UV-Vis Analysis - Determination of Iron in Drinking

Water

Prelab questions to consider, don’t forget the online quiz!

1. Draw out a simple block diagram of a UV-Visible Spectrophotometer. What type of
parameter will you actually be measuring with the instrument (i.e., concentration, mass,
energy, length, current, etc.)?

2. Pretend that you just ran a spectroscopic determination of Fe in a water sample from your
faucet. If the molar absorptivity of Fe (which you would have determined using a calibration
curve ahead of time) were 1435 M-1 cm-1, your path length were 1 cm, and you measured
an absorption of 0.10278 A.U. would your water meet the U.S. regulations for water purity?

3. As with all experiments you’ll perform there are a lot of chances for introducing error in
your measurement. Think about the different types of interference that can occur… The
instrument itself and your operating technique with it, the sample matrix itself and any
preparation you may need to stabilize your analyte. List at least two potential interferences
for this experiment, with at least one from each category above.

4. Write out your lab procedure in your notebook. Be explicit enough to be able to perform
the experiment with only your notebook as a guide.
5. Prepare an excel (or other software) spreadsheet ahead for use with your data to solve for
unknown concentrations. This means you should prepare a linear regression analysis for
your calibration curve so that when you measure your unknowns you can plug in their
absorbance and immediately receive a concentration.
Introduction
The safety of drinking water is a very important public health issue. The United States and
World Health Organization have established well-defined standards for drinking water
purity. For example, U.S. Federal regulations limit the amount of iron to less than 0.3 ppm ( 0.3
mg/L) in municipal drinking water. Although iron is only toxic at very high concentrations, it acts
as a useful surrogate for other heavy metals, whose presence in drinking water is a real danger
to public health. In this experiment we will determine the levels of iron present in the tap water
(both cold and hot) and mineral water to determine whether or not the water meets the
standards. We will provide condensed samples of the “real-world” samples. You will also
determine a challenge sample that you prepare in the lab to test your calibration curve.

A commonly used method for the determination of trace amounts of iron involves the
complexation of Fe2+ with 1,10-phenanthroline (phen) to produce an intensely red-orange
colored complex:
Fe2+ + 3 phen → 2Fe(phen)2+ 3

Since the iron present in the water predominantly exists as Fe3+, it is necessary to first reduce
Fe3+ to Fe2+. This is accomplished by the addition of the reducing agent hydroxylamine. An
excess of reducing agent is needed to maintain iron in the +2 state (because dissolved oxygen
will reoxidize Fe2+ to Fe3+).

2Fe3+ + 2NH2 OH ∙ HCl + 2OH − → 2Fe2+ + N2 + 4H2 O + H + + Cl−

Fe2+ is quantitatively complexed by 1,10-phenanthroline in the pH range from 3 to 9. Sodium

acetate is used as a buffer to maintain a constant pH at 3.5 (this is the same buffer you made in
the first week). If the pH is too high, the Fe2+ will be oxidized to Fe3+; if the pH is too low, H+ will
compete with Fe2+ for the basic 1,10-phenanthroline (to form phenH+). Either way, you won’t
get complete complexation. You should discuss these possible problems and their impact in the
Introduction section of your report. The determination of the iron-phen complex is performed
with a UV-VIS spectrophotometer at a fixed wavelength of 508 nm using external calibration
based on standard solutions. Although iron is the metal we have selected for complexation and
analysis here, this method can be expanded to the quantitative determination of many
transition metals including nickel and silver.
Methods
Apparatus
25 mL pipet (1)
plastic cuvette (1)
25 mL grad cylinder (1)
500 mL volumetric flask (1 for entire class)
100-200 mL beakers (6)
50 mL volumetric flasks (10)
50 mL beaker for H2SO4 (1)
1000 μL automatic pipettor (1)

5 mL automatic pipettor (1)

Important Instrumentation and Parameters for Report

WPA Biowave II UV-Visible spectrophotometer

Wavelengths (see procedure)

Solutions available
a) 0.29 M hydroxylamine hydrochloride (NH2OH∙HCI): How much do you need?
b) 5.0 x10-3 M 1,10-phenanthroline: How much do you need?
c) 1.2 M sodium acetate: How much do you need?
d) 2 M H2SO4 (Caution: strong oxidizing acid): How much do you need?
e) Unknown “real-world” samples (shake bottle before taking aliquot):
1) Hot Tap Water, note the dilution factor!
2) Cold Tap Water, note the dilution factor!
3) Mineral Water, note the dilution factor!

Solutions to be prepared
One student will make this solution for the entire class :

Standard iron solution (5.0 x10-4 M). Accurately weigh out about 0.100 g Fe(NH4)2(SO4)2-6H20
(FW = 392.14). Transfer quantitatively into a 500-mL volumetric flask. Add about 10 mL of 2 M
H2SO4 and 50-mL deionized water to the flask to dissolve the Fe(NH4)2(SO4)2∙6H2O completely
before diluting to volume. Fill the flask to the mark with deionized water, wipe neck of flask to
remove excess water, cap and mix thoroughly. Improper mixing is one of the most common
mistakes we see in labs.
Procedure
1) Preparing the working standard solutions
Use the 1000 μL automatic pipettor to pipet 0, 0.1, 0.25, 0.5, 0.75, and 1.0 mL of the standard
iron solution into a series of six 50 mL volumetric flasks. Using the appropriate automatic
pipette the following solutions into each of the six volumetric flasks:

a) 1 mL of the hydroxylamine solution

b) 5 mL of the sodium acetate solution
c) 5 mL of the 1,10-phenanthroline solution

Fill each flask to the mark with deionized water, dry the neck and mix thoroughly. Allow the
solutions to stand for 10 min. Mix the solutions again before measuring the absorbance.

2) Measuring the blank

To eliminate any possible absorbance from the matrix (i.e., the other reagents), the solution
with 0 mL will be used as the blank. Rinse the cuvette several times with DI water, fill it with
deionized water, place it the holder, and “zero” the spectrometer with the blue: Blank button.
Dump the water, rinse with the blank solution, fill with the 0mL standard and measure/record
the absorbance. If the number is close to zero, press the blue: Blank to “zero” the
spectrometer with the same matrix as the standards.

3) Measuring the standards

Rinse the cuvette three times with the solution that you are about to measure, and then fill it
with the solution, place it in the holder, and measure the absorbance. Repeat the measurement
with a fresh aliquot of the solution (no need to rinse this time). The two absorbance values
should be similar, otherwise measure another one. Work from lowest iron concentration to
highest, to minimize carryover.

4) Measuring the unknown water samples

Prepare a challenge unknown sample that will end up near mid-range (6.5x10-6 M or 360 ppb)
on the calibration curve, to test for method recovery and accuracy. Pipet 25 mL aliquots of a
mineral water sample, the cold tap water and hot tap water samples (all already obtained by
the TAs) and the challenge unknown sample into four 50 mL volumetric flasks,
respectively. Finish preparing the samples as described in the procedure (1). Measure the
absorbance of the solutions. Be sure to document any deviations from the suggested
procedure in the Experimental section of your report. Note the dilution factor on the sample
bottles- the samples have been boiled to increase the concentration of minerals present and
this dilution factor must be accounted for in your analysis/report.

Report
In preparing the report for this part of the experiment, you should consider/discuss the
following:

1) Calculate the analytical concentration of iron in the standard iron solutions. Tabulate the
observed absorbance and the derived concentrations (in ppm or ppb, whichever is
appropriate) of iron for all the standards, tap water samples, mineral water and unknown
samples. (Do not forget to consider the dilution or condensation factors in your
calculations.) Comment on the recovery and accuracy of the determination, based on the
challenge unknown. Calculate the limits of detection and quantitation of the method based
on the standard error of the intercept. If any of your unknowns are below these limits, how
should their concentration be reported?
2) Plot the absorbance vs. iron concentration for the standards (including the iron-free
solution.) Use the method of least-squares (i.e., linear regression) to derive the Beers law
equation in the form of A = m[Fe]+b. Be sure to report the calibration equation in the
Results and Abstract sections of your report. If the intercept has a non-zero value, explain
why, and comment on the R2 value.
3) Calculate the molar absorptivity of Fe(phen)32+ at 508 nm. Compare your value with the
literature value, 11,100 M-1cm-1. Explain possible causes for the difference, if any is
observed.
4) Find the standard error of the calibration curve, Sc, to determine the error associated with
the unknowns. Note the distance of the “unknown” from the centroid of the curve and the
magnitude of the respective error.
5) Comment on whether or not the water samples meet the Federal standards for drinking
water and any differences observed between the cold and hot water from the same tap.

Revised 1/11/16 -KNH