EXPERIMENT NO. 7 SPECTROPHOTOMETRIC DETERMINATION OF THE STOICHIOMETRY OF A COMPLEX De Galicia, Bryan Paul D. Jorge, Jessica Ann B. I.

ABSTRACT Spectrophotometry is a useful tool in determining the composition of complex ion in solution and their formation constants. The primary advantage of this method lies in the fact that quantitative absorption measurements can be performed without disturbing the equilibria under study. A complex, the subject of this experiment, when subjected into the spectrophotometer absorbs maximally when given the proper ratio in concentrations and in the proper wavelength. In this experiment, the composition and formation constant of iron(II) to a non-absorbing ligand such as 1,10 phenanthroline was determined by measuring the absorbance decrease and increase that occur when solutions are mixed in different amounts. The reliability of this approach depends on the well-known values of the formation constant,Kf 21 = 2x10 , and the natural characteristic of the 1,10 phenanthroline complex of iron(II). To determine the stoichiometry of the complex formed by Iron(II) and phenanthroline, three methods under spectrophotometry were used continuous variation method (CV) , mole-ratio method (MRM) and the slope-ratio method (SRM). The solutions were mixed in different ratios and each was placed in a spectrophotometer to measure the absorbance for each solution. For each method, a graph was created to determine the stoichiometry of the 2+ complex - absorbance against mole fraction of Fe for CV, absorbance against the ratio of moles of phenenthroline to the moles of iron for MRM, and absorbance against concentration of iron and absorbance against the concentration of phenanthroline for SRM. The ratio of Fe(II) to 1,10 phenanthroline is known theoretically to be 3:1 (L:M), and thus having the 2+ formula, Fe(C12H8N2)3 . However, in this experiment, one method only gave out this correct ratio. The other two, MRM and SRM gave a 3:2 (L:M) ratio. This may be due to photo decay or improper dilution or even personal error in preparing the reagents. II. KEYWORDS: stoichiometric ratio, mole-ratio, slope-ratio, continuous variation, complexation, extrapolation, mole fraction WEJ1, Group 7, Mr. Dmitri Cordova October 2, 2013

III. INTRODUCTION Spectroscopy is the branch of science that is concerned with electromagnetic waves at a particular wavelength or range of wavelengths that are used for qualitative analyses as well as quantitative ones. UV-Vis spectrophotometry, a more specific term and under spectrometry is a method of quantitative analysis that can be used to find out the concentration of a specific analyte or analytes in a sample. How a spectrophotometer works is a beam of light at a particular wavelength, preferably the optimal absorption wavelength of the analyte if the identity is known, is passed through the sample in a cuvette with the clear side facing the

detector and the light source. The primary data that is collected at the detector is the transmittance of the analyte and is converted to -A the absorbance by the relation 10 = T wherein A is absorbance and T is transmittance. An absorption spectrum can be quantitatively related to the amount of material present using the Beer-Lambert Law: A = b[MxLy] where A is the absorbance of the solution, is the molar absorptivity, b is the path length of the cell, and [MxLy] is the concentration of the complex. Absorption spectroscopy is widely used in quantitative and qualitative chemistry, clinical chemistry, biology, forensic science, engineering, agriculture, and molecular and

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atomic physics. It is an analytical chemistry tool which determines the presence or absence of a particular substance and quantifies the amount of the substance present. It can be used to identify the presence of pollutants in the air, distinguishing the pollutant from the nitrogen, oxygen, water, and the other expected constituents. However, one downside of this method is that a high purity of the analyte is necessary or other substances in solution would also absorb light giving rise to a different, specifically higher reading of absorbance. Another is that a spectrophotometer, the machine itself, is expensive although that is most of the time, the price of accuracy. In this experiment, the absorbance of the phenanthroline iron(II) complex was measured in three methods: continuous variation, mole-ratio and slope-ratio. These data were then analyzed graphically wherein intersecting lines were extrapolated for a computation of the stoichiometric ratio. Knowledge of linear regression and/or algebra is necessary in this experiment as the linear equation is widely used in the graphical analyses. Theoretically, the complex has a 3:1, ligand to metal ratio, phenanthroline as the ligand and the ferrous ion as the metal and this is the answer the experiment aims to produce. IV. METHODOLOGY A. Continuous Variation Method In six 50-mL volumetric flasks labelled A to F, the following solutions were prepared according to this specific order-iron solution, 5 mL acetate buffer, 1 mL hydroxylamine hydrochloride and 0.0007 M phenanthroline solution. For each flask, the amount of iron solution and hydroxylamine hydrochloride varied based on the table provided. The solutions were then diluted to mark and mixed thoroughly after which they were set aside for 10 minutes; then the absorbance of each solution was read at 508 nm using distilled water as the reference or blank solution. The calculated mole fraction for each component in the solution was acquired. The mole fraction of iron as the x-axis was plotted with absorbance as the y-axis. Finally, from the graph, the stoichiometry of the complex was computed for.

Table 1: Dilution table for continuous variation method.

Flask mL iron solution mL phenanthroline

A 0 10

B 2 8

C 4 6

D 6 4

E 8 2

F 10 0

B. Mole-Ratio Method Once again, 50-mL volumetric flasks were acquired and endowed with the same labelling system. The following solutions were mixed according to this order 2 mL iron solution, 5 mL acetate buffer, and 1 mL hydroxylamine hydrochloride. 0.0007 M phenanthroline was added based on the amounts in the table below, diluted the solution to mark and set aside for 10 minutes. The group then measured the absorbance at 508 nm using the same reference as in part A. Afterwards, the moles of phenanthroline and the moles of iron, both in solution were computed for. Finally absorbance against the ratio of the moles of phenanthroline to the moles of iron was plotted. Data was recorded and the indicated stoichiometry of the complex based on the graphical extrapolation analysis was acquired.
Table 2: Dilution table mole-ratio method.

Flask mL phenanthroline

A 1

B 3

C 5

D 8

E 12

F 15

C. Slope- Ratio Method In 50-mL volumetric flasks, A to E were put on with labels and the following solutions were added according to this specific order 5 mL iron solution, 5 mL acetate buffer, and 1 mL hydroxylamine hydrochloride. The group then added the amount of 0.0007 M phenanthroline based on the instructions in the table below, diluted the solution to mark and was set aside for 5 minutes. Afterwards, the absorbance at 508 nm was measured using the same reference in part A. The steps were repeated but instead, the reagent constant used was 0.00210 M phananthroline and varying amounts of the iron solution were added based on the instructions in the lab manual. Finally, the data in the first set up with its absorbance against the concentration of the iron were plotted. The same was done for the second set up and then the

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group calculated for the stoichiometry of the complex using the two graphs.
Table 3: Dilution table for slope ratio method with varying phenanthroline.

Graphical Analysis
1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 Series1 0.5 Series2 1 Series3 1.5

Flask mL phenanthroline

A 1

B 2

C 3

D 4

E 5

Table 4: Dilution table for slope ratio method with varying ferrous ion.

Flask mL iron solution

A 0.5

B 1.0

C 1.5

D 2.0

E 2.5

V. RESULTS A. Results for Continuous Variation Method

Table 5. Results of continuous variation method.

V(mL) Soluti on A B C D E F Fe
2+

0 2 4 6 8 10

C12H 8N2 10 8 6 4 2 0

Mole Fraction 2+ Fe C12H 8N2 0 1.0 0.2 0.8 0.4 0.6 0.6 0.4 0.8 0.2 1.0 0

Absorbance 0 0.321 0.591 0.347 0.166 0.079

Figure 2. Graphical method extrapolation analysis of continuous variation method.

Stoichiometric Ratio (M:L): 1:3 Sample Computations: Flask A: iron(II) = 0mL/10mL = 0 phenanthroline = 10mL/10mL = 1.0 Flask B: iron(II) = 2mL/10mL = 0.2 phenanthroline = 8mL/10mL = 0.8 Flask C: iron(II) = 4mL/10mL = 0.4 phenanthroline = 6mL/10mL = 0.6 Flask D: iron(II) = 6mL/10mL = 0.6 phenanthroline = 4mL/10mL = 0.4 Flask E: iron(II) = 8mL/10mL = 0.8 phenanthroline = 2mL/10mL = 0.2 Flask F: iron(II) = 10mL/10mL = 1.0 phenanthroline = 0mL/10mL = 0 B. Results for Mole-Ratio Method
Table 6. Results of mole-ratio method.

Continuous Variation Method

0.7 0.6

Absorbance

0.5 0.4 0.3 0.2 0.1 0 0 0.5 1 1.5

Mol Fraction of

Fe2+

Figure 1. Graphical plot of absorbance versus mole fraction of the ferrous ion.

Solutio n A B C D E F

No. of moles 2+ C12H8N2 Fe 7E-7 1.4E-6 2.1E-6 1.4E-6 3.5E-6 1.4E-6 5.6E-6 1.4E-6 8.4E-6 1.4E-6 1.05E-5 1.4E-6

Absor bance 0.107 0.397 0.466 0.318 0.308 0.306

Mole Ratio 0.5 1.5 2.5 4 6 7.5

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Mole Ratio Method

0.5 0.4

Absorbance

Flask E: -6 nphenanthroline = (0.0007M)(.012L) = 8.4x10 -6 -6 mole ratio = 1.4x10 /8.4x10 = 6.0 Flask F: -5 nphenanthroline = (0.0007M)(.015L) = 1.05x10 -6 -5 mole ratio = 1.4x10 /1.05x10 = 7.5 C. Slope-Ratio Method Results
Table 7. Results of slope-ratio method (constant ferrous ion).

0.3 0.2 0.1 0 0 2 4 6 8

Solution A B C D E

Mole Ratio
Figure 3. Graphical plot of absorbance versus mole ratio of the complex.

mL of C12H8N2 1 2 3 4 5

[C12H8N2] 1.4E-5 2.8E-5 4.2E-5 5.6E-5 7E-5

Absorbance 0.116 0.247 0.359 0.447 0.565

Graphical Analysis
1.2 1 0.8 0.6 0.4 0.2 0 0 2 Series1 4 Series2 6 Series3 8 0.6 0.5 0.4 0.3 0.2 0.1

Slope-Ratio Method [Constant Fe2+]

Absorbance

y = 7842.9x + 0.0174 R = 0.9961

0 0.00E+00 2.00E-05 4.00E-05 6.00E-05 8.00E-05

[C12H8N2]
Figure 5. Graphical plot of absorbance against concentration of phenanthroline. Table 8. Results of slope-ratio method (constant phenanthroline)

Figure 4. Graphical method extrapolation analysis of moleratio method.

Stoichiometric Ratio (M:L): 2:3 Sample Computations: niron(II) = (0.0007M)(.002L) = 1.4x10

-6

Solution A B C D E

Flask A: -7 nphenanthroline = (0.0007M)(.001L) = 7.0x10 -6 -7 mole ratio = 1.4x10 /7.0x10 = 0.5 Flask B: -6 nphenanthroline = (0.0007M)(.003L) = 2.1x10 -6 -6 mole ratio = 1.4x10 /2.1x10 = 1.5 Flask C: -6 nphenanthroline = (0.0007M)(.005L) = 3.5x10 -6 -6 mole ratio = 1.4x10 /3.5x10 = 2.5 Flask D: -6 nphenanthroline = (0.0007M)(.008L) = 5.6x10 -6 -6 mole ratio = 1.4x10 /5.6x10 = 4.0

mL of 2+ Fe 1 2 3 4 5

[Fe ] 7.00E-06 1.40E-05 2.10E-05 2.80E-05 3.50E-05

2+

Absorbance 0.08 0.154 0.235 0.309 0.382

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Slope-Ratio Method [Constact C12H8N2]

0.5

Absorbance

0.4 0.3 0.2 0.1

y = 10843x + 0.0043 R = 0.9997

0 0.00E+00 1.00E-05 2.00E-05 3.00E-05 4.00E-05

[Fe2+]
Figure 6. Graphical plot of absorbance against concentration of ferrous ion.

[Fe(II)] = 7.0 x 10 M Flask B: [Fe(II)] = (0.0007mol Fe(II)) L -5 [Fe(II)] = 1.4 x 10 M Flask C: [Fe(II)] = (0.0007mol Fe(II)) L -5 [Fe(II)] = 2.1 x 10 M Flask D: [Fe(II)] = (0.0007mol Fe(II)) L -5 [Fe(II)] = 2.8 x 10 M Flask E: [Fe(II)] = (0.0007mol Fe(II)) L -5 [Fe(II)] = 3.5 x 10 M

-6

(1.0mL Fe(II)) (50mL solution)

(1.5mL Fe(II)) (50mL solution)

(2.0mL Fe(II)) (50mL solution)

(2.5mL Fe(II)) (50mL solution)

Sample Computations (C1): Flask A: [C12H8N2] C12H8N2)

Computation for Stoichiometric Ratio: x Fe + y C12H8N2 = Fex(C12H8N2)y

2+

(0.0007mol C12H8N2) (1mL L (50mL

solution) -5 [C12H8N2] = 1.4 x 10 M Flask B: [C12H8N2] = (0.0007mol C12H8N2) L solution) -5 [C12H8N2] = 2.8 x 10 M Flask C: [C12H8N2] = (0.0007mol C12H8N2) L solution) -5 [C12H8N2] = 4.2 x 10 M Flask D: [C12H8N2] = (0.0007mol C12H8N2) L solution) -5 [C12H8N2] = 5.6 x 10 M Flask E: [C12H8N2] = (0.0007mol C12H8N2) L solution) -5 [C12H8N2] = 7.0 x 10 M Sample Computations (C2):

Mass balance equations: 2+ MBE1: CFe2+ = [Fe ] + x[Fex(C12H8N2)y] MBE2: CC12H8N2 = [C12H8N2] + y[Fex(C12H8N2)y] If phenanthroline is in excess, [Fe ] 0 MBE1: CFe2+ = x[Fex(C12H8N2)y] CFe2+/x = [Fex(C12H8N2)y] If ferrous ion is in excess, [C12H8N2] 0 MBE2: CC12H8N2 = y[Fex(C12H8N2)y] CC12H8N2/y = [Fex(C12H8N2)y]
2+

C12H8N2) (2mL (50mL

C12H8N2) (3mL (50mL

Inputting into Beers law, A1 = bCFe2+/x A2 = bCC12H8N2/y mFe2+ / mC12H8N2 = (b/x)/(b/y) = y/x = 10843 / 7842.9 1.5 Stoichiometric Ratio (M:L): 2:3 VI. DISCUSSION Regarding the reagents used, the Fe(NH4)2(SO4)26H2O (ammonium ferrous sulfate), was the source of the ferrous ion used in the complexation reaction as the C12H8N2 (phenanthroline) was the source of the ligand. 2+ This reaction is theoretically Fe (aq) + 3 2+ C12H8N2(aq) = Fe(C12H8N2)3 (aq), which yields a

C12H8N2) (4mL (50mL

C12H8N2) (5mL (50mL

Flask A: [Fe(II)] = (0.0007mol Fe(II)) (0.5mL Fe(II)) L (50mL solution)

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3:1 (L:M) complex with a deep orange-red color. The NH2OHHCl (hydroxylamine hydrochloride) countered the rapid oxidation of the ferrous ion to the ferric ion which may also complex with the ligand but has a different wavelength and a pale color of blue. If some ferric ions were in solution, hydroxylamine hydrochloride reacts with it in: 4 3+ 2+ + Fe + 2 NH2OHHCl = 4 Fe + N2O + 4H + H2O + Cl . This is to ensure that all of the complex will have a ferrous central cation. The acid-base buffer at a pH of 4 prevented ferric and/or ferrous hydroxides from precipitating out of solution; however a strongly acidic solution is also not advised since the iron competes with hydronium because the ligand may be protonated, leaving no bounding areas for the metal cation. The wavelength at 508 nm was used since this is where the complex optimally absorbs. The flasks used in preparing the different reagents were immediately covered with foil to inhibit photo decay which makes the reagent prematurely absorb light. Incubation is also necessary to ensure the completeness of reaction. In the three parts of the experiment, the continuous variation, the mole-ratio and the slope-ratio methods were used to determine the stoichiometric ratio between phenanthroline and the ferrous ion. This is theoretically 3:1 (L:M), 2+ forming the complex ion Fe(C12H8N2)3 . In the first part, the continuous variation method was used, varying mole fractions of the ferrous ion and the phenanthroline were mixed in different flasks and each were measured for their respective absorbances. The basis for this method is because of the fact that maximum absorbance occurs at the correct combining ratio of the complex since the complex has maximum concentration in there as well, given the assumption that the free components do not absorb significantly. The maximum absorbance is taken as the intersection of the extrapolated straight-line portions of the curve to avoid uncertainty caused by rounding of the curve near the maximum absorbance. This extrapolation may be simulated using the linear regression program of a scientific calculator wherein the first three points are inputted, leading to its equation in the y=mx+b form; then the input of the latter points are followed giving rise to their equation as well. These two equations are a system and solving for x gives the mole fraction of the ferrous ion as well. One has a negative slope and the other a positive

which makes sure that the intersection of these exist. Based from the graphical data (figure 2), 2+ the mole fraction of Fe where absorbance peaks is at 0.34, implying a 1:3 ratio. This is where the two extrapolated lines intersect. In the mole-ratio method, the plot of the absorbance against the mole ratio is expected to be a graph that increases until it peaks, then flatlines. This is because the absorbance is maxed out upon the formation of the proper complex thus the flatline. This was prepared by having a constant concentration of phenanthroline with a varying amount of ferrous ion added to different flasks. The experimental graph somehow deviates from the theoretical graph of a moleratio plot which gave rise to the 2:3 ratio. This method is solved via line extrapolation or through the two-part linear regression, as well, just like in the previous method. The graphical way seemed ambiguous because the graph did not immediately flatline after the peak. Therefore, the linear regression method was preferred, which gave an x value (mole ratio) of approximately 1.5 which when transformed to a fraction gives 3:2 (L:M). The slope-ratio method was simply a plot wherein ones concentration is held constant as the other is varied and eventually, the reverse is also done. A straight line per plot is expected in an ideal experiment but in this case, a best fit line was acquired via linear regression and its slope was used. The slopes of the two plots were put in a ratio which gave rise to a stoichiometric ratio of 3:2 (L:M). This method has three assumptions, that the system obeys Beers law; that the reaction proceeds to completion because of excess of one of the components of the complex; that the complex is the only species that absorbs at the chosen wavelength. The computation is shown on the results part and it can be seen from here that the concept of massbalance equations is the basis for this method wherein the free species are accounted for as negligible since the presence of the other component is in excess so one reactant is all used up. The slope of the constant phenanthroline, varying metal vs. the constant iron, varying phenanthroline was the ratio

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acquired in this method, which gave rise to a L:M ratio as seen in the algebraic manipulations above. VII. ANSWER TO GUIDE PROBLEM 2. The accompanying data were obtained in a slope-ratio investigation of the complex formed between Ni2+ and 1-cyclopentene-1dithiocarboxylic acid (CDA). The measurements were made at 530 nm in 1.00-cm cells. Determine the stoichiometry of the complex.

The linear regression of the table on the left and of the table on the right gave rise to: Right Table: A vs CCDA [Constant Ni] mL = 3426.57 Left Table: A vs. CNi2+ [Constant Phenanthroline] mM = 10277.12 m M / mL = 3 Formula: Ni(CDA)3 Stoichiometric Ratio: 3:1 (L:M) VIII. CONCLUSIONS AND RECOMMENDATIONS One way to acquire the proper stoichiometry of a complex is through a method known under spectrophotometry where UV-Vis electromagnetic waves are used and the absorbance as well as the transmittance of the complex are acquired. The reaction is 2+ theoretically: Fe (aq) + 3 C12H8N2(aq) = 2+ Fe(C12H8N2)3 (aq). The complex formed has an optimum wavelength of 508 nm which should give the maximum absorbance once maximum concentration of the substance is formed, given the same premise of equal volumes. In continuous variation, the concentration of both
2+

components are varied and when a proper ratio of concentration is achieved, it should yield maximum absorbance making a somehow inverse parabolic graph when an absorbance vs. mole fraction of ferrous ion is prepared. The second method, one component, in this case phenanthroline was held constant while the other is varied which should give rise to a graph that increases, peaks and finally flatlines when absorbance vs. mole ratio is plotted. However, in this experiment, the graph did not immediately flatline which is why a wrong ratio of 2:3 was attained. Lastly, the slope ratio was done by preparing ferrous ion concentration constant, then eventually phenanthroline concentration was the one held constant. Afterwards, two graphs were prepared, absorbance vs. phenanthroline concentration, when ferrous ion was constant and then absorbance vs. ferrous ion concentration was plotted when phenanthroline concentration was constant. A ratio of the slopes of these two increasing lines were acquired and this when algebraically manipulated, yielded the proper ratio of the two components. In this experiment, a phenanthroline iron(II) complex was formed which is a 3:1 (L:M) complex, theoretically. However, only the continuous variation method yielded this correct; the other two resulted in the observation of a 3:2 (L:M) complex species. This may be because of photo decay, undetected oxidation of iron, personal errors in reagent preparation or possibly instrument error. IX. REFERENCES Department of Chemistry (2005), EXPERIMENT 5 Molecular Absorption Spectroscopy: Determination of Iron With 1,10Phenanthroline, Retrieved from: http://www.chem.uky.edu/courses/che22 6/labs/050-fe_absorption.pdf (adapted from: D. A. Skoog, et. al, Analytical Chemistry: An Introduction, 7th ed., Chapters 21 and 22, pp. 547-592.) Organo, V. (n.d.), Spectrometry [PDF document]. Retrieved from Lecture Notes Online Web site: http://dpsm.cas.upm.edu.ph/courses/co urse/view.php?id=25

We hereby certify that we have given substantial contribution to this report.

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Bryan Paul D. De Galicia

Jessica Ann B. Jorge

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