ENGINEERING SCIENCE DEPARTMENT
Water and Environmental Technology Program
Name _________________
SPRING TERM
Aquatic Chemistry II, WQT-134
Total Iron Laboratory #6
Modified Iron STM 3500 Fe (B) Phenanthroline Method (Applied Water and Spentwater
Manual Ch 38)
Purpose: In this lab you will perform a total iron test on a water/waste water sample. This will
involve the use of a spectrophotometer. Iron (Fe) is the second most abundant metal, after
aluminum, and the forth most abundant element in the earths crust. first element in Group VIII of
the periodic table; it has an atomic number of 26, an atomic weight of 55.85, and common
valences of 2 and 3 (and occasionally valences of 1, 4, and 6). The average abundance of Fe in
the earth’s crust is 6.22%; in soils Fe ranges from 0.5 to 4.3%; in streams it averages about 0.7
mg/L; and in groundwater it is 0.1 to 10 mg/L. Iron occurs in the minerals hematite(Fe2O3),
magnetite (Fe3O4), ferrihydrite (FeOOH), aresenopyrite (FeAsS), siderite (FeCO3),
aluminosilicates, and pyrite (FeS). It is widely used in steel, alloys, as well as for environmental
remediation of nitrates and arsenic. Unfortunately, iron in residual waters after treatment can
cause laundry and fixture staining, corrosion, aesthetic taste problems and iron bacterial blooms
in drinking water. When iron reacts with carbonic acid or oxygen it can corrode, tuberculate,
and rust water distribution systems. Thus, the U.S. EPA secondary drinking water standard
MCL is 0.3 mg/L.
The solubility of ferrous ion (Fe 2+) is controlled by the carbonate concentration. Because
groundwater is often anoxic, any soluble iron in groundwater is usually in the ferrous state. On
exposure to air or addition of oxidants, ferrous iron is oxidized to the ferric state (Fe 3+) and may
hydrolyze to form red, insoluble hydrated ferric oxide. In fact, the reddish brown precipitate that
you oftentimes see in springs or near ponds is the oxidation of Fe from its reduced to its oxidized
state. In the absence of complex-forming ions, ferric iron is not significantly soluble unless the
pH is very low. Elevated iron levels in water can cause stains in plumbing, laundry, and cooking
utensils, and can impart objectionable tastes and colors to foods. The United Nations Food and
Agriculture Organization recommended level for irrigation waters is 5 mg/L.
Factoids/Conversions
 Presence of elevated iron in a water system may indicate corrosion.
 Above 5 mg/L Fe accuracy decreases due to over production of orange color.
Advantages
 Accuracy of dissolved or total concentrations of iron as low as 10 μg/L can be determined
with a spectrophotometer using cells with a 5 cm or longer light path.
 No pretreatment necessary for well or potable water samples
Disadvantages/Interferences
 Preliminary treatment is a must for waste or organic rich stream water
 Cyanide, nitrite, and phosphates (polyphosphates more so than orthophosphate),
chromium, zinc in concentrations exceeding 10 times that of iron, cobalt and copper in
excess of 5 mg/L, and nickel in excess of 2 mg/L.
 Bismuth, cadmium, mercury, molybdate, and silver precipitate phenanthroline.
 Color or organic matter may necessitate digestion before use of the extraction procedure.
Sample collection: Plan in advance the methods of collecting, storing, and pretreating samples.
Clean sample container with acid and rinse with reagent water. Equipment for membrane
filtration of samples in the field may be required to determine iron in solution (dissolved iron).
Dissolved iron, considered to be that passing through a 0.45-μm membrane filter, may include
colloidal iron.
SUPPLIES/SAFTEY

PLEASE WEAR LAB COATS, SAFETY GOGGLES, AND GLOVES AT ALL TIMES FOR
THIS LAB PROCEDURE. USE THE FUME HOOD WHEN NECESSARY
Lab Materials:
1. Spectrophotometer, for use at 510 nm, providing a light path of 1 cm or longer.
2. Nessler tubes, matched, 100-mL, tall form and rack.
3. Acid-washed glassware: Wash all glassware with conc hydrochloric acid (HCl) and rinse
with reagent water before use to remove deposits of iron oxide.
4. Hot plate
5. 5-10 ml graduated pipettes for reagent addition and making of the standards
6. Beakers 125-, 250 mL
7. 1000 ml, 100, and 50 ml volumetric flasks
REAGENTS/STORAGE Mandatory Use of Gloves and Goggles!:
Hydroxylamine solution: Dissolve 10 g NH2OHHCl in 100 mL water.
Ammonium acetate buffer solution (pH 4): IN FUME HOOD! In 1000 ml flask, dissolve 250 g
NH4C2H3O2 in 150 mL water. Add 700 mL conc (glacial) acetic acid. DO NOT BRING TO
VOLUME, store in a liter bottle …IN FUME HOOD
Phenanthroline solution: Dissolve 100 mg (or 0.100 grams) 1,10-phenanthroline monohydrate,
C12H8N2H2O, in 90 mL DI water on a magnetic stir plate with a stir bar, heat to 90oC to
dissolve crystals and add 2 drops conc (12 M) HCl to the water (this will dissolve crystals), bring
to 100 ml volume with DI water. (NOTE: One milliliter of this reagent is sufficient for no more
than 100 μg Fe.)
Stock iron solution: IN THE FUME HOOD! wearing goggles and gloves slowly add 20 mL
conc (18 M/36 N) H2SO4 to 50 mL deionized water and dissolve 1.404 g ferrous ammonium
sulfate Fe(NH4)2(SO4)26H2O. Dilute to 1000 mL with water; 1.00 mL = 200 μg Fe or 200
mg/L Fe. Note: color should be light green
Standard iron solutions: Prepare daily for use.
10 mg/L Standard iron solutions; Pipet 5.00 mL stock
iron solution into a 100mL volumetric flask and dilute to mark with boiled and cooled deionized water; 1.00 mL = 10.0
μg Fe or 10 mg/L Fe.
Table 1. Dilution for Fe calibration curve using the 10 mg/L Standard iron solutions.
Label Sample ID
Volume of Standard Iron Sol
2- std 0.50 mg/L Fe 2.5 ml of 10 mg/L Std Fe solution, 10 ml acetate buffer, 2 ml of phenanthroline solution
and dilute to 50 ml (wait 10 min)
3- std 1.0 mg/L Fe
5.0 ml of 10 mg/L Std Fe solution , 10 ml acetate buffer, 2 ml of phenanthroline
solution and dilute to 50 ml (wait 10 min)
4- std 1.50 mg/L Fe 7.5 ml of 10 mg/L Std Fe solution, 10 ml acetate buffer, 2 ml of phenanthroline solution
and dilute to 50 ml (wait 10 min)
5- std 2.0 mg/L Fe
10 ml of 10 mg/L Std Fe solution, 10 ml acetate buffer, 2 ml of phenanthroline solution
and dilute to 50 ml (wait 10 min)
6- std 2.5 mg/L Fe
12.5 ml of 10 mg/L Std Fe solution, 10 ml acetate buffer, 2 ml of phenanthroline
solution and dilute to 50 ml (wait 10 min)
7- std 3.0 mg/L Fe
15.0 ml of 10 mg/L Std Fe solution, 10 ml acetate buffer, 2 ml of phenanthroline
solution and dilute to 50 ml (wait 10 min)
8- std 4.0 mg/L Fe
20.0 ml of 10 mg/L Std Fe solution, 10 ml acetate buffer, 2 ml of phenanthroline
solution and dilute to 50 ml (wait 10 min)
9- std 5.0 mg/L Fe
25.0 ml of 10 mg/L Std Fe solution, 10 ml acetate buffer, 2 ml of phenanthroline
solution and dilute to 50 ml (wait 10 min)
SUMMARY PROCEDURE
Modified Iron STM 3500 Fe (B) Phenanthroline Method (Applied Water and Spentwater
Manual Ch 38)
Total Iron Measurement: Iron is brought into solution, reduced to the ferrous state by boiling
with acid and hydroxylamine, and treated with 1,10-phenanthroline at pH 3.2 to 3.3. Three
molecules of phenanthroline chelate each atom of ferrous iron to form an orange-red complex.
The colored solution obeys Beer’s law; its intensity is independent of pH from 3 to 9. A pH
between 2.9 and 3.5 insures rapid color development in the presence of an excess of
phenanthroline. Color standards are stable for at least 6 months.
Step-by-step Procedure
Modified Iron STM 3500 Fe (B) Phenanthroline Method (Applied Water and Spentwater
Manual Ch 38)
Part 1 Total Iron Unknown Sample (Potable and Groundwater Samples/Skip These Steps).
1. Mix (your stream or wastewater sample) sample thoroughly and measure 50.0 mL into a
125-mL erlenmeyer flask.
2. Wearing gloves and goggles, add 2 mL conc (12 M) HCl and 2 ml hydroxylamine
hydrochloride.
3. To insure dissolution of all the iron boil sample.
4. Cool to room temperature and transfer 38 ml into a 50 mL volumetric flask.
5. Add 10 mL ammonium acetate (NH4C2H3O2) buffer solution and 2 mL phenanthroline
solution, and dilute to 50 ml mark with water.
6. Mix thoroughly and allow a minimum of 10 min for maximum color development.
Modified Iron STM 3500 Fe (B) Phenanthroline Method (Applied Water and Spentwater
Manual Ch 38)
Part 1 Creation of Fe Standards/Calibration Curve/Unknown Sample/ Using the
Spectronic 20D
1. Warm up the colorimeter and set the wavelength to 510 nm with the Wavelength control
knob.
2. Set the filter switch at bottom left to read (350-599 nm).
3. Close sample door and without any sample in spec adjust the to 0%T with the Zero
Control knob (Left hand knob).
4. Set the display mode to Absorbance by hitting the Mode control button
5. Make up a Spec blank (38 ml of DI water,10 ml acetate buffer, 2 ml of phenanthroline
solution (wait 10 min)).
6. Insert Spec blank to zero instrument by using the 100%T/OA right knob button (note:
you may have to turn it counterclockwise a few times if you are getting +1999 or
clockwise if it reads -1999) before numbers start to drop to 0 (Periodically re-analyze the
blank throughout the procedure as part of your internal Quality Control Procedure to
determine if there is any instrument drift=readjust if necessary).
7. Arrange standards Labeled correctly from high to low as follows:
Label Sample ID
1-Spec blank
2- std 0.50
3- std 1.0
4- std 1.50
5- std 2.0
6- std 2.5
7- std 3.0
8- std 4.0
9- std 5.0
10-unknown sample
Volume of Standard Iron Sol
38 ml of DI water,10 ml acetate buffer, 2 ml of phenanthroline
solution (wait 10 min)
2.5 ml of 10 mg/L Std Fe solution, 10 ml acetate buffer, 2 ml of
phenanthroline solution and dilute to 50 ml (wait 10 min)
5.0 ml of 10 mg/L Std Fe solution , 10 ml acetate buffer, 2 ml of
phenanthroline solution and dilute to 50 ml (wait 10 min)
7.5 ml of 10 mg/L Std Fe solution, 10 ml acetate buffer, 2 ml of
phenanthroline solution and dilute to 50 ml (wait 10 min)
10 ml of 10 mg/L Std Fe solution, 10 ml acetate buffer, 2 ml of
phenanthroline solution and dilute to 50 ml (wait 10 min)
12.5 ml of 10 mg/L Std Fe solution, 10 ml acetate buffer, 2 ml of
phenanthroline solution and dilute to 50 ml (wait 10 min)
15.0 ml of 10 mg/L Std Fe solution, 10 ml acetate buffer, 2 ml of
phenanthroline solution and dilute to 50 ml (wait 10 min)
20.0 ml of 10 mg/L Std Fe solution, 10 ml acetate buffer, 2 ml of
phenanthroline solution and dilute to 50 ml (wait 10 min)
25.0 ml of 10 mg/L Std Fe solution, 10 ml acetate buffer, 2 ml of
phenanthroline solution and dilute to 50 ml (wait 10 min)
38 ml of unknown sample, 10 ml acetate buffer, 2 ml of
phenanthroline solution and dilute to 50 ml (wait 10 min)
8. Read the absorbance at 510 nanometers using the colorimeter, working from low to high
concentration then to your unknown sample. Wipe the Cuvette each time!! Record data
in table on next page.
Table 2. Raw data
Label Sample ID
Absorbance at 510 Nm
Expected
Concentration
0
0.5 mg/L Fe
1.0 mg/L Fe
1.5 mg/L Fe
2.0 mg/L Fe
2.5 mg/L Fe
3.0 mg/L Fe
4.0 mg/L Fe
5.0 mg/L Fe
1-Spec Blank
2- std 0.50
3- std 1.0
4- std 1.50
5- std 2.0
6- std 2.5
7- std 3.0
8- std 4.0
9- std 5.0
10-unknown sample
9. Plot the curve of absorbance vs. concentration. Use the curve to determine the final iron
concentration in the unknown sample.
Example of Iron Standard Curve From Niazi Fe(II) determination paper
Absorbance at 510 nm
Fe II Calibration Curve at 510 nm From Niazi paper
1.4
1.2
y = 0.1896x - 0.1031
R2 = 0.9974
1
0.8
0.6
0.4
Series1
Linear (Series1)
0.2
0
0
2
4
6
8
Concentration (mg/L)
Part 3 Using Excel to plot the data.
1. Plot absorbance or percent transmission (on the vertical or y-axis) vs. iron concentration
(on the x or horizontal axis) in mg/l as a linear graph. The absorbance should range from
0.0000 to 0.7000 absorbance units, and the concentration should range from 0.200 mg/l
to 2 mg/l as total iron. A straight line should pass through the origin(set intercept to =0).
2. To make a graph in Excel go to Insert and then Chart or the Chart Icon
3. Now select the X Y (Scatter) Chart Type with data/line points Chart subtype and click
Next
4. Now we need to select our data to make a graph. Our data are in columns (hit column
button). Click in the data range and then highlight the columns (of data only=no text) for
Concentration (X axis) and AVG 510 Absorbance (Y axis). Note: your raw data should
be ='Raw data'!$E$5:$F$15. Click Next
5. Now label your Chart Titles as Total Fe Method 3500, X axis: Concentration Fe mg/L,
and Y Axis: Absorbance at 510 nm. Click Next then Finish
6. Voila you have made a graph in Excel (see below: Note your graph will have the data). We
still need to add a slope to the line.
7. To do this mouse click on one data point and the right click mouse click and select Add a
Trendline. Select keep default as Linear (it should obey Beers Law and be straight if
absorption is linear with concentration), and then select the Options tab and select display
equation on chart, SET INTERCEPT TO =0, and display R2 value on chart. We made it!
Part 4 Calculations
mg/L as Fe = mg/l as read from standard curve
mg/L as Fe = (curve value)(dilution factor=necessary only for high Fe [])
Example: If 25 ml of sample has been diluted to 100 ml, and then 50 ml of this solution was used
for analysis, the dilution factor to multiply times the curve value (which is in terms of
concentration- would be 4.0). Suppose the diluted sample produced an absorbance equivalent to
0.145 mg/L then the actual co0ntration would be.
mg/l as Fe = (0.145)(4)= 0.580 mg/l as Fe
http://www.cofc.edu/~kinard/221LCHEM/CHEM221L%20Spectrophotometric%20Determinatio
n%20of%20Iron.htm