analytical chemistry

2. Prepared by:
Karwan O. Ali
Yousif T. Maaroof
Mzgin. M. Ayoob
UV- VISIBLE SPECTROSCOPY

3. What is spectroscopy?
The interactions of radiation and matter are the subject of the
science called spectroscopy. Spectroscopic analytical methods
are based on measuring the amount of radiation produced or
absorbed by molecular or atomic species of interest

4. The Electromagnetic Spectrum

5. Light exhibits wave property during its propagation and energy particle
during its interaction with matter. The double nature of light (waves and
particles) is known as dualism.
Dual nature of light

6. • Light consist of energy packets, known as photons.
• The energy (E) of photons is proportional to the frequency i.e.
related to c and . It can be expressed by max plank relation:
• E = h  ( = C /)
• where h = max plank constant = 6.63 x 10-27 erg., sec.)
• i.e. E   or E  1/   

7. For analytical purposes we use the region of I.R, visible and U.V
radiations.
UV radiation region is classified into : far UV from (10nm-200nm) and
near UV from (200nm-380nm

8. Why Is a Red Solution Red?

10. • When a molecule interact with radiant energy, the molecule is said to
be excited , because the outer valence electrons undergo transition
from original energy level ground state (E g) to an exited state (E s).
Interaction of a substance with EMR
Excited state Es
Ground state E g

11. When a molecule in the ground state absorbs
EMR, 3 energy state transitions will take place.
•These types of transition are:
1) Electronic
2) Vibrational
3) Rotational.

12. • When the molecule absorb Visible and U.V region.
Raising electrons to a higher energy level, raising the Vibration of
molecule, and increasing rotation of the molecule (Electronic transition
energy + Vibrational transition energy + Rotational transition energy)
1) Electronic

13. • When the molecule absorb I.R region.
Raising the Vibration of molecule and increasing rotation of the
molecule (Vibrational transition energy + Rotational transition energy)
2) Vibrational

14. • When the molecule absorbs F.I.R and Microwave regions.
Increasing rotation of the molecule (Rotational transition
energy).
3) Rotational

16. • Absorption measurements based upon ultraviolet and visible radiation find widespread
application for the quantitative determination of a large variety species [1].
• Beer’s Law:
• A = -logT = logP0/P = abc = bc = 2 - log%T
• Where T = transmittance, T = percentage transmittance, P = transmitted power of
radiation, Po = incident power of radiation, A = absorbance, a = absorptivity,
b = path length, c = concentration,  = molar absorptivity, extinction coefficient
An Introduction to Ultraviolet/Visible
Molecular Absorption Spectrometry

17. The Quantitative Picture
Where the absorbance A has no units, since A = log10 P0 / P
 Is the molar absorbtivity with units of L mol-1 cm-1
b is the path length of the sample in cm
c is the concentration of the compound in solution, expressed in mol L-1 (or M,
molarity)

18. The Beer-Lambert Law (Beer’s Law): A =  b c

19. • Monochromatic incident radiation (all molecules absorb light of one )
• Absorbers independent (Absorbing molecules act independently of one another i.e., low c)
• Path length is uniform (all rays travel the same distance in sample)
• No scattering
• Absorbing medium is optically homogeneous
• Incident beam is not large enough to cause saturation
• All rays should be parallel to each other and perpendicular to surface of medium
Assumptions in derivation of Beer’s Law

20. Deviations from Beer’s Law
• Real Limitations
• Beer’s law is successful in describing the absorption behavior of dilute solutions only ; in this sense it is a
limiting law. At high concentrations ( > 0.01M ),the average distance between the species responsible for
absorption is diminished to the point where each affects the charge distribution of its neighbors
This interaction, in turn, can alter the species’ ability to absorb at a given wavelength of radiation thus leading
to a deviation from Beer’s law [2].
A similar effect is sometimes encountered in solutions containing low absorber concentrations and high
concentrations of other species, particularly electrolytes.
Beer’s law is valid at low concentrations,
but breaks down at higher concentrations
For linearity, A < 1

21. Deviations from Beer's law also arise because ε is dependent upon the
refractive index of the solution. Thus, if concentration changes cause
significant alterations in the refractive index η of a solution, departures from
Beer's law are observed. A correction for this effect can be made by
substitution of the quantity εη /(η2 + 2)2 for ε in the Beer equation, as shown
below [2]:
A= [εηb /(η2 + 2)2 ] bC
In general, this correction is never very large and is rarely significant at
concentrations less than 0.01 M.

22. • Chemical deviations from Beer’s law are caused by shifts in the position of a chemical or physical
equilibrium involving the absorbing species. A common example of this behavior is found with
acid/base indicators. Deviations arising from chemical factors can only be observed when
concentrations are changed
Chemical Deviations
HIn
Ka
H+ + In-
Red,  =600nm Colorless
Phenolphthalein:
If solution is buffered, then pH is constant and [HIn]
is related to absorbance.
But, if un buffered solution,
equilibrium will shift depending on
total analyte concentration
C
HIn
Expected
Actual

23. • instrument may be caused by fluctuations in the power-supply voltage, an
unstable light source, or a non-linear response of the detector-amplifier system
• Polychromatic Radiation
• All monochromators, regardless of quality and size, have a finite resolving
power and therefore minimum instrumental bandwidth. Polychromatic
radiation (i.e., light of more than one )
Instrumental Factors

24. Band A shows little deviation, because ε does not change greatly throughout the band. Band
B shows marked deviations because ε undergoes significant changes in this region

25. The width of the image produced is thus an important measure of the quality of the
performance of a spectrometer. The figure below shows the loss of detail that accompanies the
use of wider slits. It is evident that an increase in slit width brings about a loss of spectral
detail
Effect of Slit Width on Absorbance Measurements

26. • quantitative measurement of narrow absorption bands demand the use of narrow slits widths. Unfortunately, a
decrease in slit width is accompanied by a second-order power reduction in the radiant energy; at very narrow settings
spectral detail may be lost owing to an increase in the signal-to-noise ratio. In general, it is good practice to narrow
slits no more than is necessary for good resolution for the spectrum at hand
Another effect of slit width is the change of absorbance values that accompany a change in the slit width. The
figure below illustrates this effect. Note that the peak absorbance values increase significantly (by as much as
70% in one instance) as the slit width decreases

27. The basic components of analytical instruments for absorption spectroscopy,
as well as for emission and fluorescence spectroscopy, are remarkably alike
in function and in general performance requirements whether the
instruments are designed for ultraviolet (UV), visible, or infrared (lR)
radiation. We often call the UV/visible and IR regions of the spectrum the
optical region
Instruments for Optical Spectrometry

28. 1) a stable source of radiant energy
2) a wavelength selector that isolates a limited region of the spectrum for
measurement
3) one or more sample containers
4) a radiation detector, which converts radiant energy to a measurable electrical
signal
5) a signal processing and readout unit, usually consisting of electronic hardware
and, in modern instruments, a computer.
INSTRUMENT COMPONENTS

29. To be suitable for spectroscopic studies, a source must generate a beam of radiation that is sufficiently powerful
to allow easy detection and measurement.
In addition, its output power should be stable for reasonable periods of time. Typically, for good stability, a well-
regulated power supply must provide electrical power for the source
Spectroscopic Sources
Spectroscopic sources are of two types:
A- Continuum sources, which emit radiation that changes in intensity only
slowly as a function of wavelength.
B- Line sources, which emit a limited number of spectral lines, each of
which spans a very limited wavelength range.
The distinction between these sources is illustrated in Figure bellow. Sources
can also be classified as
continuous sources, which emit radiation continuously with time, or pulsed
sources, which emit radiation in bursts

30. tungsten lamp of the type used in spectroscopy and its spectrum (b). Intensity of the
tungsten source is usually quite low at wavelengths shorter than about 350 nm. Note that
the intensity reaches a maximum in the near-IR region of the spectrum (<1200 nm in this
case).
Tungsten lamp

31. • Tungsten/halogen lamp
• Tungsten/halogen lamps, also called quartz/halogen lamps, contain a small amount of iodine
within the quartz envelope that houses the filament. Quartz allows the filament to be operated
at a temperature of about 3500 K, leading to higher intensities and extending the range of the
lamp well into the UV. The lifetime of a tungsten/halogen lamp is more than double that of an
ordinary tungsten lamp, which is limited by sublimation of tungsten from the filament. In the
presence of iodine, the sublimed tungsten reacts to give gaseous WI2 molecules. These
molecules then diffuse back to the hot filament where they decompose, redeposit W atoms on
the filament, and release iodine. Tungsten/halogen lamps are finding ever-increasing use in
spectroscopic instruments because of their extended wavelength range, greater intensity, and
longer life

33. A deuterium lamp consists of a cylindrical tube containing deuterium at
low pressure, with a quartz window from which the radiation exits
Deuterium lamp

34. The word laser originally was the upper-case LASER, the acronym from Light
Amplification by Stimulated Emission of Radiation. Lasers have become useful
as sources in certain types of analytical spectroscopy. To understand how a laser
works, consider an assembly of atoms or molecules interacting with an
electromagnetic wave. Laser radiation is highly directional. Spectrally pure,
coherent. and of high intensity. These properties have made possible many
unique research applications that cannot easily be achieved with conventional
sources
Laser Sources

35. Spectroscopic instruments in the UV and visible regions are usually equipped with one or more
devices to restrict the radiation being measured to a narrow band that is absorbed or emitted by
the analyte. Such devices greatly enhance both the selectivity and the sensitivity of an
instrument. In addition, for absorption measurements, narrow bands of radiation greatly
diminish the chance of Beer's Jaw deviations due to polychromatic radiation. Many
instruments use a monochromator or filter to isolate the desired wavelength band so that only
the band of interest is detected and measured. Others use a spectrograph to spread out, or
disperse, the wavelength so that they can be detected with a multichannel detector
Wavelength Selectors

36. Monochromators generally have a diffraction grating to disperse the radiation into
its component wavelengths, as shown in Figure bellow
By rotating the grating, different
Wavelengths can be made to pass
through an exit slit
Monochromators consist of:
1- entrance slit
2- collimating mirror or lens
3- a prism or grating
5- focal plane
6- exit slit
Older instruments used prisms for this purpose
Monochromators

37. Older instruments used prisms for this purpose

38. Radiation from a source enters the monochromator
via a narrow rectangular opening, or slit.
The radiation is then collimated by a concave
mirror, which produces a parallel beam that
strikes the surface of a reflection grating.
Angular dispersion results from diffraction,
which occurs at the reflective surface
Grating Monochromator

39. The effective band width of the monochromator depends on the size and quality of the
dispersing element, the slit widths, and the focal length of the monochromator. A high-quality
rnonochromator will exhibit an effective band-width of a few tenths of a nanometer or less in
the ultraviolet/visible region. The effective bandwidth of a monochromator that is satisfactory
for most quantitative applications is about 1 to 20 nm. Many monochromators are equipped
with adjustable slits to permit some control over the bandwidth. A narrow slit decreases the
effective bandwidth but also diminishes the power of the emergent beam. Thus, the minimum
practical bandwidth may be limited by the sensitivity of the detector For qualitative analysis,
narrow slits and minimum effective bandwidths are required if a spectrum is made up of
narrow peaks. For quantitative work. however, wider slits permit operation of the detector
system at lower amplification, which in turn provides greater reproducibility of response.

40. Most gratings in modern monochromators are replica gratings, which are obtained by
making castings of a master grating. The latter consists of a hard, optically flat, polished
surface on which a suitably shaped diamond tool has created a large number of parallel and
closely spaced grooves. A grating for the ultraviolet and visible region will typically contain
300 to 2000 grooves/mm. with 1200 to 1400 being most common. The construction of a
good master grating is tedious, time consuming, and expensive because the grooves must
be identical in size, exactly parallel, and equally spaced over the length of the grating (3 to
10 cm). Replica gratings are formed from a master grating by a liquid resin casting process
that preserves virtually perfectly the optical accuracy of the original master grating on a clear
resin surface. This surface is ordinarily made reflective by a coating of aluminum or, some
times, gold or platinum

42. Radiation Filters
Filters operate by absorbing all but a restricted band of radiation from a
continuum source. As shown in Figure bellow, two types of filters are used in
spectroscopy; interference filters and absorption filters. Interference filters are
typically used for absorption measurements, and they generally transmit a much
greater fraction of radiation at their nominal wavelengths than do absorption
filters

44. Interference Filters
Interference filters are used with ultraviolet and visible radiation, as well as with
wavelengths up to about 14 µm in the infrared region. As the name implies, an
interference filter relies on optical interference to provide a relatively narrow
band of radiation. typically 5 to 20 nm in width. As shown in Figure bellow, an
interference filter consists of a very thin layer of a transparent dielectric material
(frequently calcium fluoride or magnesium fluoride) coated on both sides with a
film of metal that is thin enough to transmit approximately half the radiation
striking it and to reflect the other half.

46. • the radiant power transmitted, fluoresced, or emitted must be detected in some
manner and converted into a measurable quantity. A detector is a device that
indicates the existence of some physical phenomenon. Familiar examples of
detectors include photographic film (for indicating the presence of
electromagnetic or radioactive radiation.
• The human eye is also a detector; it converts visible radiation into an electrical
signal that is passed to the brain via a chain of neurons in the optic nerve and
produces vision.
Detecting and Measuring Radiant Energy

47. • The term transducer is used to indicate the type of detector that
converts quantities, such as light intensity, pH, mass, and temperature,
into electrical signals that can be subsequently amplified, manipulated,
and finally converted into numbers proportional to the magnitude of
the original quantity.
• A transducer is a type of detector that converts various types of
chemical and physical quantities into electrical signals such as
electrical charge, current, or voltage.

48. There are two general types of transducers:
• Photons
All photon detectors are based on the interaction of radiation with a reactive
surface either to produce electrons (photoemission) or to promote electrons to
energy states in which they can conduct electricity (photoconduction). Only
UV, visible, and near-IR radiation possess enough energy to cause
photoemission to occur; thus, photoemissive detectors are limited to
wavelengths shorter than about 2 µm (2000 nm).
Types of Transducers

49. • Thermal detectors (Heat)
detect a temperature change in a material due to photon absorption
• Thermal detectors can be used over a wide range of wavelengths
.Their main disadvantages are slow response time and lower
sensivity relative to other types of detectors.

51. Widely used types of photon detectors include phototubes, photomultiplier
tubes, silicon photodiodes, and photodiode arrays.
Photon Detectors
Phototubes
The response of a phototube or a photomultiplier tube is based on the photoelectric effect. a
phototube consists of a semicylindrical photocathode and a wire anode sealed inside an
evacuated transparent glass or quartz envelope. The concave surface of the cathode supports
a layer of photoemissive material. such as an alkali metal or a metal oxide. that emits
electrons when irradiated with light of the appropriate energy. When a voltage is applied
across the electrodes, the emitted photoelectrons are attracted to the positively

52. charged wire anode, In the complete circuit, a photocurrent then results that is easily
amplified and measured. The number of photoelectrons ejected from the photocathode per
unit time is directly proportional to the radiant power of the beam striking the surface. With
an applied voltage of about 90 V or more, all these photoelectrons are collected at the anode
to give a photocurrent that is also proportional to the radiant power of the beam.
Photoelectrons: are electrons that are ejected
from a photosensitive surface by electromagnetic
radiation.
Photocurrent: is the current in an external
circuit that is limited by the rate of ejection of
photoelectrons.

53. The photomultiplier tube (PMT) is similar in construction to the phototube but is
significantly more sensitive. Its photocathode is similar to that of the phototube, with electrons
being emitted on exposure to radiation. In place of a single wire anode. however, the PMT has a
series of electrodes called dynodes, The electrons emitted from the cathode are accelerated
toward the first dynode. which is maintained 90 to 100 V positive with respect to the cathode.
Each accelerated photoelectron that strikes the dynode surface produces several electrons, called
secondary electrons, that are then accelerated to dynode 2, which is held 90 to 100 V more
positive than dynode l. Again, electron amplification results. By the time this process has been
repeated at each of the dynodes, 105 to 107 electrons have been produced for each incident
photon. This cascade of electrons is finally collected at the anode to provide an average current
that is further amplified electronically and measured.

55. One of the major advantages of photomultipliers is their automatic internal amplification.
About 106 to 107 electrons are produced at the anode for each photon that strikes the
photocathode of a photomultiplier tube. Photomultiplier tubes are among the most widely
used types of transducers for detecting ultraviolet/visible radiation. With modern electronic
instrumentation, it is possible to detect the electron pulses resulting from the arrival of
individual photons at the photocathode of a PMT. The pulses are counted, and the
accumulated count is a measure of the intensity of the electromagnetic radiation impinging on
the PMT. Photon counting is advantageous when light intensity, or the frequency of arrival of
photons at t1e photocathode, is low.

56. Photoconductive transducers consist of a thin film of a semiconductor
material, such as lead sulfide, mercury cadmium telluride (MCT), or
indium antimonide, deposited often on a nonconducting glass surface
and sealed in an evacuated envelope. Absorption of radiation by these
materials promotes nonconducting valence electrons to a higher energy
state, which decreases the electrical resistance of the semiconductor.
Typically, a photoconductor is placed in series with a voltage source and
a load resistor, and the voltage drop across the load resistor serves as a
measure of the radiant power of the beam of radiation. The PbS and InSb
detectors are quite popular in the near-IR region of the spectrum. The
MCT detector is useful in the mid- and far-IR regions when cooled with
liquid N2 to minimize thermal noise.
Photoconductive Cells:

57. Diode-Array Detectors
Silicon photodiodes have become important recently because 1000 or more can
be fabricated side by side on a single small silicon chip. (The width of individual
diodes is about 0.02 mm). With one or two of the diode-array detectors placed
along the length of the focal plane of a monochromator. All wavelengths can be
monitored simultaneously, thus making high-speed spectroscopy possible. Silicon
photodiode detectors respond extremely rapidly, usually in nanoseconds.

58. They are more sensitive than a vacuum phototube but considerably less
sensitive than a photomultiplier tube.

59. Sample containers, which are usually called cells or cuvettes must have windows that are
transparent in the spectral region .Thus as shown in the figure below, quartz or fused silica is
required for the UV region (wavelengths less than 350nm nm) and may be used in the visible
region and out to about 3000 nm (3 µm) in the IR region. Silicate glass is ordinarily used for
the 375 to 2000 nm region because of its low cost compared with quartz. Plastic cells are also
used in the visible region. The most common window material for IR studies is crystalline
sodium chloride, which is soluble in water and in some other solvents.
Sample Containers

60. The best cells have windows that
are perpendicular to the direction
of the beam in order to minimize
reflection losses. The most
common cell path length for
studies in the UV and visible
regions is 1 cm; matched,
calibrated cells of this size are
available from several
commercial sources. Many other
cells with shorter and longer path
lengths can be purchased.

61. For reasons of economy, cylindrical cells are sometimes used. Fingerprints, grease, or other
deposits on the walls markedly alter the transmission characteristics of a cell. Thus,
thorough cleaning before and after use is need , and care must be taken to avoid touching
the windows after cleaning is complete. Matched cells should never be dried by heating in
an oven or over a flame because this may cause physical damage or a change in path
length..

62. Photometers provide simple, relatively inexpensive tools for performing
absorption measurements. Filter photometers are often more convenient and
more rugged and are easier to maintain and use than the more sophisticated
spectrophotometers. Furthermore, photometers characteristically have high
radiant energy throughputs and thus good signal-to-noise ratios even with
relatively simple and inexpensive transducers and circuitry. Photometers have
the advantages of simplicity, ruggedness, and low cost.

63. Spectrophotometers
offer the considerable advantage that the wavelength can be varied
continuously, thus making it possible to record absorption spectra and is a
scanning instrument (i.e. a spectrophotometer has a monochromator for
separating the individual wavelengths of light). Several dozen models of
spectrophotometers are available commercially. Most spectrophotometers
cover the UV /visible and occasionally the near-infrared region, while
photometers are most often used for the visible region.. Both photometers and
spectrophotometers can be obtained in single- and double-beam varieties.

64. Single beam spectrometers
Single beam spectrometers are relatively cheap, simple, portable & ideally
suited to quantitative analysis .It is not possible to scan through the entire
spectrum with such an instrument because both the source intensity & the
detector response vary with the wavelength. To record an accurate value of
the absorbance it is necessary to zero the instrument on a reference/blank
before every measurement. Thus, this is essentially a single wavelength
measurement of absorbance.

66. Voltage fluctuations and changes in light source present a problem
When a heavy load is placed on the electric power system, lights dim and later brighten
*If measurements are being taken on the spectrophotometer at the same time, the
readings will be unreliable
*Aging lamp source may momentarily flicker and cause the readings to be unstable and
errorneous
So, a single-beam instrument requires a stabilized voltage supply to avoid
errors resulting from changes in the beam intensity during the time required to
make the 100% T measurement and determine %T for the analyte.
Disadvantages of single-beam spectrophotometer

67. The second type of double-beam instrument. Here the beams are separated in time by a
rotating sector mirror that directs the entire beam from the monochromator first through the
reference cell and then through the sample cell. The pulses of radiation are recombined by
another sector mirror, which transmits one pulse and reflects the other to the transducer. As
shown by the insert labeled "front view", the motor-driven sector mirror is made up of pie-
shape segments, half of which are mirrored and half of which are transparent. The mirrored
sections are held in place by blackened metal frames that periodically interrupt the beam and
prevent its reaching the transducer. The double-beam-in-time approach is generally preferred
because of the difficulty in matching the two detectors needed for the double-beam-in-space
design
Double-Beam in time spectrophotometers

69. Double-Beam in space spectrophotometers
• Many modern photometers and spectrophotometers are based on a double-beam
design. A double-beam-in-space instrument in which two beams are formed in
space by a V-shape mirror called a beamsplitter. One beam passes through the
reference solution to a photodetector, and the second simultaneously traverses
the sample to a second, matched detector. The two outputs are amplified, and
their ratio (or the logarithm of their ratio) is determined electronically or by a
computer and displayed by the readout device.

71. The determination of an analyte’s concentration based on its absorption of ultraviolet or visible radiation is one of the most
frequently quantitative analytical methods. One reason for its popularity is that many organic and inorganic compounds
have strong absorption bands in the UV/Vis region of the electromagnetic spectrum.
There are many application
• Environmental Applications
• Clinical Applications
• Industrial Analysis
• Forensic Applications
Quantitative Applications

72. • Methods for the analysis of waters and wastewaters relying on the
absorption of UV/Vis radiation are among some of the most frequently
employed analytical methods.
Environmental Applications

73. • UV/Vis molecular absorption is one of the most commonly employed
techniques for the analysis of clinical samples, several examples of
which are listed in Table below. The analysis of clinical samples is often
complicated by the complexity of the sample matrix, which may
contribute a significant background absorption at the desired wavelength
.
Clinical Applications

74. The Application of UV/Vis Molecular Absorption to the
Analysis of Clinical Samples

75. • UV/Vis molecular absorption is used for the analysis of a diverse array of
industrial samples, including pharmaceuticals, food, paint, glass, and
metals.
• In many cases the methods are Products that have been analyzed in this
fashion include antibiotics, hormones, vitamins, and analgesics.
• One example of the use of UV absorption is in determining the purity of
aspirin tablets.
Industrial Analysis

76. • UV/Vis molecular absorption is routinely used in the analysis of
narcotics and for drug testing.
• One interesting forensic application is the determination of blood
alcohol using the Breathalyzer test. In this test a 52.5-mL breath
sample is bubbled through an acidified solution of K2Cr2O7. Any
ethanol present in the breath sample is oxidized by the dichromate,
producing acetic acid and Cr3+ as products.
Forensic Applications

77. • The energy at which the absorption occurs, as well as the intensity of the
absorption, is determined by the chemical environment of the absorbing moiety.
For example, benzene has several ultraviolet absorption bands due to p  p*
transitions. The position and intensity of two of these bands, 203.5 nm (e =
7400) and 254 nm (e = 204), are very sensitive to substitution. For benzoic acid,
in which a carboxylic acid group replaces one of the aromatic hydrogens, the
two bands shift to 230 nm (e = 11,600) and 273 nm (e = 970). Several rules
have been developed to aid in correlating UV/Vis absorption bands to chemical
structure.
Qualitative Applications

78. 1-Limited since few resolved peaks
• Unambiguous identification not usually possible.
Why we cannot use UV / visible Spectroscopy in
Qualitative Analysis ?
Loss of fine structure for
acetaldehyde when
transfer to solvent from
gas phase
Also need to consider
absorbance of solvent

79. 2. Solvent can affect position and shape of curve .
• polar solvents broaden out peaks, eliminates fine structure.
•Loss of fine structure
for 1,2,4,5-tetrazine as
solvent polarity
increases

80. 3.Solvent can also absorb in UV-vis spectrum.
• Solvent for the ultraviolet and visible regions

81. • Molecular absorption, particularly in the UV/Vis range, has been used for a variety of
different characterization studies, including determining the stoichiometry of metal–
ligand complexes and determining equilibrium constants.
• Stoichiometry of a Metal, Ligand Complex
• The stoichiometry for a metal–ligand complexation reaction of the following general
form
• can be determined by one of three methods: the method of continuous variations,
•the mole-ratio method, and the slope-ratio method
Characterization Applications

82. • also called Job’s method, is the most popular. In this method a series of
solutions is prepared such that the total moles of metal and ligand, ntot, in
each solution is the same. Thus, if (nM)i and (nL)i are, respectively, the
moles of metal and ligand in the i-th solution, Then
• The relative amount of ligand and metal in each solution is expressed as the mole
• fraction of ligand, (XL)i, and the mole fraction of metal, (XM)i,
Method of continuous variations

83. A plot of A vs volume ratio (volume ratio =
mole fraction) gives maximum absorbance when
there is a stoichiometric amount of the two.

84. • A procedure for determining the stoichiometry between two reactants by
preparing solutions containing different mole ratios of two reactants . In
the mole-ratio method the moles of one reactant, usually the metal, are
held constant, while the moles of the other reactant are varied. The
absorbance is monitored at a wavelength at which the metal–ligand
complex absorbs. A plot of absorbance as a function of the ligand-to-
metal mole ratio (nL/nM) has two linear branches that intersect at a mole
ratio corresponding to the formula of the complex.
Mole-ratio method

85. • Figure (a)shows a mole-ratio plot for the formation of a 1:1 complex
in which the absorbance is monitored at a wavelength at which only
the complex absorbs.

86. • Figure (b) shows a mole-ratio plot for a 1:2 complex in which the
metal, the ligand, and the complex absorb at the selected wavelength.

87. • A procedure for determining the stoichiometry between two reactants by
measuring the relative change in absorbance under conditions when each reactant
is the limiting reagent . In the slope-ratio method two sets of solutions are
prepared. The first set consists of a constant amount of metal and a variable
amount of ligand, chosen such that the total concentration of metal, CM, is much
greater than the total concentration of ligand, CL. Under these conditions we
may assume that essentially all the ligand is complexed. The concentration of a
metal–ligand complex of the general form MxLy is
Slope-ratio method

88. • Determine endpoint by following change in absorbance of:
1) reactant (decrease)
2) product (increase)
3) titrant (increase after endpoint)
• Example Titration curves for
• 𝑺 + 𝑻 → 𝑷
• where S = analyte being titrated, T = titrant, P = product
Photometric Titrations

89. where S = analyte being titrated, T = titrant, P = product

90. References
1- Douglas A. skoog, Donald M. West, F. james Holler, Stanley R. Crouch. (2013).
Fundamentals of analytical chemistry. 9th ed . Belmont, USA: Mary finch. pp:651-674.
2- Douglas A. Skoog, F. James Holler, Stanley R. Crouch. (2007). Principles of instrumental
analysis. 6th ed. Belmont, USA: David Harris.pp:335-367.
3- Tony O.(2000). Fundamentals of modern Uv-Visible spectroscopy. 1st ed. Germany:
Agilent technologies .pp:36-43.
4- David H.(2000). Modern analytical chemistry. 1st ed . London: James M smith. Pp: 394-
407.