3500-Fe IRON*

3500-Fe A. Introduction

1. Occurrence and Significance

Iron (Fe) is the 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, magnetite, taconite,

and pyrite. It is widely used in steel and in other alloys.

The solubility of ferrous ion (Fe2) 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 (Fe3) and may hydrolyze to form red, insoluble hydrated ferric oxide. 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. The U.S. EPA secondary drinking water standard MCL

is 0.3 mg/L.

2. Selection of Method

Sensitivity and detection levels for the atomic absorption

spectrometric methods (3111B and C), the inductively coupled plasma method (3120), and the phenanthroline colorimetric procedure described here (B) are similar and generally

adequate for analysis of natural or treated waters. Lower

detection levels can be achieved with electrothermal atomic

absorption spectrometry (3113B) when an appropriate matrix

modifier is used. The complexing reagents used in the colorimetric procedures are specific for ferrous iron but the atomic

absorption procedures are not. However, because of the instability of ferrous iron, which is changed easily to the ferric

form in solutions in contact with air, determination of ferrous

iron requires special precautions and may need to be done in

the field at the time of sample collection.

The procedure for determining ferrous iron using 1,10-

phenanthroline (3500-Fe.B.4c) has a somewhat limited applicability; avoid long storage time or exposure of samples to light. A

rigorous quantitative distinction between ferrous and ferric iron

can be obtained with a special procedure using bathophenanthroline. Spectrophotometric methods using bathophenanthroline1– 6 and other organic complexing reagents such as ferrozine7

or TPTZ8 are capable of determining iron concentrations as low

as 1 g/L. A chemiluminescence procedure9 is stated to have a

detection limit of 5 ng/L. Additional procedures are described

elsewhere.10 –13

3. Sampling and Storage

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-m membrane filter, may include colloidal iron.

The value of the determination depends greatly on the care taken

to obtain a representative sample. Iron in well or tap water

samples may vary in concentration and form with duration and

degree of flushing before and during sampling. When taking a

sample portion for determining iron in suspension, shake the

sample bottle often and vigorously to obtain a uniform suspension of precipitated iron. Use particular care when colloidal iron

adheres to the sample bottle. This problem can be acute with

plastic bottles.

For a precise determination of total iron, use a separate container for sample collection. Treat with acid at the time of

collection to place the iron in solution and prevent adsorption or

deposition on the walls of the sample container. Take account of

the added acid in measuring portions for analysis. The addition

of acid to the sample may eliminate the need for adding acid

before digestion (3500-Fe.B.4a).

4. References

1. LEE, G.F. & W. STUMM. 1960. Determination of ferrous iron in the

presence of ferric iron using bathophenanthroline. J. Amer. Water

Works Assoc. 52:1567.

2. GHOSH, M.M., J.T. O’CONNOR & R.S. ENGELBRECHT. 1967. Bathophenanthroline method for the determination of ferrous iron.

J. Amer. Water Works Assoc. 59:897.

3. BLAIR, D. & H. DIEHL. 1961. Bathophenanthroline-disulfonic acid

and bathocuproine-disulfonic acid, water soluble reagents for iron

and copper. Talanta 7:163.

4. SHAPIRO, J. 1966. On the measurement of ferrous iron in natural

waters. Limnol. Oceanogr. 11:293.

5. MCMAHON, J.W. 1967. The influence of light and acid on the

measurement of ferrous iron in lake water. Limnol. Oceanogr.

12:437.

6. MCMAHON, J.W. 1969. An acid-free bathophenanthroline method

for measuring dissolved ferrous iron in lake water. Water Res.

3:743.

7. GIBBS, C. 1976. Characterization and application of ferrozine iron

reagent as a ferrous iron indicator. Anal. Chem. 48:1197.

8. DOUGAN, W.K. & A.L. WILSON. 1973. Absorbtiometric determination of iron with TPTZ. Water Treat. Exam. 22:110.

9. SEITZ, W.R. & D.M. HERCULES. 1972. Determination of trace amounts

of iron (II) using chemiluminescence analysis. Anal. Chem. 44:2143.

10. MOSS, M.L. & M.G. MELLON. 1942. Colorimetric determination of

* Approved by Standard Methods Committee, 1997. Editorial revisions, 2011.

Joint Task Group: 20th Edition—See Section 3500-Al.

1
iron with 2,2-bipyridine and with 2,2,2-tripyridine. Ind. Eng.

Chem., Anal. Ed. 14:862.

11. WELCHER, F.J. 1947. Organic Analytical Reagents. D. Van Nostrand

Co., Princeton, N.J., Vol. 3, pp. 100 –104.

12. MORRIS, R.L. 1952. Determination of iron in water in the presence

of heavy metals. Anal. Chem. 24:1376.

13. DOIG, M.T., III & D.F. MARTIN. 1971. Effect of humic acids on iron

analyses in natural water. Water Res. 5:689.

3500-Fe B. Phenanthroline Method

1. General Discussion

a. Principle: 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.

b. Interference: Among the interfering substances are strong

oxidizing agents, 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. The initial boiling with acid converts polyphosphates to

orthophosphate and removes cyanide and nitrite that otherwise

would interfere. Adding excess hydroxylamine eliminates errors

caused by excessive concentrations of strong oxidizing reagents.

In the presence of interfering metal ions, use a larger excess of

phenanthroline to replace that complexed by the interfering

metals. Where excessive concentrations of interfering metal ions

are present, the extraction method may be used.

If noticeable amounts of color or organic matter are present, it

may be necessary to evaporate the sample, gently ash the residue, and redissolve in acid. The ashing may be carried out in

silica, porcelain, or platinum crucibles that have been boiled for

several hours in 6N HCl. The presence of excessive amounts of

organic matter may necessitate digestion before use of the extraction procedure.

c. Minimum detectable concentration: Dissolved or total concentrations of iron as low as 10 g/L can be determined with a

spectrophotometer using cells with a 5 cm or longer light path.

Carry a blank through the entire procedure to allow for correction.

d. Quality control (QC): The QC practices considered to be an

integral part of each method can be found in Section 3020.

2. Apparatus

a. Colorimetric equipment: One of the following is required:

1) Spectrophotometer, for use at 510 nm, providing a light

path of 1 cm or longer.

2) Filter photometer, providing a light path of 1 cm or longer

and equipped with a green filter having maximum transmittance

near 510 nm.

3) Nessler tubes, matched, 100-mL, tall form.

b. Acid-washed glassware: Wash all glassware with conc

hydrochloric acid (HCl) and rinse with reagent water before use

to remove deposits of iron oxide.

c. Separatory funnels: 125-mL, Squibb form, with groundglass or TFE stopcocks and stoppers.

3. Reagents

Use reagents low in iron. Use reagent water (see Sections 1080

and 3111B.3c) in preparing standards and reagent solutions and

in procedure. Store reagents in glass-stoppered bottles. The HCl

and ammonium acetate solutions are stable indefinitely if tightly

stoppered. The hydroxylamine, phenanthroline, and stock iron

solutions are stable for several months. The standard iron solutions are not stable; prepare daily as needed by diluting the stock

solution. Visual standards in nessler tubes are stable for several

months if sealed and protected from light.

a. Hydrochloric acid, HCl, conc, containing less than 0.5 ppm

iron.

b. Hydroxylamine solution: Dissolve 10 g NH2OHHCl in 100

mL water.

c. Ammonium acetate buffer solution: Dissolve 250 g

NH4C2H3O2 in 150 mL water. Add 700 mL conc (glacial) acetic

acid. Because even a good grade of NH4C2H3O2 contains a

significant amount of iron, prepare new reference standards with

each buffer preparation.

d. Sodium acetate solution: Dissolve 200 g NaC2H3O2  3H2O

in 800 mL water.

e. Phenanthroline solution: Dissolve 100 mg 1,10-phenanthroline monohydrate, C12H8N2  H2O, in 100 mL water by stirring

and heating to 80°C. Do not boil. Discard the solution if it

darkens. Heating is unnecessary if 2 drops conc HCl are added

to the water. (NOTE: One milliliter of this reagent is sufficient for

no more than 100 g Fe.)

f. Potassium permanganate, 0.02M: Dissolve 0.316 g KMnO4

in reagent water and dilute to 100 mL.

g. Stock iron solution: Use metal (1) or salt (2) for preparing

the stock solution.

1) Use electrolytic iron wire, or “iron wire for standardizing,”

to prepare the solution. If necessary, clean wire with fine sandpaper to remove any oxide coating and to produce a bright

surface. Weigh 200.0 mg wire and place in a 1000-mL volumetric flask. Dissolve in 20 mL 6N sulfuric acid (H2SO4) and dilute

to mark with water; 1.00 mL  200 g Fe.

2) If ferrous ammonium sulfate is preferred, slowly add 20

mL conc H2SO4 to 50 mL water and dissolve 1.404 g

Fe(NH4)2(SO4)2  6H2O. Slowly add potassium permanganate (¶

f above) until a faint pink color persists. Add the last few

milliliters of the solution dropwise. Approximately 50 mL of the

IRON (3500-Fe)/Phenanthroline Method

2
potassium permanganate will be required. Dilute to 1000 mL

with water and mix; 1.00 mL  200 g Fe.

h. Standard iron solutions: Prepare daily for use.

1) Pipet 50.00 mL stock solution into a 1000-mL volumetric

flask and dilute to mark with water; 1.00 mL  10.0 g Fe.

2) Pipet 5.00 mL stock solution into a 1000-mL volumetric

flask and dilute to mark with water; 1.00 mL  1.00 g Fe.

i. Diisopropyl or isopropyl ether. CAUTION: Ethers may form

explosive peroxides; test before using.

4. Procedure

a. Total iron: Mix sample thoroughly and measure 50.0 mL

into a 125-mL erlenmeyer flask. If this sample volume contains

more than 200 g iron use a smaller accurately measured portion

and dilute to 50.0 mL. Add 2 mL conc HCl and 1 mL

NH2OH  HCl solution. Add a few glass beads and heat to

boiling. To insure dissolution of all the iron, continue boiling

until volume is reduced to 15 to 20 mL. (If the sample is ashed,

take up residue in 2 mL conc HCl and 5 mL water.) Cool to room

temperature and transfer to a 50- or 100-mL volumetric flask or

nessler tube. Add 10 mL NH4C2H3O2 buffer solution and 4 mL

phenanthroline solution, and dilute to mark with water. Mix

thoroughly and allow a minimum of 10 min for maximum color

development.

b. Dissolved iron: Immediately after collection filter sample

through a 0.45-m membrane filter into a vacuum flask containing 1 mL conc HCl/100 mL sample. Analyze filtrate for total

dissolved iron (¶ a above) and/or dissolved ferrous iron (¶ c

below). (This procedure also can be used in the laboratory if it is

understood that normal sample exposure to air during shipment

may result in precipitation of iron.)

Calculate suspended iron by subtracting dissolved from total

iron.

c. Ferrous iron: Determine ferrous iron at sampling site because of the possibility of change in the ferrous-ferric ratio with

time in acid solutions. To determine ferrous iron only, acidify a

separate sample with 2 mL conc HCl/100 mL sample at time of

collection. Fill bottle directly from sampling source and stopper.

Immediately withdraw a 50-mL portion of acidified sample and

add 20 mL phenanthroline solution and 10 mL NH4C2H3O2

solution with vigorous stirring. Dilute to 100 mL and measure

color intensity within 5 to 10 min. Do not expose to sunlight.

(Color development is rapid in the presence of excess phenanthroline. The phenanthroline volume given is suitable for less

than 50 g total iron; if larger amounts are present, use a

correspondingly larger volume of phenanthroline or a more

concentrated reagent.)

Calculate ferric iron by subtracting ferrous from total iron.

d. Color measurement: Prepare a series of standards by accurately pipetting calculated volumes of standard iron solutions

use solution described in 3500-Fe.B.3h2) to measure 1- to

10-g portions] into 125-mL erlenmeyer flasks and diluting to

50 mL by adding measured volumes of water. Add 2 mL conc

HCl and 1 mL NH2OH  HCl solution. Carry out the steps in ¶ a

above beginning with transfer to a 100-mL volumetric flask or

nessler tube.

For visual comparison, prepare a set of at least 10 standards,

ranging from 1 to 100 g Fe in the final 100-mL volume.

Compare colors in 100-mL tall-form nessler tubes.

For photometric measurement, use Table 3500-Fe:I as a rough

guide for selecting proper light path at 510 nm. Read standards

against water set at zero absorbance and plot a calibration curve,

including a blank (see Sections 1080 and 3111B.3c).

If samples are colored or turbid, carry a second set of samples

through all steps of the procedure without adding phenanthroline. Instead of water, use the prepared blanks to set photometer

to zero absorbance and read each sample developed with

phenanthroline against the corresponding blank without phenanthroline. Translate observed photometer readings into iron values

by means of the calibration curve. This procedure does not

compensate for interfering ions.

e. Samples containing organic interferences: Digest samples

containing substantial amounts of organic substances according

to the directions given in Sections 3030G or H.

1) If a digested sample has been prepared according to the

directions given in Section 3030G or H, pipet 10.0 mL or other

suitable portion containing 20 to 500 g Fe into a 125-mL

separatory funnel. If the volume taken is less than 10 mL, add

water to make up to 10 mL. To the separatory funnel add 15 mL

conc HCl for a 10-mL aqueous volume; or, if the portion taken

was greater than 10.0 mL, add 1.5 mL conc HCl/mL sample.

Mix, cool, and proceed with ¶ e3) below.

2) To prepare a sample solely for determining iron, measure

a suitable volume containing 20 to 500 g Fe and carry it

through the digestion procedure described in either Section

3030G or H. However, use only 5 mL H2SO4 or HClO4 and omit

H2O2. When digestion is complete, cool, dilute with 10 mL

water, heat almost to boiling to dissolve slowly soluble salts,

and, if the sample is still cloudy, filter through a glass-fiber,

sintered-glass, or porcelain filter, washing with 2 to 3 mL water.

Quantitatively transfer filtrate or clear solution to a 25-mL volumetric flask and make up to 25 mL with water. Empty flask into

a 125-mL separatory funnel, rinse flask with 5 mL conc HCl and

add to the funnel. Add 25 mL conc HCl measured with the same

flask. Mix and cool to room temperature.

3) Extract the iron from the HCl solution in the separatory

funnel by shaking for 30 s with 25 mL isopropyl ether (CAUTION). Draw off lower acid layer into a second separatory funnel.

Extract acid solution again with 25 mL isopropyl ether, drain

acid layer into a suitable clean vessel, and add ether layer to the

ether in the first funnel. Pour acid layer back into second separatory funnel and re-extract with 25 mL isopropyl ether. Withdraw and discard acid layer and add ether layer to first funnel.

Persistence of a yellow color in the HCl solution after three

extractions does not signify incomplete separation of iron beTABLE 3500-FE:I. SELECTION OF LIGHT PATH LENGTH FOR VARIOUS IRON

CONCENTRATIONS

Fe

g

Light

Path

cm

50-mL

Final

Volume

100-mL

Final

Volume

50–200 100–400 1

25–100 50–200 2

10–40 20–80 5

5–20 10–40 10

IRON (3500-Fe)/Phenanthroline Method

3
cause copper, which is not extracted, gives a similar yellow

color.

Shake combined ether extracts with 25 mL water to return iron

to aqueous phase and transfer lower aqueous layer to a 100-mL

volumetric flask. Repeat extraction with a second 25-mL portion

of water, adding this to the first aqueous extract. Discard ether

layer.

4) Add 1 mL NH2OH  HCl solution, 10 mL phenanthroline

solution, and 10 mL NaC2H3O2 solution. Dilute to 100 mL with

water, mix thoroughly, and let stand for a minimum of 10 min.

Measure absorbance at 510 nm using a 5-cm absorption cell for

amounts of iron less than 100 g or 1-cm cell for quantities from

100 to 500 g. As reference, use either water or a sample blank

prepared by carrying the specified quantities of acids through the

entire analytical procedure. If water is used as reference, correct

sample absorbance by subtracting absorbance of a sample blank.

Determine micrograms of iron in the sample from the absorbance (corrected, if necessary) by reference to the calibration

curve prepared by using a suitable range of iron standards

containing the same amounts of phenanthroline, hydroxylamine,

and sodium acetate as the sample.

5. Calculation

When the sample has been treated according to 3500-Fe.B.4a, b,

c, or 4e2):

mg Fe/L  g Fe (in 100 mL final volume)

mL sample

When the sample has been treated according to 4e1):

mg Fe/L  g Fe (in 100 mL final volume)

mL sample



100

mL portion

Report details of sample collection, storage, and pretreatment

if they are pertinent to interpretation of results.

6. Precision and Bias

Precision and bias depend on the method of sample collection

and storage, the method of color measurement, the iron concentration, and the presence of interfering color, turbidity, and

foreign ions. In general, optimum reliability of visual comparison in nessler tubes is not better than 5% and often only 10%,

whereas, under optimum conditions, photometric measurement

may be reliable to 3% or 3 g, whichever is greater. The

sensitivity limit for visual observation in nessler tubes is approximately 1 g Fe. Sample variability and instability may affect

precision and bias of this determination more than will the errors

of analysis. Serious divergences have been found in reports of

different laboratories because of variations in methods of collecting and treating samples.

A synthetic sample containing 300 g Fe/L, 500 g Al/L, 50

g Cd/L, 110 g Cr/L, 470 g Cu/L, 70 g Pb/L, 120 g Mn/L,

150 g Ag/L, and 650 g Zn/L in distilled water was analyzed

in 44 laboratories by the phenanthroline method, with a relative

standard deviation of 25.5% and a relative error of 13.3%.

7. Bibliography

CHRONHEIM, G. & W. WINK. 1942. Determination of divalent iron (by

o-nitrosophenol). Ind. Eng. Chem., Anal. Ed. 14:447.

MEHLIG, R.P. & R.H. HULETT. 1942. Spectrophotometric determination

of iron with o-phenanthroline and with nitro-o-phenanthroline. Ind.

Eng. Chem., Anal. Ed. 14:869.

CALDWELL, D.H. & R.B. ADAMS. 1946. Colorimetric determination of

iron in water with o-phenanthroline. J. Amer. Water Works Assoc.

38:727.

WELCHER, F.J. 1947. Organic Analytical Reagents. D. Van Nostrand Co.,

Princeton, N.J., Vol. 3, pp. 85–93.

KOLTHOFF, I.M., T.S. LEE & D.L. LEUSSING. 1948. Equilibrium and

kinetic studies on the formation and dissociation of ferroin and

ferrin. Anal. Chem. 20:985.

RYAN, J.A. & G.H. BOTHAM. 1949. Iron in aluminum alloys: Colorimetric determination using 1,10-phenanthroline. Anal. Chem. 21:1521.

REITZ, L.K., A.S. O’BRIEN & T.L. DAVIS. 1950. Evaluation of three iron

methods using a factorial experiment. Anal. Chem. 22:1470.

SANDELL, E.B. 1959. Chapter 22 in Colorimetric Determination of

Traces of Metals, 3rd ed. Interscience Publishers, New York, N.Y.

SKOUGSTAD, M.W., M.J. FISHMAN, L.C. FRIEDMAN, D.E. ERDMANN & S.S.

DUNCAN. 1979. Methods for determination of inorganic substances

in water and fluvial sediment. Chapter A1 in Book 5, Techniques of

Water Resources Investigations of the United States Geological

Survey. U.S. Geological Surv., Washington, D.C.

IRON (3500-Fe)/Phenanthroline Method

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