Spectrophotometry Lab Report

EXPERIMENT 10: SPECTROPHOTOMETRYDetermination of Iron with 1,10-Phenanthroline
Name: _________________________________
Date: _______________ Unknown Number: ______________
Assigned Balance: _______________ Accuracy: ________________ Grade: ___________________
A. Absorption Spectrum
Submit a plot of your absorption spectrum of the Fe-phenanthroline complex with all data
points on a single plot.
Do not worry about uncertainties for Part A, but do include units.
Literature max for the Fe-phenanthroline complex: ________________
Wavelength used for assay (your max): ________________
How would you explain a difference between the literature value of max and the value that you
used to make your calibration curve?
B. Calibration Curve
Submit the calibration plot based on your data. Make sure to include error bars on the data
points.
Molar absorptivity of Fe-phenanthroline complex (with units & uncertainty): ___________________________
Slope of the calibration curve (with units & uncertainty): __________________________
Y-intercept of the calibration curve (with units & uncertainty): __________________________
R2 value of the calibration curve: ________________________
C. Determination of Fe in Unknown
Diluted Volume of Sample
(mL)
Signal of Fe in Sample
Solution
(Absorbance Units)
Mass of Fe in Original Sample
(g)
Show a typical calculation (using your actual data) for the mass of iron in the
original sample:
Show the calculations used to find the uncertainty in the mass of iron in your
original sample:
In a sentence, use the 95% confidence level to express the likely true value of the
amount of iron (in grams) in your unknown sample:
CHEM 300: Analytical Chemistry
E10-1
Experiment #10
Absorption Spectrophotometry:
Determination of Iron with 1,10-Phenanthroline
Purpose: the objective of this experiment is to learn the principles and practice of absorption
spectrophotometry. Specifically, the experiment consists of the spectrophotometric determination
of iron in unknown samples after complexation with 1,10-phenanthroline to form a visible complex.
The tasks to be performed in Experiment #10 include:
A.
B.
C.
D.
E.
F.
Preparation of the standard solutions.
Zeroing the spectrophotometer.
Determination of the lambdamax of the Fe-phenanthroline complex.
Preparation of a calibration curve from the standard solutions.
Determination of iron in an unknown sample.
Questions.
Introduction: Atoms or group of atoms called chromophores within a molecule are responsible for
the selective absorption of illuminating light by that molecule. Different materials have different
absorption characteristics because they contain different chromophores. The percentage of
illuminating light absorbed by a certain material is proportional to its concentration in solution
(Beer-Lambert’s law). This law can be employed for the determination of the concentration of a
solution containing analytes with chromophoric properties.
For example, the spectrophotometric method, which we will use for determination of iron, is based
on the formation of the orange-red 1,10-phenanthroline complex of Fe2+. 1,10-phenanthroline is a
weak base; in acidic solution the principal species is the phenanthrolinium ion, PhH +. The complex
is formed in the following reaction:
Fe2+ + 3 PhH+ + 3 H2O  Fe(Ph)32+ + 3 H3O+
The equilibrium constant for this reaction is 2.5×106 at 25°C. Quantitative formation of the complex
is observed in the pH region between 2 and 9; a pH of ~3.5 is recommended to prevent the
precipitation of various iron species. An excess of reducing agent (in this experiment, hydroxylamine
hydrochloride) is added to keep the iron in the +2 oxidation state.
4 Fe3+ + 2 NH2OH  4 Fe2+ + N2O + 4 H+ + H2O
In many situations the spectrophotometric determination of a particular metal ion via the
introduction of a color-forming reagent is complicated by the presence of other ions, which may
interfere through the formation of light-absorbing species. For example, Ni, Ag, Hg, Bi, Mo and W can
provide complexes with 1,10-phenanthroline, which interfere with the determination of
iron. Elimination of the interference is most often accomplished by the addition of a masking agent,
CHEM 300: Analytical Chemistry
E10-2
which ties up the interfering species and prevents it from forming a light-absorbing species with the
color-forming reagent. Alternatively, interferents can be removed by a separation method.
Experimental Section
1. Reagents and Solutions
a. standard iron (II) solution: prepare a standard iron solution by weighing 0.0702 g ferrous
ammonium sulfate, Fe(NH4)2(SO4)2•6 H2O. Quantitatively transfer the weighed sample to a 1
L volumetric flask and add sufficient water to dissolve the salt. Add 2.4 mL of conc. sulfuric
acid, dilute exactly to the mark with distilled water, and mix thoroughly. This solution
contains 10.0 mg iron per liter (10 ppm); if the amount weighed is other than specified above,
the calculate the correct concentration of the stock with all the necessary significant figures.
b. 1,10-phenanthroline solution: dissolve 100 mg 1, 10-phenanthroline monohydrate in 100 mL
water. Store in a plastic bottle.
c. hydroxylamine hydrochloride solution: dissolve 10 g hydroxylamine hydrochloride in 100 mL
water.
d. sodium acetate solution: dissolve 10 g sodium acetate in 100 mL of water.
e. 6 M HCl.
f. unknown: a solution of iron in water.
2. Apparatus
a. spectrophotometer (analog or digital)
b. a variety of volumetric pipets (1, 2, 4, 5, and 10 mL)
c. 100 mL volumetric flasks
d. pH-test paper (acidic range)
A. Preparation of the Standard Solutions
1. Obtain from your TA a sample of the iron standard solution (ca. 10 ppm Fe). Be sure to record
the exact concentration.
2. To make the least concentrated sample, use a pipet to accurately transfer 1.00 mL of the standard
iron solution into the 100 mL volumetric flask provided.
Dilute to about 50 mL with distilled water.
Add 1.0 mL of the hydroxylamine hydrochloride solution to the volumetric flask.
Add 5.0 mL of the 1,10-phenanthroline solution to the volumetric flask.
Buffer the solution by adding 8.0 mL of the sodium acetate solution.
Using pH test paper, make sure that the solution pH is between 3 and 5.
If the pH is above 5, add HCl solution to the volumetric flask.
Dilute to the mark with distilled water. Transfer the solution to a clean container and wait at least
15 minutes for full color development. After 15 minutes the sample can be placed in the
spectrophotometer.
10. Clean the volumetric flask thoroughly with distilled water. Repeat the above procedure with 2.00,
4.00, 5.00, and 10.00 mL aliquots of the standard iron solution. Use other beakers from your
drawer to hold the solutions while they develop, but make sure to label them.
11. To make the blank solution, repeat the above procedure, omitting step 2 (addition of iron
standard).
3.
4.
5.
6.
7.
8.
9.
CHEM 300: Analytical Chemistry
E10-3
B. Determination of the Lambdamax of the Fe-Phenanthroline Complex
1. Prior to coming to lab, find the wavelength of maximum absorbance (lambdamax) reported in the
2.
3.
4.
5.
6.
literature for the Fe-Phenanthroline complex. (you cannot receive full lab notebook points without
looking this up and recording it in your lab notebook before you come to lab)
Zero the spectrophotometer using the blank solution you made. This only needs to be done once.
Fill the other cuvette with the standard of highest concentration.
Set the spectrophotometer to 600 nm.
Take a reading using the standard solution. Repeat steps 4, 5, and 6 (from Part B) as you change
the wavelength by 20 nm increments and zero with the blank between each wavelength change.
Stop at 400 nm.
NOTE: At this point you have completed a “scan” by recording the absorption spectrum of your
standard solution within the range 600-400 nm. A plot of signal vs. wavelength will provide a
spectrum with a 20 nm resolution (the interval between readings). In order to determine the
maximum of the spectrum with better accuracy, reduce the size of the intervals:
7. In the region of highest response (this region should be 40 nm wide), repeat steps 4, 5, and 6
(from Section B) as you change the wavelength by 5 nm increments.
8. For greater assurance that the maximum response has been obtained, repeat steps 4, 5 and 6
(from Section B) over a narrower region (20 nm wide) using 2 nm increments.
9. Plot the absorbance against the wavelength, and include this plot in your lab notebook and in your
report. Make sure all data points are visible on your plot; three separate plots may be necessary.
10. Select an iron-phenanthroline peak most suitable for the determination of iron and determine its
lambdamax (the wavelength of maximum absorbance in the spectrum).
11. Compare your experimentally determined value for lambdamax to the one you found prior to
coming to lab. Show your plot and wavelength selection to the TA to confirm that you have
selected the correct lambdamax.
C. Preparation of a Calibration Curve from the Standard Solutions
1. Set the spectrophotometer to the value of lambdamax determined in Section C.
2. Measure the absorbance of each of the standard solutions of iron. Use the blank solution as the
reference.
3. Plot the absorbance of the standards against concentration in ppm.
NOTE: The procedure necessary to construct a calibration curve is explained in more detail in Handout
#3.
D. Preparation of Unknown Solutions
1.
2.
3.
4.
5.
To the unknown sample, which is furnished in a 100-mL flask, add ca. 50 mL of distilled water.
Add 1.0 mL of the hydroxylammonium chloride solution.
Add 5.0 mL of the 1,10-phenanthroline solution.
Buffer the solution by the addition of 8.0 mL of the sodium acetate solution.
Using the pH test paper, make sure that the pH of the solution is between 3 and 5.
CHEM 300: Analytical Chemistry
6.
7.
8.
9.
E10-4
If the pH is not between 3 and 5, add HCl dropwise until the pH is in this range.
Wait at least 15 minutes for full color development.
Dilute to the mark and mix thoroughly.
Measure the absorbance of each of the unknown solutions of iron at least three times. Use the
blank solution as the reference.
Analysis of Data: Use the calibration curve prepared in Section D and the instructions in Handout
#3 to infer the amount of iron from the 3 determinations of the unknown samples. Provide the mean
(in ppm) and the RSD for the concentration of iron in the 100 mL of solution.
E. Questions
The answers to these questions should be in your lab notebook when you turn in your carbon copy pages
at the end of the lab day. The ones that can be done in advance may be completed before you come to
the lab; if the questions require to you be in lab to answer them, you should leave space for them in your
notebook and be sure you have enough time in the lab to answer them.
1. Did your experimental wavelength of maximum absorption match the literature (theoretical)
value? If not, what is the most likely cause of the discrepancy?
Sample Preparation
[Iron] (ppm)
Aliquot Volume (mL)
1
2
4
5
10
9.951812
[Iron]
Unknown MeasurementAbsorbance (AU)
1
0.17
2
0.16
3
0.16
Wavelength (nm)
600
580
560
540
520
518
516
515
514
512
510
508
506
505
504
502
500
495
490
485
480
460
440
420
400
Absorbance (AU)
0
0.01
0.03
0.1
0.18
0.18
0.19
0.19
0.19
0.19
0.19
0.19
0.19
0.19
0.19
0.19
0.18
0.18
0.18
0.18
0.17
0.15
0.13
0.1
0.07
Absorbance (AU) at 510 nm
Measurement 1 Measurement 2Measurement 3
0.01
0.01
0.01
0.03
0.03
0.03
0.07
0.07
0.06
0.09
0.09
0.10
0.19
0.19
0.18
urement 3