Grace Kim
(Lab Partners: Loreen Holstein, Mark Hayden)
Chem 506
7/31/2008
Colorimetric Determination of Iron
Introduction
In this lab, the absorbance of various solutions with known concentrations of iron (bound in a colored iron-
phenanthroline complex) were measured to determine a calibration curve. The absorbance of an unknown was then
determined both quantitatively (with a spectrometer) and qualitatively (by sight comparison) to assess the amount of
iron in the unknown. The relationship that links absorbance with concentration and path length is given by Beer-
Lambert’s law:
A = Ecl (1)
where A = absorbance of the sample
E= extinction coefficient (a.k.a constant of proportionality)
c = concentration
l = pathlength of sample
The following equations show the two reactions that take place in the lab: the conversion from ferric iron to ferrous
iron (rxn. 1) and the complexing of ferrous iron with o-phenanthroline (rxn. 2):
4 Fe+3(aq) + NH2OH (aq) 3 Fe+2(aq) + N2O(aq) + 4 H+(aq) + H2O(l) (rxn. 1)
Fe+2(aq) + 3 C12H2N2 (aq) [(C12H2N2)3Fe]+2(aq) (orange-red complex) (rxn. 2)
Procedures:
For details, see pp. 4-7 of “Pre-Lab Notes: Colorimetric Determination of Iron” MCEP UPenn Chem 506 Lab. 2-mL of the
unknown solution was diluted to 50-mL in accordance with lab procedure (1/25 dilution) and then 10-mL of that
solution was diluted out to 50-mL again (1/5) before measurements were made. Therefore, measurement of the
unknown are upon a 1/125 dilution of the original solution containing all of the unknown measured.
Materials used (beyond common lab glassware):
Spec 20, set at 510 nm
Total amount needed Chemical
+3
50-mL Stock Fe solution (0.050 mg Fe / ml)
5-mL 1 M ammonium acetate (NH4C2H3O2), buffer to maintain pH = 3.5
5-mL 10% Hydroxylamine HCl (NH3OHCl), reducing agent
50-mL 0.3% o-phenanthroline (C12H2N2)
10 drops 3 M H2SO4
~0.1 g Unknown #217
Safety:
Detailed hazard information can be found at http://www.flinnsci.com/search_MSDS.asp
Ammonium acetate solution is not considered hazardous. Hydroxylamine hydrochloride is moderately toxic by
ingestion and corrosive to body tissues. O-phenanthroline is highly toxic by ingestion and a mild skin irritant. Sulfuric
acid is corrosive to eye, skin, and all other body tissues, and generates considerable heat when diluted with water.
Since solutions used are dilute and students are wearing gloves and goggles, the hazards posed by exposure are
minimized. If exposed to solutions in lab, students should rinse area of contact with soap and water. Solutions may be
disposed of safely in the sink with plenty of running water.
Data:
Table 1: Calibration curve data
Concentration Absorbance (at λ = 510 nm)
0.0005 mg Fe/ mL 0.108
0.0010 mg Fe/ mL 0.203
0.0015 mg Fe/ mL 0.275
0.0020 mg Fe/ mL 0.393
Table 2: Unknown #217 Data and Results
Unknown Number #217
Mass of unknown 0.1036 g
Absorbance of diluted Trial 1: 0.173 Average: 0.1755
unknown solution (1/125
of original solution) Trial 2: 0.178
Qualitative sight Absorbance (color intensity) was the same when:
comparison data
0.0005 mg/ mL: 10.6 cm pathlength in test tube
1/125 dilute unknown: 6.10 cm pathlength in test tube
% of Fe in unknown Quanititatively (using calibration curve from Spec20 absorbances) : 5.41%
Qualitatively (using sight inspection and pathlength variation): 5.24%
Calculations:
Calculation of Fe in unknown using visual inspection and pathlength variation:
For the unknown dilute solution (1/125 of original solution in the second solution),
A1 = A2 Ec1l1 = Ec2l2
c1 = c2l2/l1 = (0.0005 mg/mg)(10.6 cm)/(6.1 cm) = 8.69 x 10-4 mg/mL in second dilution
Concentration in first dilution (5x final dilution): M1V1 = M2V2 so M1V1/V2
(8.69 x 10-4 mg Fe/mL)(50 mL)/(10mL) = 0.00434 mg Fe/mL in first dilution
Concentration in original solution (25x first dilution) : M1V1 = M2V2 so M1V1/V2
(0.00434 mg Fe/mL)(50mL)/(2 ml) = 0.109 mg Fe/mL in original solution
Mass of Fe in original solution: (0.109 mg Fe/mL)(50.0 mL) = 5.43 mg Fe = 0.00543 g Fe
% Fe in unknown = 0.00543 g Fe/ 0.1036 g unknown x 100 = 5.24%
The trendline for the data is: Absorbance at 510 nm = 190.6 (Fe concentration in mg/ mL) + 0.0052.
This can be rearranged so that Fe concentration in mg/ mL = (1/190.6)(Absorbance at 510 nm – 0.0052)
Calculation of Fe in unknown using calibration curve:
For the unknown dilute solution (1/125 of original solution in the second solution),
Fe concentration in mg/ mL = (1/190.6)(0.1755 – 0.0052) = 8.93 x 10-4 mg Fe/ mL in second dilution
Concentration in first dilution (5x final dilution): M1V1 = M2V2 so M1V1/V2
(8.93 x 10-4 mg Fe/mL)(50 mL)/(10mL) = 0.00447 mg Fe/mL in first dilution
Concentration in original solution (25x first dilution) : M1V1 = M2V2 so M1V1/V2
(0.00447 mg Fe/mL)(50mL)/(2 ml) = 0.112 mg Fe/mL in original solution
Mass of Fe in original solution: (0.112 mg Fe/mL)(50.0 mL) = 5.60 mg Fe = 0.00560 g Fe
% Fe in unknown = 0.00560 g Fe/ 0.1036 g unknown x 100 = 5.41%
Conclusions/ Discussion:
Unknown #217 was determined to have 5.41% Fe with the calibration curve method and 5.24% Fe with the visual
inspection/ pathlength variation method.
The calibration curve method is more likely to be accurate because it uses absorbances precisely determined by a
spectrometer and because it uses four point of measurement and a blank to determine the linear relationship
between absorbance and concentration rather than a single observation. Using a precise instrument and
increasing the number of observations decreases error and makes this method more accurate unless a single
observation skews the trendline.
The visual inspection/ pathlength variation method is fairly accurate, but there is significant error if the pathlengths
of the test tube are not long enough to be far away from the curved bottom of the test tube, which will
disproportionately affect the color intensity observed of the sample with the shorter pathlength. Also, care must
be taken to select two test tubes that are similar enough that differences do not affect the color intensity observed.
Since the unknown was diluted twice, there is an introduction of error with each dilution. This error, however,
cannot be avoided since to take a direct 1/125th dilution of the original solution would make the aliquot used so
small that the % error introduced by the use of a 10-mL graduated cylinder would be significant.
Post-lab Questions:
1. Fe+3 + 3 H2O Fe(OH)3
Iron (III) hydroxide precipitates out of solution, decreasing the amount of iron dissolved in solution that is
available to complex with the o-phenanthroline.
2. The hydroxylamine HCl is the reducing agent that intercepts oxygen and prevent oxidation of ferrous iron to
ferric iron. If omitted, a different complex with a different color and optimum absorption could form. Or,
perhaps no colored complex would form.
3. A1 = A2 Ec1l1 = Ec2l2 c1 = c2l2/l1
The visual inspection demonstrates this law because absorbances can be made equal by adding or removing
colored solution (increasing or decreasing path length). When the color intensities are the same, the
absorbances are assumed to be equal. Since the extinction coefficient E is the same, the concentration of the
unknown solution can be determined by calculation using the known concentration and the pathlengths
measured.
4. The rounded bottoms on the test tubes may have made the % calculated in this lab less than it should have
been. While the absolute error introduced by the rounded error is the same for both test tubes, the % error (%
the absolute error comprises of the measurement) is greater in the sample with the smaller pathlength and the
rounded bottom would disproportionately affect the sample with the smaller path length. This would make
the sample seem lighter in color intensity than it should. In this lab, this may help explain the minor
discrepancy between 5.41% (quantitative) and 5.24% (qualitative).
