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PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 1
Spectrophotometry
Spectrophotometry is one of the spectroscopic methods in which the study of interaction between light energy and matter is used for qualitative and quantitative determination of different substances.
Advantages of spectrophotometry: 1. Wide applications. 2. Ease and convenience. 3. High selectivity. 4. High sensitivity.
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 2
Electromagnetic Radiation (EMR)
• -rays
• X-rays
• UV (Ultraviolet)
• Visible light (Vis)
• IR (Infrared)
• Microwaves
• Radiowaves
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 3
Electromagnetic Spectrum
-ray x-ray Visible IR w Rw
10-9 10-7 10-5 10-3 10-1 102
Wavelength (, cm )
Frequency ( , Hz )
108 1012 1014 1016 1018 1020
UV
Wavelength (nm)
400 500 600 700 800
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 4
The UV- Visible Radiation
• The UV region of spectrum extends from about 100 - 200 nm (far or vacuum UV) and from about 200 - 400 nm (near UV).
• The visible region of spectrum extends from about 380 nm to about 780 nm. The eye can normally detect only the colors within this wavelength range, that is why it is called visible.
• The visible spectrum consists of seven colors which are; violet, indigo, blue, green, yellow, orange and red, each color has characteristic wave length region.
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 5
The UV- Visible Radiation
• When all the wavelengths or colors of the visible light are transmitted or reflected together, the light appears as white light.
• While if all wavelengths or colors of visible light are absorbed, it appears black.
• Colored substances appear colored because they selectively absorb some of wavelengths of visible light and transmitt or reflect other wavelengths or colors (apparent color).
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 6
The UV-Visible Region Wavelength nm Color Complementary
(apparent) color
200-400
400-435 Violet Yellow-green
435-480 Blue Yellow
480-490 Blue-green Orange
490-500 Green-blue Red
500-560 Green Purple
560-580 Yellow-green Violet
580-595 Yellow Blue
595-650 Orange Green-blue
650-750 Red Blue-green
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 7
Light and Radiation • Light can be described as a wave. This wave has an electric
component and a magnetic component which are perpendicular
to each other.
E = Energy in joules (J)
u= Frequency (Hz)
h = Plank’s constant (6.63x10-27 erg. s)
= Wavelength (m) = C/ u
C = Speed of light (3 x 108 m/s in vacuum) = u
E = h u
E = h C/
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 8
Properties of Electromagnetic Radiation (EMR)
• 1-Wave Properties:
• Wavelength (λ):
• It is distance between two successive maxima or minima of wave (nm).
• Frequency (ʋ):
It is the number of cycles (waves) per second
[Hertz (Hz)].
It is inversely proportional to the wave length
ʋ α 1 / λ
ʋ = C / λ
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 9
2-Particle properties: • EMR can be viewed as a stream of particles
known as photons. The energy of a photon depends upon the frequency of radiation and can be expressed by Max Plank relation;
E α ʋ
E = h ʋ
Where h is Plank's constant
h = 6.63 x 10-34 j/s or,
= 6.63 10-27 erg/s
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 10
Interaction of substance with EMR
• When a molecule interact with EMR, it will absorb energy and the molecule is said to be excited because electrons undergo transition from original energy level (ground state = Eg) to an excited state (Es).
• Transition energy is given by:
Et or ∆E = Es – Eg = h ʋ
M + E → M* (excitation)
• After a brief period (10-6-10-9 S) M* relaxes to its ground state.
M* → M + E (relaxation)
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 11
Interaction of photons with matter
A molecule may absorb light energy in three ways:
E total = E transitional + E vibrational + E rotational
[2] Increasing the vibration of constituent nuclei (vibrational)
when molecule absorb IR irradiation.
[3] Raising an electron to a higher energy level (transitional energy)
when molecule absorb visible and UV light.
[1] Increasing the rotation of molecule around its axis (rotational)
when molecule absorb F-IR irradiation.
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 12
Interaction of photons with matter
When a molecule absorbs photons in the UV-VIS region, the absorption
of energy results in displacing an outer electron (valence electron) in the
molecule. The molecule is said to undergo transition from the ground
state of energy level to an excited state of energy level.
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 13
Interaction of photons with matter An excited molecule loses energy and returns to the ground state.
The energy is released in the form of:
Heat: electrons return directly to ground state.
Light (fluorescence): electrons return via a second excited state.
Molecular collisions. PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 14
Types of Electronic Transition
Anti-bonding
Anti-bonding
n Non-bonding
Bonding
Bonding n -
-
n -
-
200 300
Wavelength, nm
150 250
-
-
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 15
Absorption Spectrum
Absorption spectrum: it is characteristic to substance, and the wavelength at
which the maximum absorption is recorded and used to trace the substance
strength to enhance the sensitivity.
Absorption band spectrum for some molecules
max
Amax
Is a plot of absorption intensity versus the wavelength of the absorbed light
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 16
Some Important Terms
Chromophores: (Chrom = color, phore = carrier)
They are functional groups which confer color on substances capable of absorbing
UV and/or visible light. They have unsaturated bonds.
Examples: C = C, - C = O, - N = N, and – C ≡ N ( electrons).
Auxochromes: They are functional groups which can not confer colors on substances
but have the ability to increase the coloring power of chromophores, they do not absorb
radiations longer than 200 nm, but when attached to a given chromophore, cause a
shift to a longer wavelength with increase in absorption intensity.
Examples: - OH, - NH2.
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 17
Some Important Terms
Hypsochromic Bathochromic
Hyp
erc
hro
mic
H
ypo
rch
rom
ic
Wavelength, nm
A
bso
rba
nce
APEX
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 18
Some Important Terms
• Bathochromic shift ( red shift ): it is the shift of max to a longer wavelength
due to substitution or solvent effects.
• Hypsochromic shift ( blue shift ): it is the shift of max to a shorter wavelength.
• Hyperchromic effect: enhancement of molecule absorptivity (or absorption
intensity).
• Hypochromic effect: decrease of molecule absorptivity (or absorption intensity).
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 19
Factors Affecting Absorption Spectrum
Affect what ?
Maximum wavelength ( max )
Intensity ( )
Effect of pH on absorption spectra
Effect of solvent on absorption spectra
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 20
Factors Affecting Absorption Spectrum
NH2 NH3
HCl
• • + Cl
Aniline
280 nm
1,430
Anilinium
254 nm
160
max
Aniline
Effect of pH on absorption spectra
The UV spectrum of aniline in acid medium shows: hypsochromic shift with hypochromic effect.
This shift is due to the protonation of the amino group, hence the pair of electrons is no longer
available and the spectrum becomes similar to that of benzene (thus called benzenoid spectrum).
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 21
Factors Affecting Absorption Spectrum Effect of pH on absorption spectra
Phenol
270 nm
1,450
Phenolate
290 nm
2,600
• •
• •
OH • • • •
• • Na
+ O O
OH-
H+
max
Phenol
The UV spectrum of phenol in acidic medium is completely different from its
spectrum in alkaline medium (using same concentration). The spectrum in alkaline
medium exhibits bathochromic shift with hyperchromic effect. The red shift is due
to the participation of the pair electrons in resonance with the -electrons of the
benzene ring, thus increasing the delocalization of the -electrons.
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 22
Applications of Spectrophotometry 1. Qualitative Analysis
Analysis of Morphine
since morphine is a phenolic compound,
the observation of bathochromic shift with
hyperchromic effect in KOH is consistent
with, but not definite proof of, the
presence of morphine in the sample.
HO O
HNH3C
OH
Since other phenolic compounds show similar behavior, this test is
a definite proof of the absence of morphine in the sample.
For definite proof for the presence of morphine, better method
(e.g. infrared spectroscopy) should be used. PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 23
Applications of Spectrophotometry Quantitative Analysis
1. Determination of proper max :
450 550 650 350
Wavelength, nm PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 24
Applications of Spectrophotometry 2. Generating the calibration curve at max :
Unknown Standard solutions
Concentration
Ab
sorb
an
ce
Linear equation: A = a + b C
Unknown conc. Is determined by:
Calibration curve: graphically
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 25
Laws of light absorption
It relates absorption capacity to the thickness of an absorbing solute
(path length of light).
Lambert’s Law:
Log Io / I = K b
Io Incident light
I Transmitted light
K Proportionality constant
b Light path length
It relates absorption capacity to the concentration of an absorbing
solute.
Beer’s Law:
Log Io / I = K C
Io Incident light
I Transmitted light
K Proportionality constant
C Concentration PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 26
Laws of light absorption
Log Io / I = A (absorbance)
It relates absorption capacity to the thickness of an absorbing solute
(path length of light) and the concentration. It is a combination between
Beer’s law and Lambert's law.
Beer’s - Lambert’s Law:
Log Io / I = a b C
A = a b C Usually b = 1 cm A = a C
Io Incident light
I Transmitted light
a Absorptivity
b Light path length (in cm)
C Concentration (in g/L)
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 27
Laws of light absorption
Beer’s - Lambert’s Law: A = a b C
Concentration
Ab
sorb
an
ce
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 28
Laws of light absorption
ε (Epsilon), Molar absorptivity,
if concentration (c) expressed as molar solution.
A = ε b c
Beer’s - Lambert’s Law: A = a b C
Expressions of a
A one percent one centimeter
if c is expressed in g/100 mL
A1%
1cm b c A
1%
1cm A =
a absorptivity,
if concentration (c) expressed as gram / Liter.
A = a b c
ε at max it is called εmax. = ε x 10 / molecular weight A1%
1cm PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213
29
• The amount of radiation absorbed may be measured in a number of ways:
• Transmittance, T = I / Io % Transmittance, %T = 100 T
• Absorbance,
• A = log10 Io / I A = log10 1 / T A = log10 100 / %T A = 2 - log10 %T
Laws of light absorption
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 30
• The last equation, A = 2 - log10 %T , is worth remembering because it allows you to easily calculate absorbance from percentage transmittance data.
• The relationship between absorbance and transmittance is illustrated in the following diagram
Laws of light absorption
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 31
• A drug with molar absorptivity ε = 1.6 x 102
L/mole.cm yields an absorbance of 0.73 when measured in a 1-cm cell. Calculate the concentration.
• A = ε b c c = A/ ε b
• C = 0.73/160 = 0.0046 M
Laws of light absorption
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 32
• Cytosine has a molar absorptivity of 6x103 at 270 nm at pH 7. Calculate the absorbance and percent transmission of 1x10-5 and 1x10-4 M cytosine solution in a 1-cm cell.
• A = log(I0/I) = ε b c = 6x103 x 1 x 1x10-5 = 0.06 • in percent transmission, I/I0 x 100, • T% = 10-0.06 x 100 = 101.94 (%) = 87.10 % • Similarly, for 1x10-4M, the absorbance A is given as, • A = log(I0/I) = ε b c = 6x103 x 1x 10-4 = 0.6 • in percent transmission, I/I0 * 100, • T% = 10-0.6 x 100 = 101.4 (%) = 25.12 %
Laws of light absorption
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 33
Spectrophotometer
• The instrument used for quantitative measurement of absorbance or transmittance of UV/Vis light.
• Must contain five basic components
1- Light source: required to emit the wavelength of interest.
2- Monochromator : used to split the light into the different wavelengths.
3-A sample compartment : holds the sample to be analysed.
4-A detector.
5-The readout
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 34
Essential parts of spectrophotometer
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 35
Types of spectrophotometers
Amplifier Meter Light
source
Sample
cuvette
Monochromator
Detector
Single-Beam Spectrophotometers:
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 36
Components of spectrophotometer Light source:
- UV measurement: hydrogen or deuterium discharge lamp
(190 – 375 nm)
- Visible measurement: Tungsten lamp
(350 – 1000 nm)
Monochromator:
Function: To select light beam of certain wavelength.
- Filter
- Prisms
- Grating
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 37
Components of spectrophotometer
Filters: function via selective absorption of unwanted wavelength and
transmitting the complementary color. It consists of colored glass, or
dye suspended in gelatin and sandwiched between two glass plates.
Monochromators:
Prisms: function via refraction of light.
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 38
Components of spectrophotometer Monochromators:
Gratings: Consist of large number of parallel ruled very close to each
other on a highly polished surface, e.g. aluminium, or aluminized glass
(600 groove/mm). Each ruled groove functions as a scattering center
for light falling on its edge and through diffraction and interference the
grating disperses the light beam into almost single wavelength.
Incident
light Diffracted
light
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 39
Components of spectrophotometer
• Transparent
• Quartz for UV measurements
• Glass or Quartz cell for VIS measurements
• Pathlength: usually 0.5, 1 or 1 cm
Sample Cell (Cuvette) :
Sample cuvette
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 40
Components of spectrophotometer
Receive light emerged from the sample,
which excite electrons and generate an
electric current that proportional to the
received light intensity.
Detectors :
Anode (iron)
Electrons
Cathode (selenium) Light
beam
Photocell Photomultiplier tube PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213
41
Components of spectrophotometer
The function of the meter to translate the received current (current
intensity proportional to light emerged from sample) to signals on
paper or computer (integration of data).
Recorders :
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 42
Types of spectrophotometers Double-Beam Spectrophotometers:
Light
source
Monochromator
Amplifier Meter
Blank cuvette
Sample
cuvette
Detector 1
Detector 2
Beam
splitter
PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 43