Experiment 23 Determination of the Fe3+ Concentration of Water with Spectrophotometry
Purposes
1．Learn to determine Fe3+ concentration in water sample with spectrophotometry
2．Learn to operate 721 Spectrophotometer
Principles
The Lambert-Beer law provides the mathematical correlation between absorbance and
concentration. It is usually stated as
A=εbc
A is the absorbance, ε is the molar absorptivity or extinction coefficient (L·mol-1·cm-1) that is
characteristic of the absorbent solute. Because the pathlength of the radiation through the cell is
identical with the cell thichness b andεis constant , the absorbance A will depend linearly on the
concentration of the absorbent solute that allow the proportionality A∝C to be converted to an
－
equation. When the concentration is in molarity units(g·L 1),then A＝abc, here a is called
－
－
absorptivit (L·g 1·cm 1).
The relationship between absorbance and concentration serves as the basis for the quantitative
analysis of a great many substances.
In the experiment, we should determine the absorbance of an unknown concentration of
absorbent medium and a known concentration of standard absorbent medium respectively. Then
set the following equation:
A(standard )
abc(standa rd)

A(unknown)
abc(unknow n)
Because all the cuvette in the spectrophotometer have the same thickness, that is, both the
absorbent medium have the same thickness b, and both the absorbent mediums are the same
absorbent, so we can calculate:
A(standard )
c(standard )

A(unknown)
c(unknown)
c(unknown) 
A(unknown)
 c(standard )
A(standard )
The way of treating date above is called comparison method.
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In practice, in order to omit the calculation and increase the accuracy of the determination, the
absorbances of a series of solutions of known concentrations are measured and a plot of
absorbance versus concentration is prepared. Such a plot represents a standard curve (or working
curve) for the particular system being studied. An unknown solution containing the same
absorbent substance may then be analyzed by measuring its absorbance, locating it’s a value on
the working curve, and reading the corresponding concentration. That concentration of sample
solution is measured by working curve this way is called working curve method.
Different substances have different ability to absorb the different light of different wavelength.
According to the shape of the curve of absorption spectrum, the λ of the absorption peak, and the
relationship between the absorbance and the concentration, and spectrophotomatry can be the
qualitative analysis.
The spectrophotometry usually uses the optimum wavelength. A graphical plot of absorbance
versus wavelength is referred to as an absorption spectrum. These are prepared by measuring the
light absorbed by a solution with different known wavelength. By referring to your plots of
absorbance versus wavelength, select λmax to study the relationship between the absorbance and
concentration.
Moreover, the acidity of the solution, the amount of the color developing reagent, the
temperature of coloration, the time and the substances, and so on. All these facts can affect the
determination. You can choose a proper condition of determination.
1 O-phenanthroline Method
In spectrophotometric determination of trace of Fe3+ ions, 1, 10-phenanthroline is a sensitive
color-developing agent, with which a complex of Fe2+ is formed to give orange red color
(lgKs=21.3). As this colored solution is measured with a spectrophotometer, maximum absorbance
is observed at 510nm. Molar absorption coefficient is equal to 1.1×104L·mol-1·cm-1. In the range
of pH 3 to 9, the complex is very stable. Iron must be in ferrous state and hence a reducing agent
is added before the color is developed. Hydroxylamine hydrochloride can be used to reduce Fe3+
to Fe2+. These reactions are given below:.
－
2Fe3++2NH2OH·HCl=2Fe2++N2↑+2H2O+4H++2Cl
2+
N
N
+
3
2+
=
Fe
N
Fe
N
3
1.1 Apparatus and Reagents
Apparatus: 721-Spectrophotometer, volumetric flask (50mL×7), measuring pipets (1mL,
2mL×2, 5mL, 10mL), analytical balance, sucker
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Reagents: 8mmol·L-10-Phenanthroline (fresh), 1.5mol·L-1 hydroxylamine hydrochloride
(fresh), 1mol·L-1 sodium acetate, standard ferrous sulfate solution (2.000mmol·L-1)
1.2 Procedure
1. Preparation of standard solutions and sample solutions of iron.
According to the volumes of various reagents listed in the table below. Pipet reagent solution into
each of the seven volumetric flasks. Fill them with distilled water to the assigned volume and mix
thoroughly.
According to the table listed below to prepare seven solutions with volumetric flash and
measuring pipet. The seven solutions are the blank solution, a serial of standard solution and the
sample solution.
Table Ⅱ-1 Preparation of the working curve and determination of trace Fe3+ ions in a sample
solution
Experiment No.
Fe2+standard/mL
NaOH/mL
HCl/mL
NaAc/mL
c(Fe2+ diluted)/μg·L-1
1
（blank）
2
3
4
5
6
7
0
0.40
0.80
1.20
1.60
2.00
1.0
2.0
5.0
1.0
2.0
5.0
1.0
2.0
5.0
1.0
2.0
5.0
1.0
2.0
5.0
1.0
2.0
5.0
10.00mL
sample
1.0
2.0
5.0
Absorbance (A)
2. Determination of absorption spectrum
Before the experiment, be sure to read the operation manual of spectrophotometer carefully. Set
the wavelength dial at 450nm, and adjust the instrument to read 0% T with no cuvette and 100%T
when the reagent blank (No. 1 solution)-filled cuvette is in the sample holder. Place No. 4 of table
Ⅱ-8-1 standard solution in your second cuvette and insert it into the sample holder. Read the A of
the standard solution from the dial. Repeat this procedure at 10-nm intervals from 450nm to
560nm. Be sure to set the instrument to 0% absorbance and 100%T with the blank solution after
each change of the wavelength.

Weight 0.7842g of (NH4)2Fe(SO4)2·6H2O accurately, put it into a beaker, add 120mL of
hydrochloric acid (6 mol·L-1) and small volume distilled water to dissolve it. Then transfer the
solution to a 1000mL volumetric flask and fill to mark with distilled water. Homogenize the
solution by shaking the flask.
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Table Ⅱ-2 The determination of absorption spectrum
λ（nm）
A
λmax
Absorption spectrum:
3. Preparation of the working curve
Transfer the standard solutions and blank solution to clean cuvettes, and determine the
absorbance for each of the five standard solutions at the wavelength that corresponds to maximum
absorption for the absorption spectrum, use the blank solution as reference. Prepare a working
curve by plotting absorbance (ordinate) versus the concentration of Fe2+ (abscissa).
4. Determination of trace Fe3+ ions in a sample solution
Under the same conditions with Determination of the working curve, determine the A of the
sample solution, and then find out the concentration of Fe3+ ions by using the comparison method
and the working curve method respectively.
1.3 Questions
1. Why is the absorbance of the solution better to be controlled in the range of 0.2-0.8? How
to controlled it?
2. Is the concentration of Fe3+ ions found out on the working curve the concentration of Fe3+
ions of the original sample solution?
3. According to this experiment, point out the advantage and disadvantage of comparison
method and working curve method.
2 Sulfocyan (MCNS) Method
Principle
Visible spectrometry only apply to the colored materials, but dilute solution of Fe3+ ions is
almost colorless. KCNS is used as a color development reagent in our experiment. Fe3+ ion can
react with CNS- ion and produce red [Fe (CNS) 6]3- complex ion (lgKs=6.4):
Fe3+ + 6 CNS-
[Fe (CNS) 6]3-
The gradation of color of the solution will be in proportion to the concentration of Fe3+ ions
In this experiment, the concentration of CNS- must be much larger than the concentration it needs
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to react with Fe3+ ions. Some HNO3 should be added into the sample solution to prevent the Fe3+
ions from hydrolysising. Furthermore, Fe3+ ions will be reduced to Fe2+ ions by CNS- ions slowly,
so (NH4)2S2O8 also should be added into the sample solution as a strong oxidant to prevent Fe3+
ions from being reduced.
2.1 Apparatus and Reagents
Apparatus: 721-type spectrophotometer, volumetric flasks (50mL, 1000mL), measuring
pipets (5mL, 10mL), sucker
Reagents: 4mol·L-1 KCNS, HNO3 (1:1), concentrated H2SO4, Fe3+ standard solution
2.2 Procedure
1. Prepare standard solutions and a water sample solution
Get 7 volumetric flasks (50mL) and use the measuring pipets to transfer those solutions into
the volumetric flasks (according to the tableⅡ- 8-3). Add distilled water until the meniscus
descends to the ring mark. Now we get a series of standard solutions, blank solution and water
sample solution, each of them has a different concentration of Fe3+ ions.
TableⅡ-8-3: Preparation of Fe3+ standard solutions and a water sample solution
Experiment NO.
Fe3+
standard solution /mL
1
(Blank solution)
2
3
4
5
6
0
0.50
1.00
1.50
2.00
2.50
1.0
5.00
1.0
5.00
1.0
5.00
1.0
5.00
1.0
5.00
1.0
5.00
2.5%NH4Fe(SO4)2 /drop
1
1
1
1
1
1
c(Fe3+)/mg·L-1
0
1.00
2.00
3.00
4.00
5.00
HNO3 /mL
4mol·L-1KCNS /mL
7
10.00
(water
sample)
1.0
5.00
2. Determination of absorption spectrum
Here we use 721-type spectrophotometer to determine the absorbance of NO.4 of tableⅡ-8-3
standard solution by the homogeneous light from 400nm to 560nm. Use blank solution as
reference solution. Determine the absorbance of No. 4 standard solution once per 10nm-20nm

Preparation of 0.1mg /1.00mL Fe3+ standard solution: Weigh 0.8640g NH4Fe (SO4)2·12H2O,
dissolve it with a little distilled water and then add 5mL concentrated H2SO4 to the solution. Allow
the solution to cool to room temperature, pour it into a volumetric flask (1000mL), and add
distilled water until the meniscus descends to the ring mark. (0.1mg /1.00mL Fe3+solution)
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from 400nm-560nm. Record the results and draw an absorption spectrum. Find the maximum
absorption of the wavelength (λmax).
TableⅡ-8-4 Determination of absorption spectrum
λ/nm
Absorbance (A)
λmax
3. Determination of absorbance (A)
According to the usage of 721-type spectrophotometer, select the λmax from the absorption
spectrum which you have determined above, or use λ=480nm (this wavelength is from some
reference books). Select suitable sensitivity of the spectrophotometer and use blank solution as
reference solution to determine absorbance of each standard solution and water sample solution.
Record the results.
4. Draw a working curve
＋
Draw a working curve. The abscissa is the concentration of Fe3
ordinate is the absorbance of the standard solutions.
of standard solutions, the
5. Determine the [Fe3+] of water sample solution.
We can look up the concentration of the unknown solution from the working curve after its
absorbance was determined by comparison method and working curve method.
2.3 Questions
＋
1. Why should we add excess KCNS solution during the color reaction of Fe3
ion?
3 Sulfosalicylic Acid Method
Principle
Fe3+ ions can react with sulfosalicylic acid (H2Ssal) and produce many kinds of complex ion.
The complex ion has different composition and different color under different pH environment.
For example, a violet red [FeSsal]+ ion is formed under pH=1.8-2.5, a brown [Fe (Ssal) 2]- ion is
formed under pH=4-8 and a stable yellow [Fe (Ssal) 3]3- ion is formed under pH = 8.0～11.5. Fe3+
ion is prone to hydrolysis and produce precipitation, Fe (OH) 3 when pH>12. Colorimetric
determination is impossible to perform under this condition. So this method should be used under
pH<12 and pH should be kept as a constant. In this experiment, we use Fe3+ ions to react with
sulfosalicylic acid and produce brown [Fe (Ssal) 2]- ions under a condition of HAc-NaAc buffer
solution (pH=5). Determine the absorbance with λ=466nm
3.1 Apparatus and Reagents
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Apparatus: 721-type spectrophotometer, volumetric flasks (50mL×8), measuring pipets
(5mL, 10mL), sucker
Reagents: 0.1g·L-1 Fe3+ standard solution (, formulation is same as ‘sulfocyan method’), 10%
sulfosalicylic acid solution, pH=5HAc-NaAc buffer solution
3.2 Procedure
1. Prepare standard solutions and a water sample solution
Get 8 volumetric flasks (50mL) and use the measuring pipets to transfer those solutions into
the volumetric flasks (according to the tableⅡ- 8-5). Add HAc-NaAc buffer solution (pH=5) until
the meniscus descends to the ring mark. Now we get a series of standard solutions, blank solution
and water sample solution.
TableⅡ-8-5 Preparation of Fe3+ standard solutions and a water sample solution
Experiment NO.
Fe3+
standard
solution /mL
sulfosalicylic acid
solution /mL
c(Fe3+)/mg·L-1
1(Blank)
2
3
4
5
6
7
8
0
0.50
1.00
1.50
2.00
2.50
3.00
10.00mL
(water sample)
5.00
5.00
5.00
5.00
5.00
5.00
5.00
5.00
0
1.00
2.00
3.00
4.00
5.00
6.00
2. Determination of absorption spectrum
Here we use 721-type spectrophotometer to determine the absorbance of NO. 4 standard
solution by the homogeneous light from 400nm to 560nm. Use blank solution as reference
solution. Determine the absorbance of No. 4 standard solution once per 10nm-20nm from 400nm
to 560nm. Record the results and draw an absorption spectrum. Find the maximum absorption of
the wavelength (λmax).
TableⅡ-8-6 Determination of absorption spectrum
λ/nm
Absorbance (A)
λmax
3. Determination of absorbance (A)
According to the usage of 721-type spectrophotometer, select the λmax from the absorption
spectrum which you have determined above, or use λ=466nm (this wavelength is from some
reference books).Select suitable sensitivity of the spectrophotometer and use blank solution as
reference solution to determine absorbance of each standard solution and water sample solution.
Record the results.
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4. Draw a working curve
＋
Draw a working curve. The abscissa is the concentration of Fe3
ordinate is the absorbance of the standard solution.
of standard solutions, the
5. Determine the [Fe3+] of water sample solution.
We can look up the concentration of the unknown solution from the working curve after its
absorbance is determined by comparison method and working curve method.
3.3 Questions
1. Why should we add buffer solution to all volumetric flasks until the meniscus descends to
the ring mark?
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