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Faculty of pharmacy &Medical Science
Petra University
DR. WAEL ABU DAYYIH pharmaceutical ANALYSIS
(501722)
2012

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 S pectrometric 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.


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

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.

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


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

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
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
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

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 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.

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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.

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 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.

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 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.

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 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.

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 Multiplication of the frequency in cycles per second by the wavelength in meters per cycle gives the
ν i second:
i in meters per
=
i
 It is important to realize that the frequency of the beam of radiation is determined by the source and
In contrast, the velocity of radiation depends upon the composition of the medium through which it passes
.

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

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
× 10 8 m/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 × 10 8 m/s = 3.00 × 10 10 cm/s

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 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 .

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
ύ
which is defined as the reciprocal of the wavelength in centimeters, is yet another way of describing electromagnetic radiation. The unit for
ύ
-1
ave number is widely used IR- infrared spectroscopy. The wave number ύ
s a useful unit because, in contrast to wave length, its is
proportional to the frequency, and thus the energy, of radiation. Thus, we may write :

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
ύ = k ν


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 .

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Monochromator (Monochromatic) beam: is a beam of radiation whose rays have identical wavelengths
Polychromatic beam is made up of rays of different wavelengths
The common unit of frequency is reciprocal second S
-1
Hertz(Hz) which corresponds to one cycle per second.
or
The units commonly used for describing wavelength differ considerably in the various spectral regions
A o
: (Angstrom)unit is suitable for X-ray and short ultraviolet radiation

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The nanometer (nm) is employed with visible and ultraviolet radiation
The micrometer (µm)( micron) is useful for the infrared region .
A o
=(10
-10 m) nm =(10
-9 m)
µm=(10
-6 m)
1 A o
= 10
-10 m = 10
-8
1nm = 10
-9 m = 10
-7
1 µm = 10
-6 m = 10
-4 cm cm cm

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No. Type Spectroscopy
1
2
6
7
3
4
5
8
GAMMA – RAY EMISSION
X- RAY
ABSORPTION,EMISSION,
FLUORESCENCE,
DIFFRACTION
VACUUM ULTAVIOLET
ABSORPTION
UV-VIS.ABSORPTION
,EMISSION,FLUORES.
INFRARED ABS& RAMAN
SCATTERING
MICROWAVE ABS.
ELECTRON SPIN
RESONANCE
NUCLEAR MAGNETIC
RESONANCE
Usual Wavelength
Rang
Usual
Wavenumber
Range,cm -1
0.005 – 1.40 A o
0.10-100 A o
-----------
------------
Type of Quantum
Transition
NUCLEAR
INNER
ELECTRON
10-180 nm
180-780 nm
0.78-300 µm
0.75-3.75 mm
3 cm
0.6-10 m
1*10
13-27
0.33
6 -5*10
1.3*10
3.3*10
4
1
-
4
5*10 4 -1.3*10 4
1.7*10 -2 -1*10 3
BONDING
ELECTRON
B.E
ROTATION/VIBR
ATION OF
MOLECULES
ROTATION OF
MOLECULES
SPIN OF
ELECTRONS IN A
MAGNETIC FIELD
Spin of nuclei in magnetic field

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 Note that the visible portion of the spectrum to which the human eye is sensitive is tiny when compared with other spectral regions.

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 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

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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 : ŋ = c / ν i i is the velocity of the radiation in the medium, and c is its velocity in a vacuum. i
Where ŋ i is the refractive index at a specified frequency, ν
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

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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 θ
1
/ Sin θ
2
= ŋ
2
/ ŋ
1
= ν
1
/ ν
2

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 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 :
 I r
I
0
= ( ŋ
2
( ŋ
2
ŋ
1
) 2
+ ŋ
1
) 2
Where I
0 is the intensity of the incident beam and reflected intensity; ŋ
1 two media
& ŋ
2
I r is the re the refractive indexes of the

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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.

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 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.

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 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.

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
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.

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 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:

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

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

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 E
1
- E
0
= hv = hc / λ
 When E
1 s the energy of the higher state and E
0 the energy of the lower state. The terms c and h are the speed of light and the Planck constant, respectively.

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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.

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 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:

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1.
2.
3.
► 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

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 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

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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.

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

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.

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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.

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




1.Bathochromic shift; the shift of absorption to longer wavelength due to substitution
( a red shift )
∆ λ = λ
2
– λ
1 while λ
2
> λ
1
“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
λ effect ( a blue shift )shorter wavelengths and higher frequency
= λ
1
– λ
2 while λ
1
3. Hyperchromic effect
> λ
2
An increase in absorption intensity
4. Hypochromic effect
A decrease in absorption intensity

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 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.

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
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
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.

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A molecule also possess normally unoccupied orbital called antibonding 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

 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).
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 An Auxochrome a saturated group with non-bonded electrons
(OH, NH
2
,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
(NO
2
,OH).

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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.

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 pka=pH + log (Ai-A)/(A-Au)
 A= - log T, T=10 -A
 A = log 1/T, A= 2- log T%


A = A
1
+A
2
+….A
n
--- A=
C = A / (A(1%,1cm)
Σ abc
 T= P/P
0
…. I/I
0

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 The quantitative absorption methods require two power measurements; one before a beam has passed through the medium that contains the analyte (P
0
) and the other after
(P). Two terms, which are widely used in absorption spectrometry and are related to the ration of (P
0
) and
(P), are transmittance and absorbance .

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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 / P
0
`
Transmittance is often expressed as percentage or:
 %T = P / P
0
× 100%

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

The absorbance A of a medium is defined by the equation:
A = - log
10
T = log P0 /P

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 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

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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 -1 cm -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,

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A=
bc
 Where
has the units Lmol-1cm-1 .
expressions of
,

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 Nature of solvent
 pH of solution
 The temperature
 High electrolytes concentration
 Presence of interfering substances

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




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.

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 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

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 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 .

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
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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.

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

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.

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 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 .

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 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.

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
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 .

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

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.