Justin Barry

Inorganic Chemistry
Dr. Berry

Colorimetric Determination of Iron

Introduction

As an essential nutrient in the human diet, iron is soluble in certain situations. Many
people take iron supplements in the form of vitamins. In this lab we will determine the
amount of iron present in a sample supplement using colorimetric determination. Iron
surrounds itself with Lewis bases (ligands) and forms a red colored complex. The red
colored complex forms quantitatively and can be measured using a Spectrometer 20.

The ligand used to form a colored complex is 1,10-phenanthroline, or o-phenanthroline.

Fe+2 reacts exclusively with o-phenanthroline and absorbs in the visible spectrum
(around 510 nm). This reaction occurs under acidic conditions. Ammonium acetate will
be used to buffer the solution that the complex will form in and hydroxylamine HCl will
be used to prevent oxidation.

Formation of the soluble Iron:

4 Fe3+(aq) + 2 NH2OH(aq)
4 Fe2+(aq) + N2O(aq) + 4 H+(aq) +H2O(l)

Complexing of Fe2+ with C12H2N2(l) (o-phenanthroline):

Fe2+(aq) + 3 C12H2N2(aq)
[(C12H2N2)3Fe]2+(aq) an orange-red complex

After making colorimetric measurements on various concentrations of solutions, a

calibration curve will be constructed. An unknown will be analyzed for iron as well. The
Beer-Lambert Law can be used to determine that absorption (A=ε εlc) of the unknown
and iron supplement. The concentration of the unknown will be determined by
extrapolation to the calibration curve and by using visual inspection.

Materials

 10mL graduated pipette  (2) Cuvettes for Spec20

 (2) 50mL volumetric flask  Stock Fe solution: 1mL =
 100mL volumetric flask 0.050mg Fe (III)
 50mL burette (1 per group)  1M NH4C2H3O2
 (4) equivalent test tubes (to be  10% Hydroxylamine HCl
distributed)  0.3% o-phenanthroline.
 Hotplate  3M H2SO4
 Millimeter ruler  6M HCl

Page 1 of 11
MSDS Important Information

Compound MSDS Information

Ammonium Acetate May cause eye and skin
irritation. May cause
respiratory and digestive tract
irritation. May cause central
nervous system effects.
Hygroscopic (absorbs
moisture from the air).
Hydroxylamine HCl Very hazardous in case of skin
contact (irritant), of eye
contact (irritant), of ingestion,
of inhalation. Slightly
hazardous in case of skin
contact (corrosive, sensitizer),
of eye contact (corrosive).
o-phenanthroline May be harmful by inhalation,
ingestion,or skin absorption.
Causes eye and skin irritation.
Material is irritating to
mucous membranes and upper
respiratory tract.
3M H2SO4 Hazardous in case of skin
contact (irritant), of eye
contact (irritant). Slightly
hazardous in
case of ingestion.
6M HCl POISON! DANGER!
CORROSIVE. LIQUID AND
MIST CAUSE SEVERE
BURNS TO ALL BODY
TISSUE. MAY BE FATAL IF
SWALLOWED OR
INHALED.

Page 2 of 11
PreLab Questions: (see scanned document below)
1. (a) The standard recipe used in this exercise uses 3.0 mL of 0.25% ophenanthroline
solution in 100mL of final solution to generate the red ironphenanthroline
complex which is determined colorimetrically. What is the initial
molarity of o-phenanthroline in the diluted solution? [0.25% means 2.50 g. of
ophenanthroline
(molar mass 180) per liter of solution.]

(b) What is the maximum concentration of iron-phenanthroline complex that can

be formed from this amount of o-phenanthroline, assuming complete
complexation?

(c) What does this concentration of iron correspond to in mg/L?

(d) Is this enough for the amounts of iron (up to 20mL of stock solution) we are
using? (i.e.: What is the limiting reagent in these reactions?)

2. Determine the concentration of the iron complex for the known iron solutions
used in this lab.

3. A Russian scientist poses the following question:

.Take a ruble and put it in the bottom of a tall coffee mug, such that the coin
completely covers the bottom of the mug. Add just enough coffee so that the coin
is completely obscured by the coffee and cannot be seen any more. How much
vodka would I need to add to the coffee in order to see the coin again:

a) one drop,
b) an equal volume of vodka to the volume of coffee that had been added, or
c) all the vodka produced in Russia, and more.

Using your knowledge of Beer’s Law, answer and be prepared to discuss in class
the day after lab.

Page 3 of 11
Page 4 of 11
Pre-Lab questions continued:
3. (cont.) Normally, on a Spec 20 the length of the sample stays the same. Therefore, absorbance
and concentration would be linearly related on a graph. However, when the length of the tall
coffee mug increases, the absorbance stays constant.

Figure 1: Structure of Iron Complex

Iron (II)-o-phenanthroline

N N

N Fe +2 N

Page 5 of 11
Prodecure/Flow Chart

Preparation Add 1 mL of 1M
NH4C2H3O2, 1mL
of 4 10% Hydroxylamine Mix thoroughly
solutions HCl, 10mL of 0.3% with H2O and
ophenanthroline
dilute to a total
volume of
50mL.
Allow 4 separate vials
to stand for 20 min.

5 mL stock soln 20 mL stock soln

10 mL stock soln 15 mL stock soln

PART A

PART B
Take 5 mL of each
solution and transfer to
50 mL volumetric and Using Spec 20
dilute. Blank DI water
(0%, 510 nm) Take diluted solutions
and measure absorbance
PART C-Unknown 1 of each solution. (cal
curve)
Take unknown 1 solution and
measure absorbance
Weigh out approximately
0.10xx g of unknown.

Cool to room
PART C-Unknown 2 temp, add to Pipette 2.5 mL
10 drops of 3M H2SO4 volumetric and of Solution A
dilute into a 50 mL
Thoroughly mix and dilute volumetric flask,
with DI H2O to total (SOLUTION A)
Confirm amount of Iron in and dilute.
volume of 50mL supplement tablet from (SOLUTION B)
(Solution U) label.

Pipette 5.00 mL of Solution B

into a 50mL volumetric flask,
2mL of Solution U Put the tablet in a and add the appropriate amount
and pour it into the 100mL beaker, of NH4C2H3O2 as posted by
the TA, along with 1.0 mL of
clean 50mL along with 25 mL 6 hydroquinone solution and 3.0
volumetric M. HCl. mL of phenanthroline solution.
Dilute to 50 mL to make
Solution C.

Add 1 mL of 1M Boil Solution for 15

NH4C2H3O2, 1mL minutes. SLOWLY!,
10% Hydroxylamine with no splatter.
HCl, 10mL of 0.3%
ophenanthroline

Dilute the mixture with 5

Mix thoroughly with mL of water, and filter
while still hot directly into
H2O and dilute to a a 100 mL volumetric
total volume of 50mL. flask. Wash filtrate.
Calculate the amount
of Iron in the tablets
Transfer to 50 mL beaker, Take unknown 2
Wait 30 min… Measure Iron conc. By solution and measure
visual inspection (see lab absorbance
notes)

Page 6 of 11
(Working in groups of 2 for Part A of the lab, and individually thereafter. Each group
can share one burette, which can be rinsed with the stock Fe (III) solution before using)

(You will work in pairs instead of groups of four for Part A. This will help us get the
same set of data points and move through the lab a bit faster. Each pair will randomly
draw the two known concentrations for which they will be responsible.)

PART A: Preparation of Fe(II) solution for calibration curve data

1. Mix thoroughly with H2O and dilute to a total volume of 50mL.

2. Each member of the group of 2 will make a solution using 5,10,15,20 mL of

stock Fe solution.
3. Add 1mL of 1M NH4C2H3O2, 1mL 10% Hydroxylamine HCl, 10mL of 0.3%
ophenanthroline to each sample. Mix thoroughly with H2O and dilute to a total
volume of 50mL.
4. Transfer to a clean, DRY beaker and label with the concentration of the iron
complex. Allow each solution you prepare to stand for at least 20 minutes at
room temperature, to provide time for formation of the iron-phenanthroline
complex.

(start on Part C while you wait)

5. After allowing the iron complex to form, take 5mL of this solution into a 50mL
volumetric flask, and dilute to the total volume of 50mL. This solution will be used
for the remainder of Part B. Put the concentrated solution to one side until Part C.

(Each member of the group should have made TWO of 4 solutions. Distribute
these dilute solutions in the group for Part B.)

PART B: Determination of the Calibration Curve

1. Mark the top of a cuvette with a marker, and add DI water to the cuvette as a
blank.
2. Align the mark at the top with the mark in the Spec 20 cuvette holder. This will be
your blank solution. Use the blank solution to set the 0% absorbance on the
spectrophotometer at 510nm.
3. Using another cuvette, also marked at top for alignment, measure the
absorbance of each solution at 510nm, using some of the solution to be tested as
a rinse for the cuvette between uses. Be sure to align the cuvette the same way
each time. (Establish calibration curve in Data Section)

Page 7 of 11
PART C: Unknowns (Unknown B was chosen for the lab analysis)
Unknown 1: Preparation and colorimetric determination of Unknown 1

1. Weigh out approximately 0.10xx g of unknown and place into a clean 50mL
volumetric flask.
2. Add 10 drops of 3M H2SO4 (from reagent bottles on shelf.)
3. Thoroughly mix and dilute with DI H2O to total volume of 50mL. This will be called
Solution U to prevent confusion later on. This may be transferred to a clean,
DRY, and labeled beaker.
4. Using a graduated pipette, take 2mL of Solution U and pour it into the clean
50mL volumetric flask.
5. Follow the steps to complex the iron by adding 1mL of 1M NH4C2H3O2, 1mL 10%
Hydroxylamine HCL, and 10mL of 0.3% o-phenanthroline.
6. Mix thoroughly with DI H2O and dilute to a total volume of 50mL.
7. Once thoroughly mixed, the 50mL solution can be transferred to a clean, DRY,
and labeled beaker, where the Colorimetric Determination of Iron reaction will
develop over half an hour (30 mins).
8. Repeat this step once using the same Solution U
9. Once the iron in Solution U has developed for half an hour, measure the
concentration of the iron in solution by visual inspection.
10. First, prepare two cylindrical paper sleeves, which can be slipped over the large
test tubes to exclude side lighting, by wrapping strips of paper around a test tube
and securing the edges with gummed labels. In a test tube, add enough of the
designated concentration of iron complex as your referent. (Your TA will indicate
which solution from Part A should be used for this referent. )
11. Add enough to cover 5cm length of the test tube. With the paper sleeves in
place, arrange the referent test tube and another test tube so as to look
lengthwise through the solution toward a good diffuse light source (preferably a
light box.)
12. Measure one of the other solutions by adding small amounts to the comparison
test tube until the color intensities appear exactly the same when viewed
lengthwise through the tube.
13. Reverse the viewing positions of the tubes for better comparison, and make final
adjustments with a pipette.
14. Measure the depth of each solution to the nearest millimeter, taking all
measurements from the bottom of the test tube. Save your referent for the
second unknown.
15. After you have finished with the colorimetric inspection, check the absorbance of
your unknown with the Spec 20, using the same procedure as in Part B. (Note: It
is recommended that you measure the absorbance of a second sample of the
same diluted unknown solution (Solution U). If the absorbance is the same as the
first (within the precision of the absorbance measurement), that is evidence both
that you waited the appropriate length of time for the formation of the complex
and that you mixed the solution to a uniform composition. If the two do not agree,
one or the other of the above may not be true. In the latter case, you may wish to
prepare a new dilution of your unknown solution.

Page 8 of 11
Data

Concentration Transmittance (%) Absorbance = 2-log*T Length of column (cm)

with visual inspection
10 mL stock soln 79.0 0.102 41.0
(assigned)
15 mL stock soln 57.4 0.241 n/a
(partner)
Unknown 1 77.3 0.112 56.0

Unknown # B
0.1048 g

Figure 2: The calibration curve graph

Absorbance vs Iron Concentration

1.200

y = 15660x - 0.1107
2
1.000 R = 0.9827

0.800
Absorbance

absorbance
0.600 Linear (absorbance)

0.400

0.200

0.000
0.00E+00 1.00E-05 2.00E-05 3.00E-05 4.00E-05 5.00E-05 6.00E-05 7.00E-05 8.00E-05
Fe+2 Concentration (mol/L)

Slope = 15660 = ε1 l1
Equation = y = 15660x-0.1107

My unknown Absorbance = 0.112

-5
Concentration of unknown sample from extrapolation: 1.42 * 10 mol/L

Data Analysis
Visual Inspection Method:
Known Absorbance of Stock 10 mL Solution = Unknown Absorbance
ε1 l1 c1 = ε2 l2 c2
l1 c1 = l2 c2
-4
(41.0 cm)(1.79*10 mol/L) = (56.0 cm)*([Fe unknown])
-4
[Fe unknown] = 1.31*10 mol/L

Page 9 of 11
Questions

1. Iron (III) reacts with water by a hydrolysis reaction. In order to prevent this
hydrolysis, acid has been added to the standard solution (and you add H2SO4 to
the unknown sample). How would your results change if no acid had been added
to the standard iron solution or to the unknown solution?

If no acid was added to the standard iron solution or the unknown solution, the Fe+3
would not be soluble. Adding the acid will allow the Fe+2 to form, thus allowing it to form
the complex.

2. A MCE student, Matt, omitted the hydroxylamine HCl from the solution he
prepared to determine the amount of iron in his unknown. What effect is that
omission likely to have on his result?

Hydroxylamine HCl, (NH3OH+Cl-) was added as a reducing agent to intercept oxygen

and prevent oxidation of ferrous iron to ferric iron. If Matt omits hydroxylamine HCl,
oxidation will occur and the Iron complex would not form.

3. Beer.s Law states that A=ε l c. How does the visual inspection colorimetric
method demonstrate this Law?

By visual inspection colormetric method, two of the same solutions are compared under
a light box and the amount of solution (l) are adjusted so that the absorbance of solution
#1 (standard) = absorbance #2 (unknown). Also, each solution has a defined
concentration. As the length of solution varies in each test tube the amount of light
traveling through each solution will be equal to each other.. The length of each test
tube will be proportionate to the concentrations of each solution. We know l1,c1, l2.
Therefore, we can determine the concentration of the unknown solution. (Refer to data
section)

A=ε l c ε1 l1 c1 = ε2 l2 c2

4. During our colorimetric determination by visual inspection, we are using test

tubes with rounded bottoms. How does this affect your results?

Round test tubes do not affect our results greatly because we are consistently using
round test tubes for each solution. The visual inspection method is comparing two
samples to each other not to a standard. Therefore the rounded bottom, which will
affect the length of the solution column, does not affect our calculations of
concentration. Also, if one measures the length of the test tubes from the same bottom
point on each test tube, the roundness should not affect our results greatly. Both tubes
were treated as “identical”. The light was allowed to enter the filled tubes the same way
and the length was measured the same way using a millimeter ruler.

Page 10 of 11
Conclusion

In this lab, Iron (III) was acidified in order to produce a soluble Iron (II) solution. The Iron (II)
was then added to o-phenathroline to form a stable soluble complex, Iron (II)-o-phenanthroline
(See Figure 1). This soluble complex was made in several solutions with known concentrations.
The Beer-Lambert Law (A=εεlc) was used to determine the absorption of the unknown. A
calibration curve was constructed using data from 13 other sources and used to compare to the
concentration of an unknown sample (See Figure 2). The concentration via extrapolation of the
unknown compound was 1.42 * 10-5 mol/L. Visual inspection was used as well to compare the
known concentration to the unknown concentration. The length of the solutions in the test tubes
were adjusted to give similar concentrations. The concentration for the unknown using visual
inspection was 1.31*10-4 mol/L. The visual inspection method and extrapolation method gave
values for the unknown that were off by a power of 10. Human error must have played a role in
the visual inspection method. Reasons why the numbers were so far off cannot be fully
explained at this time. The extrapolation method is much more reliable in determining the
concentration of the unknown.

References

Atkins, R.C. (1975) .Colorimetric determination of iron in multivitamins. Journal of Chemistry

Education. 52 (550).

Department of Chemistry (2000) General chemistry laboratory manual. University of

Pennsylvania.

Frantz, H. & Maim, L. (1970) .Reversible reactions and chemical equilibrium.. Laboratory
studies in general chemistry. Freeman. 1041.

Nelson, J. & Kemp, K. (2000) Chemistry the central science. Brown, LeMay, & Bursten.

Page 11 of 11