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Lab 1:

UV-Vis Spectroscopy and Determination of


Ligand:Metal Ratios via Mole Ratio and Slope Ratio
Methods

Date of Experiment: February 4, 2015


Date Due: February 11, 2015

Abstract
Through the use of the mole ratio and slope ratio methods, metal:ligand ratios were found for
two different iron organometallic complexes to be three. The theory behind instrumentation and
the two methods, as well as materials, chemicals, procedures, and derivations/calculations of molar
absorptivity coefficients are all contained in this lab report.

Contents
1 Introduction

2.3

Procedure for Slope-Ratio Method

1.1

UV-Vis Theory . . . . . . . . . .

1.2

UV-Vis Spectrophotometer . . .

1.2.1

4 4 Discussion
5
4.1 Mole Ratio Method . . . . . . . .
5
4.1.1 Post Lab Questions . . .

Schematics . . . . . . . .

1.3

Mole Ratio Method . . . . . . . .

1.4

Slope Ratio Method . . . . . . .

3 Results

4.2
2 Procedure

8
10
10
10

Slope Ratio Method . . . . . . .

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4.2.1

11

Post Lab Questions

2.1

Chemicals and Labware . . . . .

2.2

Procedure for Mole-Ratio Method

6 5 Conclusion

. . .

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Introduction
The purpose of this lab was to determine the ligand to metal ratio of iron complexes via

two methods: The Mole-Ratio Method and Slope-Ratio Method. These methods rely on UV-Vis
spectroscopy in determining the peak absorbance of organometallic solutions at different molarities.
The theories, benefits, and methods are discussed below.
The organometallic complexes that are studied in this report are Iron-2,2-Bipyridine for the
Mole Ratio Method and Iron-1,10-Phenanthroline for the Slope Ratio Method. The Iron-2,2bipyridine was created by reacting (Fe(SO4 )2 (NH4 )2 )6H2 O, known as ferrous ammonium sulfate
hexahydrate, or FAS with 2,2-bipyridine. The Iron-1,10-phenanthroline was created with FAS,
1,10-phenanthroline, hydroxylamine hydrochloride, and a pH=5 buffer of acetic acid and sodium
acetate.

1.1

UV-Vis Theory

Apart from any lab/glassware that is commonly used, a UV-VIS Spectrophotometer was necessary in order to determine the peak absorbance of the organometallic solutions. UV-VIS Spectroscopy is reliant on the absorption of ultraviolet and visual regions of the electromagnetic spectrum, generally in the range of 200-800nm. These energies of light allow for the transition of
bonding electrons between quantized energy levels, which allows them to be studied. The effective
area, size, electronic structure, and some quantum considerations all have an effect in how well a
certain compound absorbs light. Large conjugated multi-cyclic structures are often the source of
beautiful and vibrant colors in nature because of this. For simplicity, these electronic, structural,
and quantum considerations manifest themselves in what is known as the molar absorbance coefficient or molar absorptivity, or even the more archaic molar extinction coefficient. Since simplicity
is often preferred, chemists have reduced the complexity of absorbance equations to a rather trivial
formula, commonly known as the Beers Law:
A = bc

(1)

Where A is the absorbance, which can be calculated by relating the initial and final intensities,
 is the molar absorptivity, b is the path length, and c is the concentration of the solution under
study. Since absorbance is a dimensionless quantity, the units of , b, and c are as follows:
Term

Units

Dimensionless

M 1 cm1

cm

While this equation works for the majority of solutions, when the concentrations get either too
low or too high, deviations can occur. The Beers Law is an approximation- which can be treated
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linearly for a range of concentrations [1] [2] [3]. Fortunately, for the duration of this experiment,
Beers Law will hold true, such that no complex equations be needed.
For this experiment, a number of ligands attached to a metal center will act as the absorbing
species. The ligands themselves do not absorb at specific wavelengths, so only the organometallic
complexes will absorb. By changing the concentration of ligands and metal, the number of ligands
attached to a metal center can be determined [4].

1.2

UV-Vis Spectrophotometer

The UV-Vis spectrophotometer used was a single beam Hewlett Packard (now Agilent) 8453
UV-VIis. This spectrophotometer has the following parts: Tungsten lamp, deuterium lamp, source
lens, shutter/stray light filter, sample cuvette, another source lens, slit, diffraction grating, and
finally, a photodiode array (PDA) sensor.
The light source for UV range is the deuterium lamp, emitting from 190nm to 800nm. The
tungsten lamp is the light source for the visual and near-IR range, emitting from 370nm-1100nm. In
this instrument, the tungsten lamp shines through the deuterium lamp via a shine through aperture.
The source lenses receives the light from both sources, collimates it, and aims at the sample. Before
the light moves to the sample, it is passed through an electro-mechanically operated shutter, which
exposes the sample to the light. There is also a stray light filter, which blocks all light under
400nm, when needed. The light is then passed through an open sample, to a second source lens
that re-collimates the beam. This re-collimated beam then passes through a slit the size of one
diode. The light is then diffracted by a concave holographic grating onto a photodiode array with
a sampling interval of approximately 0.9nm [5].
There are other sources, gratings, and detectors that can be used in UV-Vis spectrophotometers.
The reason why D2 and tungsten lamps are used is because they are relatively inexpensive contiuum
sources that cover a very large range of wavelengths (in this case, 190-1100nm). Some sources may
be better for specific ranges, such as xenon arc lamps, mercury line sources, and lasers, but cannot
be used for everything, unlike D2 and tungsten sources.
There are also other ways of analyzing wavelengths of light. This instrument uses a grating/PDA
combination that is very fast, as an entire spectrum of light can be analyzed simultaneously within
seconds. This method is very selective, but not very sensitive in the sense that PDAs can analyze
everything at once, but enough light is needed to register. Another way of analyzing light is
through the use of a monochromator (Czerny-Turner style with an echelle, echellete, or holographic
grating) and a photomultiplier tube. Instead of registering the entire spectrum simultaneously, this
combination scans through the spectrum and hence, takes more time. The monochromator, using
a grating, separates the light based on wavelength and selectively lets light through a slit. This
light goes to a photomultiplier tube (PMT). PMTs are very sensitive, but are not selective. Any
light in the UV-Vis range that hits the PMT creates a cascade effect of electrons, creating a strong
electrical current of up to 107 gain from the initial strike. The following page contains schematics
of the UV-Vis instrument and monochromator/PMT combination.
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1.2.1

Schematics

1.3

Mole Ratio Method

The Mole Ratio Method is an absorbance method used for determining the number of ligands
attached to a metal center. In this case, ferrous ammonium sulfate reacts with 2,2-bipyridine to
create an Fex (bipy)y complex, where x and y are unknowns. This creates a pink solution that is
easily analyzed via UV-Vis spectroscopy.
By creating solutions with known concentrations of the metal and incrementally adding a reacting ligand and then plotting the absorbance data, one can determine the amount of ligand attached
to each metal center. Since only organometallic complexes absorb at a specified wavelengths, the
more ligand added, the higher the absorbance of solution until all metal centers are fully reacted
and excess ligand exists in the solution. After this point, absorbance does not increase with ligand
concentration. By plotting the data and creating two trend lines: one for the increase in absorbance,
and the second for when the absorbance tapers off, the point at which the lines intercept is the
amount of ligand attached to each metal center. For example, if the absorbance increases until it
intersects a line at x=4, then there are a maximum of 4 ligands per metal center.
This method is mostly used when the metal:ligand ratio is greater than 1:2. Also, if the
dissociation constant of the complex is too large, a smooth rather than an abruptly changing slope
is obtained, making it hard to accurately determine the amount of ligand per metal center. In
this case, the slope ratio method is preferred [4]. There can be some problems, however, if several
complexes are formed in solution. These complexes can give rise to deviations from Beers law [6].

1.4

Slope Ratio Method

The Slope Ratio Method is a second absorbance method for determining the number of ligands
attached to a metal center. In this case, ferrous ammonium sulfate reacts with 1,10-phenanthroline
in order to create a Fex (phen)y complex, where x and y are unknown. This creates a red/orange
complex that is easily studied via UV-Vis spectroscopy.
In this method, two sets of solutions are created: one set where the metal is at a constant concentration and varying concentrations of ligand are added, and vice-versa, where the concentration
of ligand is constant, and concentration of metal, varying. This creates a large excess of either
ligand or metal, and when the ratio of slopes is taken, the ligand:metal ratio is found.
since, XFe2+ + Yphen FeX (phen)y2+ , assuming Beers law holds true, then Sy =  yb and
likewise Sx =  xb are the slopes of the two lines where S is the slope,  is the molar absorptivity,
and b is the path length. Taking the ratio of the slopes,

Sx
Sy

x
y:

the ligand:metal ratio is then

found.
This method is commonly used for very low concentrations of ligand and metal and if the
dissociation constant of the organometallic complex is high. This method can only be used, however,
when only one organometallic complex is formed and Beers law is obeyed [4].

Procedure

2.1

Chemicals and Labware

For the Mole Ratio Method, the following chemicals were used:
Chemical

Molar Mass (g/mol)

Ferrous ammonium sulfate hexahydrate (FAS)

392.15

2,2-bipyridine

156.18

For the Slope Ratio Method, the following chemicals were used:
Chemical

Molar Mass (g/mol)

Ferrous ammonium sulfate hexahydrate (FAS)

392.15

1,10-phenanthroline

198.22

Hydroxylamine hydrochloride

69.49

acetic acid, sodium acetate(.1M total acetate)

used as pH=5 buffer

Relevant glassware, such as volumetric flasks and burets were also used in measurements.

2.2

Procedure for Mole-Ratio Method

1. All flasks and glassware cleaned with soap and DI water


2. Labled 10-10mL flasks 1, 1.5, 2, 3, 4, 5, 6, 7, 8, and 9. This is corresponding to the ligand:metal
molar ratio
3. Weighed 0.0454g of FAS and added to 100mL volumetric flask. Volumetric flask was then
filled with water to line (after dissolving FAS in a minimal amount of water). This is a
1.16E-3M stock solution of FAS.
4. Weighed 0.0181g 2,2-bipyridine and added to 100mL volumetric flask. Volumetric flask then
filled with water to line (after dissolving bipy in a minimal amount of water). This is a
1.16E-3M stock solution of 2,2-bipyridine.
5. added 1mL of FAS solution to each of the 10 volumetric flasks
6. added 1, 1.5, 2... 9 mL of bipy solution to each of the corresponding volumetric flasks
7. Flasks filled to 10mL and capped with parafilm
8. The absorbance for each solution was then analyzed with the UV-Vis at 522nm using DI
water as a blank.

2.3

Procedure for Slope-Ratio Method

1. All flasks and glassware cleaned with soap and DI water


2. Weighed 0.0407g FAS and added to 250mL volumetric flask. Flask then filled. This is used
as a 5E-4M stock solution of FAS.
3. Weighed 0.0251g of 1,10-phenanthroline and added to 250mL volumetric flask. Flask then
filled. This is used as a 5E-4 stock solution of 1,10-phenanthroline.
4. measured 1.2576g hydroxylamine hexachloride and added water to 25mL to make a 5% stock
solution
5. Labled 10-50mL volumetric flasks 1-10
6. Flasks were filled as per the following table

Flask nr.

FAS soln (mL)

Buffer (mL)

hydroxylamine hydrochloride (mL)

phenanthroline soln (mL)

30

10

30

10

30

10

30

10

30

10

10

30

10

30

10

30

10

30

10

10

30

The FAS solution was added after the buffer and hydroxylamine hexachloride. After all additions, the solutions were left standing for 10 minutes. UV-Vis spectrometry was then performed to
find peak absorbance at 510nm, using DI water as a blank.

Results
The following table and respective plot, Figure 1, is the UV-Vis data for the Mole Ratio Method

showing peak absorbance at 522nm.


Flask nr.

L:M Ratio

Peak Absorbance

0.26956

1.5

0.46566

0.58979

0.86626

0.83426

0.88026

0.81815

0.86681

0.83747

10

0.85466

Figure 1: Data and plot of Mole Ratio data


Line one has the equation y = 0.2916x + 0.0011 with an R2 value of 0.9925. Line two (near
horizontal) has the equation y = 0.0006x + 0.8445 with an R2 value of 0.0027.

The following table and respective plot, Figure 2, is the UV-Vis data for the Slope Ratio Method,
showing peak absorbance at 510nm.
Flask nr.

Concentration of variable component

Peak Absorbance

1E-05

2.1958E-03

2E-05

4.5454E-02

3E-05

8.1673E-02

4E-05

0.11014

5E-05

0.16405

1E-05

5.6325E-02

2E-05

0.19088

3E-05

0.29214

4E-05

0.37100

10

5E-05

0.47573

Flasks 1-5 vary in 1,10-phenanthroline concentration. Flasks 6-10 vary in FAS concentration.

Figure 2: Data and plot of Slope Ratio data


Line one has the equation y = 3883.9x 0.0358 with an R2 value of 0.9911. Line two (steeper
slope) has the equation y = 10189x 0.0285 with an R2 value of 0.9919.

Discussion
Using spreadsheet software, Apple numbers (akin to excel, but made by Apple), data was

plotted and a linear regression performed in order to determine trend lines and R2 values.
The Mole Ratio Method was performed by two people in my group, including myself. The Slope
Ratio Method was performed by the other two people in my group.

4.1

Mole Ratio Method

The intercept of the two lines occurs at x=2.90. This means that three 2,2-bipyridine ligands
can attach to an iron center. There is a small deviation of .1 in this which can be due to a
variety of factors, including deviations in solution concentration, problems in laboratory practice
(e.g. filling volumetric flasks to exact volume), and variances in measurements. There were two
known problems during setup: the first being an unwillingness of the 2,2-bipyridine to dissolve in
DI water, which may have something to do with the small deviation from a perfect three. Secondly,
there may be compounded error/uncertainty from the need to use a 5mL pipet twice per solution
when additions were greater than 5mL. A combination of any of these sources of error may explain
the small deviation from the whole number value.
Another possible source of error was stated in the introduction to the mole ratio method, where,
if several complexes were formed, deviations from Beers law could have occurred. This may also
be a possible source of error in determining the number of ligands per metal center [6].
No larger problems were faced during this time.
4.1.1

Post Lab Questions

Since three ligands per metal is what makes up this organometallic complex, the formula is then
iron(II) tris(2,2bipyridine)
The molar absorptivity of organometallic complex,  can be calculated from the following equation:
A
=
bc

(2)

The concentration of FAS solution in the 10mL volumetric flask is 1.16E-04M. When all ligand
and metal is reacted, the concentration of the iron(II) tris(2,2-bipyridine) solution is also 1.16E04M. So A = 0.86626, b = 1cm, and c = 1.16E 04M , then  = 7467.75M 1 cm1

4.2

Slope Ratio Method

The ratio of the slopes given by the linear regressions is 2.62. The closest whole number value
is 3, so it is assumed that three 1,10-phenanthroline ligands can react with iron. This deviation is
quite significant, especially compared to the Mole Ratio Method. While I personally did not oversee
this part of the setup, the data given to me by the second group has some nuances. Firstly, when
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calculating the amount of FAS to make the stock solution, the result should have been 0.0490g
to make a 5E-04M solution. The amount of FAS added, however, was only 0.0407g, which only
represents a 4.15E-04M solution of FAS. This is a significant deviation from what should have been
added, and hence, the data is faulty. Secondly, the laboratory procedure said to wait 10 minutes
after adding everything and filling the volumetric flask to the line. We waited 10 minutes before
filling each of the flasks to the line, which may add error since not everything may have had the
chance to react.
These were the two main problems in this procedure that may explain the rise of this rather
large deviation of 0.38.
4.2.1

Post Lab Questions

The formula, derived from the ratio of the slopes was found to be iron(II) tris(1,10-phenanthroline).
The molar absorptivity is again given by formula 2 in section 4.1.1.
Given excess ligand, which does not absorb at 510nm, the concentration of the organometallic
complex is dependent solely on the concentration of FAS. In this case, volumetric flask 10, which
5E-05M FAS corresponds to a 5E-05M concentration of the organometallic complex. A = 0.47573,
b = 1cm, and c = 5E 05, so  = 9514.6M 1 cm1 .
Since the slope ratio methods requires that only one organometallic complex be analyzed, it is
important to ensure that this is the case. Hydroxylamine hydrochloride is a reducing reagent that
reduces any potential Fe(III) Fe(II) [7]. In the same breath, the buffer is utilized in order to
stabilize the pH of the solution to favor one organometallic complex. This is necessary since pH
can influence different iron oxidation states, and therefore complexes, as seen in this iron-water
pourbaix diagram [8]:

Figure 3: Pourbaix diagram of Iron in an Aqueous Solution

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As seen here, a pH of 5 and 0 of 0 is necessary for the stability of Fe(II) in solution. Allowing
the pH to go to 7+ may influence the creation of hydrated complexes, which would interfere in the
determination of ligand:metal ratios.

Conclusion
In conclusion, the ligand to metal ratios for both organometallic complexes were found to be 3:1.

Both ratios were found using two different methods- the mole ratio and slope ratio methods, which
have their advantages and disadvantages depending on the type of complex and concentrations.
There were some problems in the initial setup of the solutions, especially for the slope ratio method,
however the calculated values still agree with literature over iron(II) complexes [9]. The calculated
molar absorptivity values, , are approximately 7500M 1 cm1 for iron(II) tris(2,2bipyridine), and
approximately 9500M 1 cm1 for iron(II) tris(1,10-phenanthroline).

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References
[1] J Hollas. Modern Spectroscopy. Wiley, New York, 2nd edition, 1992.
[2] Heinz G. Pfeiffer and Herman A. Liebhafsky. The Origins of Beers Law. Journal of chemical
education, 28:123, 1951.
[3] Douglas A. Skoog, F. James Holler, and Stanley R. Crouch. Principles of Instrumental Analysis.
Cengage Learning, 6 edition, 12 2006.
[4] Heidi Conrad and Teresa Golden. Chemistry 4632 Instrumental Analysis Laboratory Notebook.
pages 1922, 2015.
[5] Agilent Technologies. Agilent 8453 UV-visible Spectroscopy System Service Manual. Waldbronn,
2000.
[6] Albert S. Meyer and Gilbert H Ayres. The Mole Ratio Method for Spectrophotometric Determination of Complexes in Solution. Journal of the American Chemical Society, 79(2):4953,
1957.
[7] Ulrich Camp and Oliver Seely. Colorimetric Fe Analysis.
[8] Wikipedia.
[9] Chemspider. CSID:167912.

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