Experiment 7: Spectrophotometric Determination of the Stoichiometry of a Complex Pabilane, Alma L. dela Cruz, Marie Giecel V. Group 6, Chem 27.

1, SEJ1, Ms. Noime Walican March 5, 2011 I. Abstract Stoichiometry of the complex formed by Iron (II) and 1,10-phenanthroline is determined using the three methods involving the use of spectrophotometry, namely, continuous-variations method (CVM), mole-ratio method (MRM) and sloperatio method (SRM). The solutions of the two compounds of different proportions were subjected to UV-Vis absorbance spectroscopy. The absorbance is plotted against the mole fraction (in the case of CVM) or mole ratio (in the case of MRM) or concentration of Iron (II) or phenanthroline (in the case of SRM). Based on the methods used and the experimental results, the stoichiometry of the complex of Iron (II) and phenanthroline is 1:3. II. Keywords: spectrophotometry, stoichiometry, absorbance, continuous-variations, mole-ratio, slope-ratio III. Objectives In this experiment, the students must be able to: (1) Determine the formula of a colored complex by the continuous variations method; (2) Determine the formula of a colored complex by the mole-ratio method; (3) Determine the formula of a colored complex by the slope-ratio method. IV. Introduction and Principles Several analytical techniques have been developed for the determination of metal ions in solution. Among these methods are: electrochemical oxidation reduction, ion-chromatography, atomic emission or absorption and measuring the absorbance of a complex formed between the metal and a complexing agent. Spectrophotometry is a valuable technique for this purpose because absorbance measurements can be made without perturbing the equilibrium of the system being examined. Spectrophotometric methods of determination based on UV-Vis absorption are probably the most often used than any other analytical method. Three most common methods of determining the stoichiometry of complexes using spectrophotometry are continuous- variations method, mole-ratio method and slope-ratio method. In this experiment the stoichiometry of complex formation for the metal ion Fe+2 and the complexing agent 1, 10 -phenanthroline (otherwise known as ophenanthroline C12H8N2) sometimes referred to as a ligand will be studied. Several solutions of this 2 substances will be prepared in different proportions, and the will be undergone the 3 methods mentioned above to determine the stoichiometry of the complex formed between Fe2+ and phenanthroline. Table 1. Volumes of Iron and Phenanthroline added to each flask for Continuous Variation Method Flask mL iron soln mL phenan. soln A 0 10 B 2 8 C 4 6 D 6 4 E 8 2 F 10 0 V. Methodology and Materials A. Continuous-Variations Method Six clean 50-mL volumetric flasks were labeled A to F. The reagents were pipetted into the flasks in the following order: iron solution, 5 mL acetate buffer, 1 mL hydroxylamine, hydrochloride, 0.0007 M phenathroline solution. Table 1 gives the amounts of iron solution and phenanthroline added in each flask.

The solutions in the volumetric flasks were diluted to mark and mixed thoroughly. After 10 minutes, the absorbance of each solution was measured at 508 nm using distilled water as reference. The mole fraction of each component in the solution was calculated. The mole fraction of iron was plotted against absorbance. The linear portions of the plot were extrapolated and the intersection would indicate the stoichiometry of the complex. B. Mole-Ratio Method Six clean 50-mL volumetric flasks were labeled A to F. Into each flask, the reagents were pipetted in the following order: 2 mL of the standard iron solution, 5 mL of acetate buffer, 1 mL of hydroxylamine hydrochloride solution. The solution was mixed thoroughly. Then, 0.0007 M phenanthroline was added. The amount of phenanthroline added is given by Table 2. Page 1 of 5

Chem 27.1. Spectrophotometric Determination of Stoichiometry of a Complex

Table 3. Volumes of Phenanthroline added to each flask for mole-ratio Method Flask mL phenan. soln A 1 B 3 C 5 D 8 E 12 F 15

in part two. The stoichiometry of the complex is calculated from the slopes of the two graphs. VI. Results A. Continuous Variations Method Table 5. Data for Continuous-Variations Method of Determining Stoichiometry of Complex of Iron(II) and 1,10-phenanthroline Flask A B C D E F V of Soln (mL) Iron(II) Phenan. 0 10 2 8 4 6 6 4 8 2 10 0 Mole Fraction Iron(II) Phenan. 0 1.0 0.2 0.8 0.4 0.6 0.6 0.4 0.8 0.2 1.0 0 A 0.002 0.290 0.284 0.184 0.100 0.003

The solution was diluted to the mark of the volumetric flask with distilled water and mixed thoroughly. After 10 minutes, the absorbance of each solution was measured at 508 nm using distilled water as reference. The number of moles of phenathroline and the number of moles of iron were calculated. The molar ratio of phenanthroline to iron is plotted against absorbance. The linear portions of the plot were extrapolated and the intersection would indicate the stoichiometry of the complex. C. Slope-Ratio Method Five clean 50-mL volumetric flasks were labeled A to E. Into each flask, the reagents were pipetted in the following order: iron solution, 5 mL acetate buffer, 1 mL hydroxylamine hydrochloride solution and 0.0007 M phenanthroline. For the first part, where the volume of iron solution is constant, Table 3 gives the volumes of phenanthroline added in each flask. Table 3. Volumes of Phenanthroline added to each flask for Slope-Ratio Method Flask mL phenan. soln A 1 B 2 C 3 D 4 E 5

For the second part, where the volume of phenanthroline is constant, Table 4 gives the volumes of iron solution added in each flask. Table 4. Volumes of Phenanthroline added to each flask for Slope-Ratio Method Flask mL iron soln A 0.5 B 1.0 C 1.5 D 2.0 E 2.5

Figure 1. Plot of mole fraction versus absorbance; for Continuous Variation Method

Stoichiometry of Complex : 1:3 (metal to ligand) B. Mole Ratio Method Table 6. Data for Mole-Ratio Method of Determining Stoichiometry of Complex of Iron(II) and 1,10phenanthroline Flask Number of moles Phenan. Iron(II) 0.0021 0.0063 0.0105 0.0014 0.0014 0.0014 Mole phenan mole Fe(II) 1.50 4.50 7.50 A

The solutions were diluted to mark with distilled water and mixed thoroughly. After five minutes, the absorbance of each solution was measured at 508 nm using distilled water as reference. The concentration of iron was plotted against the absorbance from data in 1st part. The concentration of phenanthroline was plotted against absorbance from data

A B C

0.051 0.148 0.241

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D E F

0.0168 0.0252 0.0315

0.0014 0.0014 0.0014

12 18 22.5

0.303 0.296 0.297

M Absorbance

x 10-5 0.042

x 10-5 0.084

x 10-5 0.127

x 10-5 0.184

x 10-5 0.229

Figure 2. Plot of molar ratio (phenanthroline: iron) vs. absorbance; for Molar-Ratio Method

Figure 3. Plot of concentration of phenanthroline vs. Absorbance for Slope-Ratio Method where concentration of Fe(II) is constant

Calculations: Moles phenanthroline = MV A= (0.0021 M)(1 mL) = 0.0021 mols B= (0.0021 M)(3 mL) = 0.0063 mols C= (0.0021 M)(5 mL) = 0.0105 mols D= (0.0021 M)(8 mL) = 0.0168 mols E= (0.0021 M)(12 mL) = 0.0252 mols F= (0.0021 M)(15 mL) = 0.0315 mols Moles Iron(II) = MV = (0.0007 M)(2 mL) = 0.0014 mols Mole ratio = mol phenan./mol Fe(II) A= (0.0021)/(0.0014) = 1.5 A= (0.0063)/(0.0014) = 4.5 A= (0.00105)/(0.0014) = 7.5 A= (0.00168)/(0.0014) = 12 A= (0.0252)/(0.0014) = 18 A= (0.0315)/(0.0014) = 22.5 Mole Ratio = 9 = (0.0021)(y)/(0.0007)(x) y/x = ligand:metal ratio = 3:1 Stoichiometry of Complex: 1:3 (metal to ligand) C. Slope Ratio Method Table 7. Data for Slope-Ratio Method of Determining Stoichiometry of Complex of Iron(II) and 1,10phenanthroline (Iron is constant) Constant Amount of Fe(II) , 0.0007 M, 5 mL Flask A B C D mL phenan. 1 2 3 4 [C12H8N2] in 5.83 1.08 1.50 1.87 E 5 2.19

Table 8. Data for Slope-Ratio Method of Determining Stoichiometry of Complex of Iron(II) and 1,10phenanthroline (phenanthroline is constant) Constant 1,10-phenanthroline (0.0021 M), 5 mL Flask A B C D mL Iron(II) 0.50 1.0 1.50 2.0 [Fe(II)] in M 3.04 5.83 8.40 1.08 x 10-5 x 10-5 x 10-5 x 10-5 Absorbance 0.085 0.187 0.209 0.457 E 2.50 1.30 x 10-5 0.372

Figure 4. Plot of concentration of Iron(II) vs. absorbance; for Slope-Ratio Method where concentration of phenanthroline is constant

Calculations: [Phenanthroline] = MV/V total A = (0.0007 M)(1 mL)/(11+1mL) = 5.83 x 10-5 M B = (0.0007 M)(2 mL)/(11+2mL) = 1.08 x 10-5 M C = (0.0007 M)(3 mL)/(11+3mL) = 1.50 x 10-5 M

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D = (0.0007 M)(4 mL)/(11+4mL) = 1.87 x 10-5 M E = (0.0007 M)(5 mL)/(11+5mL) = 2.19 x 10-5 M [Fe(II)] = MV/V total A = (0.0007 M)(0.50 mL)/(11+0.50mL) = 3.04 x 10-5 M B = (0.0007 M)(1 mL)/(11+1mL) = 5.83 x 10-5 M C = (0.0007 M)(1.50 mL)/(11+1.50mL) = 8.40 x 10-5 M D = (0.0007 M)(2.0 mL)/(11+2mL) = 1.08 x 10-4 M E = (0.0007 M)(2.50 mL)/(11+2.50mL) = 1.30 x 10-4 M Slope Ratio = Slope of Part II (constant amount of ligand) Slope of Part I (constant amount of metal) = 3719.08/1172.49 = 3.17 Stoichiometry of Complex: 1:3 (metal to ligand) VII. Discussion The three most common methods employing spectrophotometry used to determine the stoichiometry of metal to ligand in complexes are Continuous Variations Method, Mole-Ratio Method and Slope-Ratio method. Absorbance is directly proportional to concentration, that is, as concentration of the substance becomes higher, absorbance increases as well. The graph is linear. Furthermore, the plot of each of the three methods would generate a line or lines that intersect when extrapolated. Continuous Variation Method (CVM) is based on the measurement of a series of solutions in which the molar concentrations of two reactants differ but their sum is constant. The absorbance of each solution is measured at a suitable wavelength (508 nm as used in the experiment). The mole fraction of one component given by the equation, Mole Fraction of A = moles of A/ total moles, is plotted against the absorbance. The linear portion of the plot is extrapolated and the intersection corresponds to the mole fraction of the components that indicate their stoichiometry. In Figure 1, the intersection is at the point where mole fraction of Iron(II) is at 0.25 and that of phenanthroline is 0.75. If we take the ratio of Iron(II) to phenanthroline, the ratio is 1:3. The maximum absorbance occurs at the molar ratio of the combining ratio of reactants, which is also taken to be the intersection of the extrapolated lines. If the complex absorbs less strongly than the reactants, a minimum would be observed. A continuous variation plot generally does not give valid result for reactants that form more than one complex. If only one complex is formed, the maximum absorbance should be independent of the wavelength used. Therefore, if the solutions absorbance were measured at different wavelengths, and the maximum varies, it suggests that more than one complex is present.

In Mole-Ratio Method (MRM), solutions containing equal amounts of the metal are treated with increasing amounts of the ligand. The plot will also yield two intersecting lines of different slopes when extrapolated if the reaction is sufficiently complete. In Figure 2, it can be seen that the intersection of the two lines is at molar ratio 9. Molar ratio of phenanthroline to Iron (II) is given by, Molar Ratio = moles phenan. = MV phenan. moles Fe(II) MV Fe(II) The intersection of the extrapolated lines corresponds to the ligand to metal ratio, hence, the stoichiometry of metal to ligand is still 1:3, because the concentration of phenanthroline solution used is thrice the concentration of the iron(II) solution used. The Slope-Ratio Method (SRM) is commonly used for weak complexes. The reaction between the metal and the ligand is forced to completion by adding large excess of either ligand or metal. Two sets of solutions are prepared. The first set of solutions has constant amount of the metal, and varying amounts of ligand; the other one has constant amount of ligand and varying amounts of the metal. If the ligand is present in excess, the concentration of the complex is governed by the concentration of the metal. Using Beers Law, A= b[MxLy] = bCM/x The plot of A vs. concentration of metal has slope b/x. If the metal is present in excess, the concentration of the complex is dependent on the concentration of the ligand. Using Beers law, A= b[MxLy] = bCL/y The plot of A vs. concentration of ligand has slope b/y. The ratio of the slopes is equal to y/x, which is ligand to metal ratio. In the experiment the slope of the graph in Figure 4, absorbance vs. concentration of iron(II), where ligand is in excess, is 3719.08. And the slope of the graph in Figure 3, absorbance vs. concentration of phenanthroline, where iron(II) is in excess, is 1172.49. The ratio of the slopes is 3.17. The reaction of Fe2+ and phenanthroline is shown below. Fe2+ + 3 o-Phen Fe(o-Phen)32+ Because Fe2+ can be oxidized by air to Fe3+, hydroxylamine hydrochloride is added. It serves as a mild reducing agent so that the oxidized Fe3+ will be reduced back to Fe2+. The reaction is shown below. 2 Fe3+ + 2 NH2OHHCl + 2 OH- 2 Fe2+ +N2 + 4H2O + H+ + ClThe acetate buffer is used to adjust the pH of the solution between pH 6 to 9.

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VIII. Conclusion and Recommendations There are three most common methods employing spectrophotometry that can be used to determine the stoichiometry of a complex or the ratio of metal to ligand in the complex. These are Continuous Variation, Mole-Ratio, and Slope-Ratio Methods. Based on the three methods used, the complex formed by Iron (II) and 1,10phenanthroline has stoichiometry of 1:3. This means that every molecule of iron in the solution can accommodate 3 phenanthroline ligands. The Mole-Ratio Method is better to use for complexes having large ligand to metal ratio, because the results are distinctly shown in the plot. The Continuous Variation Method is not ideal if the reactants form more than one complex. It is not also very useful for complexes with many ligands. The Slope-Ratio Method is the most prone to experimental errors. It is recommended that the volumes of the reagents be measured accurately because it directly affects the results. IX. References

1. Hargis L.(1988).Analytical Chemistry. PrenticeHall Inc.New Jersey. 2. Skoog, D.,West, D, Holler, F., Crouch, S. (2004).Fundamentals of Analytical Chemistry 8th ed.Thomson Learning Brooks/Cole.Singapore. 3. http://www.chem.uky.edu/courses/che226/labs/05 0-fe_absorption.pdf March 3, 2011 4. http://tonga.usip.edu/bentzley/expt5complex.htm March 3, 2011 5. http://libinfo.uark.edu/aas/issues/1968v22/v22a17. pdf March 4, 2011

I hereby certify that I have given substantial contribution to this report.

PABILANE, ALMA L. DELA CRUZ, MARIE GIECEL V.

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