instrumental analysis spectroscopy dr. hisham e abdellatef [email protected]
TRANSCRIPT
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Definition
• Spectroscopy - The study
of the interaction of
electromagnetic radiation
with matter
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• What to be discussed – Theoretical background of spectroscopy
– Types of spectroscopy and their working principles in brief
– Major components of common spectroscopic instruments
– Applications in Chemistry related areas and some examples
Introduction to Spectroscopy
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Electromagnetic Spectrum
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Electromagnetic Spectrum
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Electromagnetic Spectrum
Co
smic
X-r
ay
Ult
ravi
ole
t
Vis
ible
Infr
ared
Mic
row
ave
Rad
io
(nm)
Hz 1021 1018 1015 1012 109 106
10-3 1 200 500 106 109 1012
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Electromagnetic Radiation
• Electromagnetic radiation (e.m.r.) – Electromagnetic radiation is a form of energy– Wave-particle duality of electromagnetic
radiation • Wave nature - expressed in term of frequency, wave-length and velocity
• Particle nature - expressed in terms of individual photon, discrete packet of energywhen expressing energy carried by a photon, we need to know the its frequency
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Definitions
• E = energy (Joules, ergs)
• c = speed of light (constant) = wavelength
• h = Planck’s constant = “nu” = frequency (Hz)
• nm = 10-9 m
• Å = angstrom = 10-10 m
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• Characteristics of wave – Frequency, v - number of oscillations per unit time, unit: hertz (Hz) - cycle per second– velocity, c - the speed of propagation, for e.m.r c=2.9979 x 108 ms-1 (in vacuum)– wave-length, - the distance between adjacent crests of the wave
wave number, v’, - the number of waves per unit distance v’ =-1
• The energy carried by an e.m.r. or a photon is directly proportional to the
frequency, i.e. where h is Planck’s constant h=6.626x10-34Js
Electromagnetic Radiation
c'vc
v
c'hvhc
hvE
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Key Formulae
• E = h• h = 6.626 x 10-34 J-s = frequency in Hz, E = energy = c/• c = 3.0 x 108 m/s = wavelength, = frequency in Hz
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Molecular Absorption
• The energy, E, associated with the molecular bands:Etotal = Eelectronic + Evibrational + Erotational
In general, a molecule may absorb energy in three ways:•By raising an electron (or electrons) to a higher energy level.•By increasing the vibration of the constituent nuclei. •By increasing the rotation of the molecule about the axis.
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Absorption vs. Emission
Eo
h
Absorption
En
Eo
h
Emission
En
h
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Rotational absorption
Vibrational absorption
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Type of EM Interactions
• Absorption - EM energy transferred to absorbing molecule (transition from low energy to high energy state)
• Emission - EM energy transferred from emitting molecule to space (transition from high energy to low energy state)
• Scattering - redirection of light with no energy transfer
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Type of electronic transitions:•Sigma () electrons: represent valence bonds They posses the lowest
energy level (i.e. most stable)•pi () electrons: pi bonds (double bonds) They are higher energy than
sigma electrons.•Non bonding () electrons: these are atomic orbital of hetero atom
(N,O, halogen or S) which do not participate in bonding. They usually occupy the highest energy level of ground state.
*
*
n
Antibonding
Antibonding
non-bonding
Bonding
En
erg
y
*
*
n
*
n
*
*
*
n
Antibonding
Antibonding
non-bonding
Bonding
En
erg
y
*
*
n
*
n
*
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UV Activity
h
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Laws of light absorption
Total light interringIo
Reflacted partIr
Absorbed partIa
Transmitted partIt
Refracted partIf
Scattered part Is
absorption
transmission
refraction
scatteringreflection
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Definitions
• Io = intensity of light through blank• IT = intensity of light through sample• Absorption = Io - IT
• Transmittance = IT/Io
• Absorbance = log(Io/IT)
Io IT
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Absorbance & Beer’s Law
Increasing absorbance
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Beer’s Law
pathlength b pathlength b
Io IT Io IT
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Beer-Lambert Law
Log I0/I = abc
A = ε. B.C
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“M
ole
cu
lar”
SP
EC
TR
UM
Absorption spectrum
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•Chromophore: C=C, C=O, N=O….•Auxchrome: e.g. -OH, NH2,-Cl …•Bathochromic shift (red shift): •the shift of absorption to a longer wavelength•Hypsochromic shift (blue shift): •the shift of absorption to a shorter wavelength•Hyperchromic effect: an increase in the absorption intensity.•Hypochromic effect; an decease in the absorption intensity
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Effect of pH on absorption spectra:
OH O O
H +
acid medium alkaline medium
Phenol
alkaline medium exhibits bathochromic shift and hyperchromic effect .
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aniline NH2NH2
+H
-H
NH3
alkaline medium acid medium
acid medium shows hypsochromic shift and hypochromic effect
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Complementary Colours
AbsorbedObserved
Absorbed colourObserved colour
400VioletYellow-green
425Dark-blueYellow
450BlueOrange
510GreenRed
550Yellow-greenPurple
575YellowViolet
590OrangeBlue
650redBlue-green
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Visible Light
Red
Orange
Yellow
Green
Blue
Indigo
Violet
R
O
Y
G
B
I
V
700 nm
650 nm
600 nm
550 nm
500 nm
450 nm
400 nm
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Single Beam Spectrophotometer
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Dual Beam Spectrophotometer
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Light source
1. Tungsten halide lamp visible molecular absorption to deliver constant and uniform
radiant energy from 350 nm up to 2400 nm.2. High pressure hydrogen or deuterium
discharged lamp are used in the UV molecular absorption to deliver continuum
source from 160-380 nm.
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Monochromator: wavelength selector
Prisms: refraction. In UV range prism can made from quartz or fused silica but in visible range
Grating: diffraction and interference. it consist of a large number of parallel line (15000 -30 000 line per inch) ruled very close to each other on a highly polished surface as aluminum or aluminized glass.
Filter,: absorption, it can be gelatin, liquid and intended glass filters.
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Cuvettes (sample holder)
• plastic or glass for determination the sample in visible rang,
• or quartz cell for determination the sample in UV. Cell usually take rectangular (cuvette)
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Light detector transducer
• convert a signal photons into an easily measured electrical signal such as voltage or current
Transducer should have the:•High sensitive
•Linear response•A fast response time
•High stability
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Light detector transducer
Types of Transducer:• 1. Barrier layer (photovoltaic cell)
• 2. Phototube
• 3. Photomulriplier
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Application of spectrophotometry
1. Quantitative analysis of a single component:
Calibration curve
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2. Quantitative analysis of multi-component mixture:
)λ(at bCεbCε A"
)λ(at bCεbCε A'"
N"NM
"M
'N
'NM
'M
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The measurement of complexation (ligand/metal ratio in a complex):
1. The mole- ratio method
(Yoe and Jones method)
2. The method of continuous
variations (Job's method)
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Deviation from Beer's law
1. Real deviations:
2. Instrumental deviations– Irregular deviations – ii. Regular deviations– Stray light:
3. Chemical deviations:
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Practical Applications
• Pharmacy Practice– Ultraquin (psoriasis med. Needs UV. Act.)– Pregnancy tests (colorimetric assays)– Blood glucose tests, Bilichek
• Pharmaceutics– pH titrations, purity measurement– concentration measurement
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pKa Measurement with UV
Titration of Phenylephrine
pKa = pH + log
i
n
Ai - A
A - An
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Pharmaceutical Apps.
• On Line Analysis of Vitamin A and Coloring Dyes for the Pharmaceutical Industry
• Determination of Urinary Total Protein Output • Analysis of total barbiturates • Comparison of two physical light blocking agents for
sunscreen lotions • Determination of acetylsalicylic acid in aspirin using Total
Fluorescence Spectroscopy • Automated determination of the uniformity of dosage in
Quinine Sulfate tablets using a Fibre Optics Autosampler• Determining Cytochrome P450 by UV-Vis
Spectrophotometry• Light Transmittance of Plastic Pharmaceutical Containers