Download An-Introduction-to-Spectrometric

An Introduction to Spectrometric Methods
Faculty of pharmacy &Medical Science
Petra University
DR. WAEL ABU DAYYIH
pharmaceutical ANALYSIS
(501722)
2012
1
Spectrometric methods
 Spectrometric methods are a large group of analytical
methods that are based on atomic and molecular spectroscopy.
Spectroscopy is a general term of the science that deals with
the interactions of various types of radiation with matter.
Historically, the interactions of interest were between
electromagnetic radiation and matter, but now spectroscopy
has been broadened to include interactions between matter
and other forms of energy. Examples include acoustic waves
and beans of particles such as ions and electrons.

2
Spectrometry and spectrometric
 Spectrometry and spectrometric methods refer to the
measurement of the intensity of radiation with a
photoelectric transducer or other type of electronic
device.
 The most widely used spectrometric methods are based
on electromagnetic radiation, which is a type of energy
that takes several forms, the most readily recognizable
being light and radiant heat. Less obvious manifestations
include gamma rays and X-rays as well as ultraviolet,
microwave and radio-frequency radiation.
3
spectroscopic methods
 Spectroscopy: is the use of absorption, emission and
scattering of electromagnetic radiation by matter to
qualitatively and quantitively study of the matter or to
study some of physical process of matter.
 Matter : atoms, molecules atomic or molecules ions.
 In spectroscopic methods the sample solutions absorbs
electromagnetic radiation from an appropriates source
and the amount absorbed is related to the concentration
of the analyte in the solution
4
Electromagnetic radiation EMR
 Is the type of energy that is transmitted through space.
 EMR is viewed as waves and on the other cases as a particles called
photons.
 EMR is used in chemical analysis in the followings:
 If EMR is absorbed by the sample the λ, ν at which absorption
occur can be used for qualitative analysis.
 The extent at which absorption occurs can be used for quantitative
 Emission of EMR :
 Intensity of emission - Quantitive
 λ ======►
Qualitative
5
Electromagnetic Radiation
 It is a form of Energy, made up of particles which are called
photons the fundamental property of the radiation is the
Frequency which is a number of waves pass in given time.
6
General properties of electromagnetic radiation
 Many of the properties of electromagnetic radiation are
conveniently described by means of a classical sinusoidal
wave model, which embodies such parameters as wavelength,
frequency, velocity, and amplitude. In contrast ot other wave
phenomena, such as sound, electromagnetic radiation
requires no supporting medium for its transmission and thus
passes readily a vacuum.
7
Wave Properties of Electromagnetic
Radiation
 For many purposes, electromagnetic radiation is
conveniently represented as electric and magnetic field
that undergo in-phase, sinusoidal oscillations at right
angles to each other and to the direction of propagation.
Figure is such representation of a single ray of planepolarized electromagnetic radiation. The term plane
polarized implies that all oscillations of either the
electric or the magnetic fields lie within a single lane.
Figure is a two –dimensional representation of the
electric component of the ray in Figure 6.
8
Wave Properties of Electromagnetic
Radiation
 The electric field strength in figure is represented as a vector
whose length is proportional to its magnitude. The abscissa of this
plot is either time as the radiation passes a fixed point in space or
distance when time is held constant. Throughout this chapter and
most of the remaining text, only the electric component of
radiation will be considered because the electric field is
responsible for most of the phenomena that are of interest to us,
including
Transmission, Reflection, Refraction, and Absorption.
Note how ever, that the magnetic component of electromagnetic
radiation is responsible for absorption of radio-frequency
waves in nuclear magnetic resonance.
9
10
Wave parameters
 In figure 6, the amplitude A of the sinusoidal wave is
shown as the length of the electric vector at a maximum
in the wave. The time in seconds required for the passage
of successive maxima or minima through a fixed point in
space is called the period, p, of the radiation. The frequency,
ν , is the number of oscillations of the field that occur
per second and is equal to 1/P. Another parameter of
interest is the wavelength, λ, which is the linear distance
between any two equivalent points on successive waves.
(e.g., successive maxima or minima.
11
Wave parameters
 Multiplication of the frequency in cycles per
second by the wavelength in meters per cycle
gives the velocity of propagation νi in meters per
second: νi= νλi
It is important to realize that the frequency of the
beam of radiation is determined by the source
and remains invariant. In contrast, the velocity of
radiation depends upon the composition of the
medium through which it passes.
12
Wave parameters
 In a vacuum, the velocity of radiation is independent of
wavelength and is at its maximum. This velocity, given
the symbol c, has been determined to be
2.99792×108m/s. It is significant that the velocity of
radiation in air differs only slightly form c (about 0.03%
less); thus, for either air or vacuum, Equation 6 can be
written to three significant figures as:
 c = νλ = 3.00 × 108m/s = 3.00 × 1010cm/s
13
Wave parameters
 In any medium containing matter, propagation of
radiation is slowed by the interaction between the
electromagnetic field of the radiation and the bound
electrons in the matter. Since the radiant frequency is
invariant and fixed by the source, the wavelength must
decrease as radiation passes from a vacuum to another
medium (Equation 6-2). This effect is illustrated in figure
6-2 for a monochromatic beam of visible radiation.
14
Wave parameters
 The wave number ύ, which is defined as the
reciprocal of the wavelength in centimeters, is yet
another way of describing electromagnetic
radiation. The unit for (ύ) is cm-1.Wave number is
widely used IR- infrared spectroscopy. The wave
number ύ is a useful unit because, in contrast to
wave length, its is directly proportional to the
frequency, and thus the energy, of radiation. Thus,
we may write :
15
Wave parameters
 ύ=kν
 Where the proportionality constant K depends on the
medium and is equal to the reciprocal of the velocity
(Equation 6-1).
 The power P of the energy of the beam that reaches a
given area per second, whereas the intensity I is the
power per unit solid angle. Theses quantities are related
to the square of the amplitude A (see figure 6-1).
Although it is not strictly correct to do so, power and
intensity are often used synonymously .
16
17
Used terms
 Monochromator (Monochromatic) beam: is a beam of




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radiation whose rays have identical wavelengths
Polychromatic beam is made up of rays of different
wavelengths
-1
The common unit of frequency is reciprocal second S or
Hertz(Hz) which corresponds to one cycle per second.
The units commonly used for describing wavelength differ
considerably in the various spectral regions
o
A : (Angstrom)unit is suitable for X-ray and short ultraviolet
radiation
Used terms
 The nanometer (nm) is employed with visible and ultraviolet
radiation
 The micrometer (µm)( micron) is useful for the infrared
region .
o
 A =(10





19
-10
m)
-9
nm =(10 m)
-6
µm=(10 m)
-10
-8
o
1 A = 10 m = 10 cm
-7
-9
1nm = 10 m = 10
cm
-4
-6
1 µm = 10 m = 10 cm
20
No.
Type Spectroscopy
Usual Wavelength
Rang
Usual
Wavenumber
Range,cm-1
Type of Quantum
Transition
1
GAMMA – RAY EMISSION
0.005 – 1.40 A
-----------
NUCLEAR
2
X- RAY
ABSORPTION,EMISSION,
FLUORESCENCE,
DIFFRACTION
0.10-100 A
------------
INNER
ELECTRON
3
VACUUM ULTAVIOLET
ABSORPTION
10-180 nm
1*106-5*104
BONDING
ELECTRON
4
UV-VIS.ABSORPTION
,EMISSION,FLUORES.
180-780 nm
5*104-1.3*104
B.E
5
INFRARED ABS& RAMAN
SCATTERING
0.78-300 µm
1.3*104 3.3*101
ROTATION/VIBR
ATION OF
MOLECULES
6
MICROWAVE ABS.
0.75-3.75 mm
13-27
ROTATION OF
MOLECULES
7
ELECTRON SPIN
RESONANCE
3 cm
0.33
SPIN OF
ELECTRONS IN A
MAGNETIC FIELD
8
NUCLEAR MAGNETIC
RESONANCE
0.6-10 m
1.7*10-2-1*103
Spin of nuclei in
magnetic field
o
o
The Electromagnetic Spectrum
 Note that the visible portion of the spectrum to which the human
eye is sensitive is tiny when compared with other spectral regions.
21
The Electromagnetic Spectrum
 It should also be noted that spectrochemical methods
that employ not only visible but also ultraviolet and
infrared radiation are often called Optical Methods
despite the fact that the human eye is sensitive to neither
of the latter two types of radiation. This somewhat
ambiguous terminology arises from the many common
features of instruments for the three spectral regions and
the similarities in the way in which we view the
interactions of the three types of radiation with matter
22
The Electromagnetic Spectrum
23
Transmission of Radiation
 The rate at which radiation is propagated through a transparent
substance is less than its velocity in a vacuum and depends upon
the kinds and concentrations of atoms, ions, or molecules in the
medium. If follows from these observations that the radiation must
interact in some way with the matter. Because a frequency change
is not observed, however, the interaction cannot involve a
permanent energy transfer.
 The refractive index of a medium is one measure of its interaction
with radiation and is defined by : ŋi = c / νi
 Where ŋi is the refractive index at a specified frequency, νi is the
velocity of the radiation in the medium, and c is its velocity in a
vacuum.
 The refractive index of the most liquids lies between 1.3 and 1.8; it is 1.3
to 2.5 or higher for solids
24
Refraction of Radiation
 When radiation passes at an angel through the interface
between two transparent media that have different densities,
and abrupt change in direction, or refraction, of the beam is
observed as a consequence of a difference in velocity of the
radiation in the two media. When the beam passes from a
less dense to a more dense environment, as in figure, the
bending is toward the normal to the interface. Bending away
from the normal occurs when the beam passes from a more
dense to a less dense medium.
 The extent of refraction is given by Snell’s law:
 Sin θ1 / Sin θ2 = ŋ2 / ŋ1 = ν1/ ν2
25
Refraction of Radiation
26
refractive index
 When radiation crosses and interface between media that
differ in refractive index, reflection always occurs. The
fraction of radiation reflected becomes greater with
increasing differences in refractive index. A beam that enters
an interface at right angles, the fraction reflected is given by :
 Ir = (ŋ2 - ŋ1) 2
I0 (ŋ2 + ŋ1) 2
Where I0 is the intensity of the incident beam and Ir is the
reflected intensity; ŋ1& ŋ2 re the refractive indexes of the
two media
27
Scattering of radiation
The transmission of radiation in mater can be pictured as a
momentary retention of the radiant energy by atoms, ions or
molecules followed by reemission of the radiation in al directions
as the particles return to their original state. With atomic or
molecular particles that are small relative to the wave length of the
radiation, destructive interface removes most but not all of the
reemitted radiation except the radiation that travels in the original
direction of the beam; the path of the beam appears to be
unaltered as a consequence of the interaction. Careful observation,
however, reveals that a very small fraction of the radiation is
transmitted at all angles from the original path and that the
intensity of this scattered radiation increases with particle size.
28
Rayleigh Scattering
 Scattering by molecules or aggregates of molecules with
dimensions significantly smaller than the wavelength of
the radiation is called Rayleigh scattering; its intensity is
proportional to the inverse fourth-power of the
wavelength, the dimensions of the scattering particles,
and the square of the polarizability of the particles. An
everyday manifestation of Rayleigh scattering is the blue
color of the sky, which results from the greater
scattering of the shorter wavelength of the visible
spectrum.
29
Scattering by Large Molecules
 with particles of colloidal dimensions, scattering I is
sufficiently intense to be seen by the naked eye (the Tyndall
effect). Measurements of scattered radiation are used to
determine the size and shape of polymer molecules and
colloidal particles.
30
Raman scattering
 The Raman scattering effect differs from ordinary
scattering in that part of the scattered radiation suffers
quantized frequency changes. These changes are the
result of vibration energy level transitions that occur in
the molecules as a consequence of the polarization
process.
 Energy of radiation in the visible region is often
expressed in kJ/mol rather than kJ/photon to aid in the
discussion of the relationships between the energy of
absorbed photons and the energy of chemical bonds.
31
Energy states of chemical Species
 The quantum theory was first proposed in 1900 by Max
Planck(h), a German physicist, to explain the properties of
radiation emitted by heated bodies. The theory was later
extended to rationalize other types of emission and
absorption processes. Two important postulates of quantum
theory include:
32
Energy states of chemical Species
 1. Atoms, ions, and molecules can exist only in certain
discrete states, characterized by definite amounts of
energy. When a species changes its state, it absorbs or
emits an amount of energy exactly equal to the energy
difference between the states.
 2. When atoms, ions, or molecules absorb or emit
radiation in making the transition from one energy state
to a second, the frequency ν or the wave length λ of the
radiation is related to the energy difference between the
states by the equation
33
Energy states of chemical Species
 E1 - E0 = hv = hc / λ
 When E1 s the energy of the higher state and E0 the energy of
the lower state. The terms c and h are the speed of light and
the Planck constant, respectively.
34
Energy states of chemical Species
 For atoms or ions in the elemental state, the energy of any given
state arises from the motion of electrons around the positively
charged nucleus. As a consequence the various energy states are
called electronic states. In addition to having electronic states,
molecules also have quantized vibrational states that are associated
with the energy of interatomic vibrations and quantized rotational
states that arise from the rotation of molecules around their
centers of gravity.
 The lowest energy state of an atom or molecule is its ground state.
Higher energy states are termed excited states.
 Generally at room temperature, chemical species are in their
ground state.
35
Emission of Radiation
 Electromagnetic radiation is produced when excited particles
(atoms, ions, or molecules) relax to lower energy levels by
giving up their excess energy as photons.
 Excitation can be brought about by variety of means,
including:
36
Emission of Radiation
1.
2.
3.
37
►Bombardment with electrons or other elementary
particles, which generally leads to the emission of Xradiation;
►Exposure to an electrical current ac spark or the
heat of a flame, and arc, or a furnace, which produces
ultraviolet, visible, or infrared radiation.
►Irradiation with a beam of electromagnetic
radiation, which produces fluorescent radiation; and
exothermic chemical reaction that produces
chemiluminescence's
types of spectra
 Three types of spectra are evident in the figure: lines, bands,
and a continuum.
o The line spectrum is made by of a series of sharp, welldefined peaks caused by excitation of individual atoms
38
types of spectra
o The band spectrum consists of several groups of lines so
closely spaced that they are not completely resolved. The
source of the bands consists of small molecules or
radicals . Finally, the continuum portion of the spectrum
is responsible for the increase in the background that is
evident above about 350 nm.
39
types of spectra
 Continuum spectra
 Truly continuum radiation is produced when solids are heated to
incandescence. Thermal radiation of this kind, which is called
black-body radiation is characteristic of the temperature of the
emitting surface rather than the material of which that surface is
composed. Blackbody radiation is produced by the innumerable
atomic and molecular oscillations excited in the condensed solid
by the thermal energy. Note that the energy peaks in Figure 6-18
shift to shorter wavelengths with increasing temperature. It is
clear that very high temperatures as needed to cause a thermally
excited source to emit a substantial fraction of its energy as
ultraviolet radiation.
40
41
Absorption of Radiation
 The Absorption of Radiation
 There are three basics processes by which a molecular can absorb
radiation; all involve raising the molecule to higher internal energy
lever.
 Rotation transition; the molecule absorb radiation and be
raised to higher rotational energy level.
 Vibrotional transition; the molecule absorb amount of energy
and be raised to higher vibrotional energy.
 Electronic transition; the electron of a molecule absorb
amount of energy and raised to a higher vibrational energy level.
42
Spectral changes can be closed as
follows;
1.Bathochromic shift;
the shift of absorption to longer wavelength due to substitution
(a red shift)
∆λ = λ2 – λ1
while λ2 > λ1
“changed spectral band position of molecule to a longer wave length and longer
frequency”

2. Hypsochromic shift ;
the shift of absorption to shorter wave length due to substitution or solvent
effect (a blue shift)shorter wavelengths and higher frequency
∆λ = λ1 – λ2
while λ1 > λ2

3. Hyperchromic effect
An increase in absorption intensity

4. Hypochromic effect
A decrease in absorption intensity


43
Electronic spectral and molecular
structure
 Electronic spectral and molecular structure
 The electronic transitions that take place in the vv-vi,
regions of the spectrum are due to the absorption of
radiation by specific types of groups, bonds, and
functional group.
 The wave length of absorption in a measure of the
energy required for the transition.
44
Kind of transition
 Kind of transition: electron in a molecule can be
classified in to 4 different types:
 Closed shell electrons that are NOT involved in bonding (high
excitation energies and don’t contribute to absorption in the in
the visible or UV-region).
 Co-valent single bond electron (б-electrons) also possess too high
and excitation energy to contribute to absorption of UV-vis
radiation {-CH2-CH2-}
 Paired non-bonding, outer shell electrons(n-electrons) N ,O, S
which can be excited by UV-vis.
 Electrons in π orbital's (double, triple bonds) excited and
responsible for electronic spectra in UV-region.
45
Kind
of
transition….
NOTE
o A molecule also possess normally unoccupied orbital called anti-
bonding orbital's; these corresponds to excited state energy level
and either б * or л * orbital's.
o Hence, absorption of radiation results in an electronic transition
to and anti-bonding orbital
46
Chromophore
 Chromophore which is a covalently unsaturated group responsible for
electronic absorption (C=C, C=O)
 Chromophore: absorption group
 Chromophore:
It is a group which is responsible for light
absorption.
Chromospheres: a chemical group with high electron
density that induces high light absorption
(benzene).
47
Auxochrome
 An Auxochrome a saturated group with non-bonded electrons
(OH, NH2,Cl) does not absorb radiation itself but if present in
molecule it can alerts both wave length and intensity of the
radiation
 Auxochrome:
It is a group that does not possess absorption
but it enhances absorption by a chromophore,
all Auxochrome contain atoms with unshared
electron pair.
 Auxochrome : a chemical group that doesn’t
have strong absorption on its own but can
enhance the absorption of adjacent chromophore
(NO2,OH).
48
Conjugated system
Conjugated system where
multiple bonds (double, triple)
can be separated by one
single bond.
49
types of absorbance
 There are three There are three t Types of
absorbance instruments used to collect
UV-vis spectra:
 1) Single beam spectrometer.
 2) Double beam spectrometer.
 3) Simultaneous spectrometer.
50
Beer’s law
 pka=pH + log (Ai-A)/(A-Au)
T=10-A
 A = log 1/T,
A= 2- log T%
 A = A1+A2+….An --- A=Σabc
 C = A / (A(1%,1cm)
 T= P/P0 …. I/I0
 A= - log T,
51
Absorption Methods
The quantitative absorption methods require two power
measurements; one before a beam has passed through the
medium that contains the analyte (P0) and the other after
(P). Two terms, which are widely used in absorption
spectrometry and are related to the ration of (P0) and
(P), are transmittance and absorbance.
52
Transmittance
o Depicts a beam of parallel radiation before and after it has passed
through a medium that has a thickness of b cm and a concentration
c of an absorbing species. As a consequences of interactions
between the photons and absorbing atoms or molecules, the
power of the beam is attenuated from P0 to P. The transmittance T
of the medium is then the fraction of incident radiation
transmitted by the medium :

T = P / P0
`
Transmittance is often expressed as percentage or:
 %T = P / P0 × 100%
53
Absorbance
 The absorbance A of a medium is defined by the equation:
 A = - log 10 T = log P0 /P
54
Beer’s law
 For monochromatic radiation, absorbance is directly
proportional to the path length b through the medium and
the concentration c of the absorbing species.
These relationships are given by :

A = abc
55
A = abc
 Where a is a proportionality constant called the absorptivity. The
magnitude of a will clearly depend upon the units used for b and c.
for solutions often absorbing species b is often given in terms of
centimeters and c in grams per liter. Absorptivity then has units of
: Lg-1cm-1
 Where the concentration is expressed in moles per liter and the
cell length is in centimeters, the absorptivity is called the molar
absorptivity and is given the special symbol ε. Thus, when b is in
centimeters and c is in moles per liter,
56
A= εbc
Where ε has the units Lmol-1cm-1 .
expressions of Beer’s law,
57
Variables that influence absorbance
 Nature of solvent
 pH of solution
 The temperature
 High electrolytes concentration
 Presence of interfering substances
58
Deviation of Lawbort Bear law
 At a high conc. The linear relationship not hold good.
 Deviation from the low because absorptivity depends on
the refractive index of the medium which is function of
concentration “see ref index”
 Association , dissociation of rxn with the solvent can
disort the linear relationship.
 Instrumental deviation with polychromic radiation
 Instrumental deviation in the presence of stray radiation.
59
Application of UV-Vis spectroscopy in
Pharmaceuticals
 A robust workhorse method for the quantification of drugs in
formulation were is no interference from excipients.
 Determination of the pka value of some drugs
 Determination of partition coefficient of and solubility of the
drugs
 The UV spectroscopy of a drug is often used as one of number of
pharmacopeia identity checks.
 Used to determine the release of drugs form formulation with
time for rxn kinetics of drug and degradation
60
General Designs of optical instruments
 Optical spectroscopic methods are based upon six phenomena :
1. absorption,
2. fluorescence ,
3. phosphorescence,
4. scattering,
5. emission, and
6. chemiluminescence.
 While the instruments for measuring each differ somewhat in
configuration, most of their basic components are remarkably
similar. Furthermore, the required properties of these
components are the same regardless of whether they are applied to
the ultraviolet, visible, or infrared portion of the spectrum.
61
Typical spectroscopic instruments contain
five components, including :
 A stable source of radiant energy.
 A transparent container for holding the sample
 A device that isolates a restricted region of the spectrum
for measurement.
 A radiation detector, which converts radiant energy to a
usable signal (usually electrical)
 A signal processor and readout, which displays the
transduced signal on a meter scale, an oscilloscope face, a
digital meter, or a recorder chart.
62
63
64
65
Types of optical instruments
 A spectroscope: is an optical instrument used for the visual
identification of atomic emission lines. It consists of a
monochromator,, in which the exit slit is replaced by an eye-piece
that can be moved along the focal plane. The wavelength of an
emission line can then be determined from the angle between the
incident and dispersed beam when the line is centered on the
eyepiece.
 We use the term colorimeter to designate an instrument for
absorption measurements in which the human eye serves as the
detector using one or more color- comparison standards.
66
Types of optical instruments
 A photometer consists of a source, a filter, and
photoelectric transducer as well as a signal processor and
readout. It should be noted that some scientists and
instrument manufacturers refer to photometers as
colorimeters or photoelectric colorimeters. Filter
photometers are commercially available for absorption
measurements in the ultraviolet, visible, and infrared
regions, as well as emission and fluorescence in the first
two wave length regions. Photometers designed for
fluorescence measurements are also called fluorometers.
67
Types of optical instruments
 A spectrograph, is similar in construction to the two
monochromators except that the slit arrangement is
replaced with a large aperture that holds a detector or
transducer that is continuously exposed to the entire
spectrum of dispersed radiation. Historically, the
detector was photographic film or plate. Currently,
however, diode arrays or charge-transfer devices are
often used as transducers in spectrographs.
68
Types of optical instruments
 A spectrometer is an instrument that provides information about the
intensity of radiation as a function of wavelength or frequency. The
dispersing modules in some spectrometers are multichannel so
that two or more frequencies can be viewed simultaneously. Such
instruments are sometimes called polychromators.
 A spectrophotometer is a spectrometer equipped with one or more
exit slits and photoelectric transducers that permit the
determination of the ratio of the power of two beams as a function
fo wavelength as in absorption spectroscopy. A spectrophotometer for
fluorescence analysis is sometimes called a spectrofluorometer.
69
Types of optical instruments
 All of the instruments named in this section thus far employ filters or
monochromators to isolate a portion of the spectrum for measurement. A
multiplex instrument, in contrast, obtains spectral information without first
dispersing or filtering the radiation to provide wavelengths of interest. The term
multiplex comes from communication theory, where it is used to describe
systems in which many sets of information are transported simultaneously
through a single channel. Multiplex analytical instruments then are singlechannel devices in which all components of an analytical response are collected
simultaneously.
 In order to determine the magnitude of each of these components, its is
necessary to modulate the analyte signal in a way that permits subsequent
decoding of the response into its components.
70