 25 mL of each calibration solution and water
sample. The solutions were shaken briefly and allowed to sit
for at least 3 min before analysis. Sequentially, the calibration
solutions and samples were added to the cuvette (with distilled
water rinses between each measurement), and the measured
voltage was recorded. Each solution was measured three times
to determine the variability of the detector response. In
addition, the measured voltage of distilled water (no reagent)
was recorded to determine V0 (see equation below). As
described by the Beer−Lambert Law, the light intensity
through a solution at a specific wavelength is
I
log10 0 = ε × b × c
I (1)
where I is the intensity of the light through the test solution, I0
is the intensity of the light through the solvent without the
compound of interest, ε is the absorptivity of the compound of
interest at the specified wavelength, b is the path length of the
cell (often 1 cm), and c is the compound concentration of the
test solution. The value on the left side of the equation is
equivalent to the absorbance (A), which is the typical response
reported by a conventional UV−vis spectrophotometer. As the
voltage produced by the LED detector is proportional to the
intensity of the light detected, the measured voltage can be
substituted for the intensities, therefore
Figure 3. Absorption spectra of 1 mg/L Fe2+ solution with TPTZ (orange solid line) and distilled water (blue dotted line).
C DOI: 10.1021/acs.jchemed.8b00515
Activity
V0
log10 =ε×b×c
V (2)
where V0 is the measured voltage response of the LED detector
from the light through the solvent (distilled water) without the
compound of interest, and V is the measured voltage response
of the LED detector from light through the solution with the
compound of interest.
Conventional UV−Vis Spectrophotometer
To evaluate the performance of the LED spectrophotometer,
the calibration and sample solutions described above were
analyzed using a Cary 100 UV−vis spectrophotometer (Agilent
Technologies, Santa Clara, CA). The absorbance of the
solutions was measured at 585 nm with spectral bandwidth of
1.5 nm and collection time of 0.1 s.
■
RESULTS
The results from the calibration solutions, using both the LED
and conventional spectrophotometers, are shown with linear
regression statistics (y = mx + b; with the slope m and the
intercept b) and calibration curves in Table 1 and Figure 4,
Table 1. Comparison of Linear Regression Results of the
Calibration Curve of the Two Spectrophotometers
Spectrophotometer
Statistic LED b
UV−Visc
a
Slope 0.36 0.46
Intercept 0.013 0.0061
Correlationd 0.99822 0.99992
a
m within y = mx + b regression. bUsing log10(V0/V) for y. cUsing
measured absorbance for y. dThe correlation value represents the
Pearson product−moment correlation coefficient (based on 4 points).
respectively. As can be noted in Table 1 by the correlation
coefficient for the linear regression, there is a good-fitting linear
response (R2 = 0.998, based on 4 points) for the LED
spectrophotometer, which is comparable to the conventional
spectrophotometer. As noted in Kvittingen et al., LED
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Figure 4. Calibration curve of the mean responses for the individual calibration solutions using the LED spectrophotometer (top) and the
conventional UV−vis spectrophotometer (bottom).

spectrophotometers do have a nonlinear response over a large Table 2. Calculated Results for Iron Concentration in the
range of concentrations;10 for the purposes of this experiment Water Samples
and within the range of the calibrants of this activity, we
Mean Fe2+ Concentration Values, mg/L (SD) by
treated the response as linear for ease of calculation. Spectrophotometer
Using the standard deviation (σ; n = 3) of the signal of the 0
Sample Source LED UV−Vis
mg/L solution and the slope of the calibration curve, the limit
of detection (LOD) and limit of quantitation (LOQ) can be Spiked unknown 0.215 (0.012) 0.204 (0.0005)
determined for both the LED and conventional spectropho- River <LODa <LODa
tometer using the following equations: Pond <LOQb 0.0276 (0.00316)
Well 0.808 (0.0074) 0.805 (0.0035)
3.3 × σ Ocean <LODa <LODa
LOD =
slope (3) a
Samples below the limit of detection, LOD. bSamples below the
limit of quantitation, LOQ.
10 × σ
LOQ =
slope (4)
spiked “unknown” water sample (0.2 mg/L), the calculated
The LOD and LOQ for the LED spectrophotometer were concentration of iron was 0.22 and 0.20 mg/L for the LED and
0.022 and 0.065 mg/L, respectively, and the LOD and LOQ conventional UV−vis spectrophotometers, respectively. This
for the conventional spectrophotometer were 0.0069 and result suggests that the accuracy of the LED spectropho-
0.0221 mg/L, respectively. tometer based on the reagent-based iron indicator is similar to
Using the linear regression of the measured voltage response the conventional UV−vis spectrophotometer using the same
(and absorbance), the concentrations of iron in the five water wavelength. For the water samples, the precision of the LED
samples were calculated and are reported in Table 2. In the spectrophotometer ranged from 0.92% to 5.6% relative
D DOI: 10.1021/acs.jchemed.8b00515
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standard deviation (RSD) for the two detected concentrations,
while the conventional UV−vis spectrophotometer precision
ranged from 0.25% to 11% RSD for the three detected
concentrations. For both spectrophotometers, the high
variability was observed at the lowest iron concentrations
(the river, pond, and ocean water samples).
As can be observed with the measurement of iron in the
pond sample (and their respective LOD/LOQ values), the
sensitivity of the LED spectrophotometer is poorer than the
conventional UV−vis spectrophotometer. The sensitivity of
the LED spectrophotometer could be improved by reducing
the impact of stray light (using an opaque cover for the
spectrophotometer and light slits on the cuvette) and changing
the resistance between the battery and the source LED. The
experiments above were conducted in laboratories with
fluorescent lighting and no windows allowing natural sunlight;
therefore, the impact of stray natural light was not determined.
■ CONCLUSIONS
The activity presented provides a modular tool for teaching
electrical circuits, analytical chemistry, and water quality to a
wide range of student audiences. For middle school and early
high school students, the LED spectrophotometer can be
preassembled and the focus of the activity can be on water
sampling and the analysis of iron in various water bodies in
their local area. As the spectrophotometer is portable, the tool
can be used outside (with shielding from natural light) to
demonstrate field laboratory measurements. Upon introduc-
tion of the assembly of the spectrophotometer, high school
students would learn the general structure of an electrical
circuit diagram, as well as general analytical chemistry related
to calibration and quantitation using Excel spreadsheet
calculations.
For undergraduate students, the activity can demonstrate the
previously noted educational goals, while also introducing the
discussion of spectroscopy, which includes the identification of
the λmax for the greatest sensitivity and optimal design of a
spectrophotometer. Design parameters such as the presence of
a light shield, orientation of the LEDs, resistance of the
resistor, and battery voltage (using a AAA, AA, or 9 V battery)
could be safely evaluated (the LEDs do have a maximum input
voltage). The use of TPTZ introduces the concept of a
coordination compound that results in a visible color change.
In addition, concepts such as linear regression and regression
statistics can be introduced when examining a calibration curve
plot.
Using drinking water sourced from an underground well (as
demonstrated in the presented activity), students can evaluate
the concentration of iron in drinking water. Iron in drinking
water can be an excellent tool to discuss the differences
between well-sourced water and municipality-supplied water;
while iron in drinking water is not toxic, it has qualitative
aspects that can be undesirable, such as color or taste. In
addition, a household that has an individual water-treatment
system for the removal of iron can be used to measure the iron
concentration before and after treatment to evaluate the
performance of the treatment system. As noted in the
demonstrated activity, waters such as ocean water or surface
waters may not have a high enough concentration of iron to
detect using the LED spectrophotometer; for a greater number
of water samples with detectable water, samples should be
taken from groundwater sources or surface water near point
sources of iron, such as mining facilities or industrial runoff.
E DOI: 10.1021/acs.jchemed.8b00515
Activity
In conclusion, the LED spectrophotometer performs well
and is comparable to the conventional UV−vis spectropho-
tometer for measuring moderate to high levels of iron in water.
Given the ease of construction and the inexpensive nature of
the materials used to build the LED spectrophotometer, the
instrument provides a unique tool to teach students about
general spectroscopy and water quality.
■
*
ASSOCIATED CONTENT
S Supporting Information
The Supporting Information is available on the ACS
Publications website at DOI: 10.1021/acs.jchemed.8b00515.
Student activity handout (PDF, DOCX)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: benjamin.place@nist.gov.
ORCID
Benjamin J. Place: 0000-0003-0953-5215
Notes
Certain commercial equipment, instruments, or materials are
identified in this communication in order to specify the
experimental procedure adequately. Such identification is not
intended to imply recommendation or endorsement by the
National Institute of Standards and Technology, nor is it
intended to imply that the materials or equipment identified
are necessarily the best available for the purpose.
The author declares no competing financial interest.
■
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F DOI: 10.1021/acs.jchemed.8b00515
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