SPECTROSCOPY

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SPECTROSCOPY
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Introduction of Spectrometric Analyses
The study how the chemical compound interacts
with different wavelenghts in a given region of
electromagnetic radiation is called spectroscopy
or spectrochemical analysis. The collection of
measurements signals (absorbance) of the compound
as a function of electromagnetic radiation is
called a spectrum.
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Energy Absorption
The mechanism of absorption energy is different
in the Ultraviolet, Infrared, and Nuclear
magnetic resonance regions. However, the
fundamental process is the absorption of certain
amount of energy. The energy required for the
transition from a state of lower energy to a
state of higher energy is directly related to
the frequency of electromagnetic radiation that
causes the transition.
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Spectral Distribution of Radiant Energy
  Wave Number (cycles/cm)
X-Ray
UV
Microwave
Visible
IR
200nm
400nm
800nm
Wavelength (nm)
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Electromagnetic Radiation
V Wave Number (cm-1) l Wave Length C
Velocity of Radiation (constant) 3 x 1010
cm/sec. u Frequency of Radiation
(cycles/sec)   The energy of photon h
(Planck's constant) 6.62 x 10-27 (Erg?sec)
C u?
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Spectral Properties, Application and Interactions
of Electromagnetic Radiation
Type Quantum Transition
Type spectroscopy
Type Radiation
Frequency ?
Wavelength ?
Wave Number V
Energy
Hz
cm
cm-1
Kcal/mol
eV
Gamma ray
Gamma ray emission
Nuclear
X-ray absorption, emission
Electronic (inner shell)
X-ray
Ultra violet
Electronic (outer shell)
UV absorption
Visible
Infrared
IR absorption
Molecular vibration
Molecular rotation
Microwave absorption
Micro-wave
Magnetically induced spin states
Nuclear magnetic resonance
Radio
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Spectrum of Radiation
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Dispersion of Polymagnetic Light with a Prism
Prism - Spray out the spectrum and choose the
certain wavelength (l) that you want by slit.
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Ultra Violet Spectrometry
The absorption of ultraviolet radiation by
molecules is dependent upon the electronic
structure of the molecule. So the ultraviolet
spectrum is called electronic spectrum.
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Electronic Excitation
The absorption of light energy by organic
compounds in the visible and ultraviolet region
involves the promotion of electrons in ?, ?, and
n-orbitals from the ground state to higher energy
states. This is also called energy transition.
These higher energy states are molecular orbitals
called antibonding.
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Electronic Molecular Energy Levels
The higher energy transitions (? ??) occur a
shorter wavelength and the low energy transitions
(???, n ??) occur at longer wavelength.
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Chromophore is a functional group which absorbs a
characteristic ultraviolet or visible region.
UV 210 nm Double Bonds 233 nm Conjugated
Diene 268 nm Conjugated Triene 315
nm Conjugated Tetraene
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Spectrophotometer
An instrument which can measure the absorbance of
a sample at any wavelength.
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Fluorometer
Instrument to measures the intensity of
fluorescent light emitted by a sample exposed to
UV light under specific conditions.
Emit fluorescent light
Antibonding
s
'
as energy decreases
p
'
Antibonding
s
n-gt
'
n-gt
p
'
Nonbonding
n
p -gtp
'
Ground state
p
Bonding
s -gts
Energy
'
s
Bonding
Electron's molecular energy levels
UV Light Source
Detector
Monochromator
Monochromator

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C
Sample
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Food Compound
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Chromophore is a functional group which absorbs a
characteristic ultraviolet or visible region.
UV 210 nm Double Bonds 233 nm Conjugated
Diene 268 nm Conjugated Triene 315
nm Conjugated Tetraene
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Beer Lambert Law
As the cell thickness increases, the transmitted
intensity of light of I decreases.
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  R- Transmittance R I0 - Original
light intensity I- Transmitted light
intensity   Transmittance 100 x
Absorbance (A) Log Log
2 - LogT Log is proportional
to C (concentration of solution) and is also
proportional to L (length of light path
through the solution).
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A ? CL ECL by definition and it is called the
Beer - Lambert Law. A ECL   A ECL E Molar
Extinction Coefficient ---- Extinction
Coefficient of a solution containing 1g molecule
of solute per 1 liter of solution
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UNITS   A ECL A No unit (numerical number
only)
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L Cm C Moles/Liter
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Steps in Developing a Spectrometric Analytical
Method

• Run the sample for spectrum
• 2. Obtain a monochromatic wavelength for the
  maximum absorption wavelength.
• 3. Calculate the concentration of your sample
  using Beer Lambert Equation A ECL

2.0
Absorbance
0.0
350
250
300
400
450
200
Wavelength (nm)
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Spectrometer Reading
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x
nm
1.0
x
A at 280
0.5
x
4
1
2
3
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Concentration (mg/ml)
There is some A vs. C where graph is
linear. NEVER extrapolate beyond point known
where becomes non-linear.
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Spectrometric Analysis Using Standard Curve
1.2
0.8
A at 540 nm
0.4
3
1
4
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Concentration (g/l) glucose
Avoid very high or low absorbencies when drawing
a standard curve. The best results are obtained
with 0.1 lt A lt 1. Plot the Absorbance vs.
Concentration to get a straight line
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Sample Cells
UV Spectrophotometer Quartz (crystalline
silica)  Visible Spectrophotometer Glass  
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Light Sources
 UV Spectrophotometer 1. Hydrogen Gas
Lamp 2. Mercury Lamp Visible Spectrophotometer 1
. Tungsten Lamp
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Chemical Structure UV Absorption
Chromophoric Group ---- The groupings of the
molecules which contain the electronic system
which is giving rise to absorption in the
ultra-violet region.
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Chromophoric Structure
Group Structure nm Carbonyl gt C
O 280 Azo -N N- 262 Nitro -NO 270
Thioketone -C S 330 Nitrite -NO2 230 C
onjugated Diene -CC-CC- 233 Conjugated
Triene -CC-CC-CC- 268 Conjugated
Tetraene -CC-CC-CC-CC- 315 Benzene 261
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UV Spectrometer Application
Protein Amino Acids (aromatic) Pantothenic
Acid Glucose Determination Enzyme Activity
(Hexokinase)
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Flurometric Application
Thiamin (365 nm, 435 nm) Riboflavin Vitamin
A Vitamin C
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Visible Spectrometer Application
Niacin Pyridoxine Vitamin B12 Metal
Determination (Fe) Fat-quality Determination
(TBA) Enzyme Activity (glucose oxidase)
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Practice Examples
  1. Calculate the Molar Extinction
Coefficient E at 351 nm for aquocobalamin in 0.1
M phosphate buffer. pH 7.0 from the following
data which were obtained in 1 Cm
cell. Solution C x 105 M Io I A 2.23
100 27 B 1.90 100 32
2. The molar extinction coefficient (E) of
compound riboflavin is 3 x 103 Liter/Cm x Mole.
If the absorbance reading (A) at 350 nm is 0.9
using a cell of 1 Cm, what is the concentration
of compound riboflavin in sample?  
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3. The concentration of compound Y was 2 x
10-4 moles/liter and the absorption of the
solution at 300 nm using 1 Cm quartz cell was
0.4. What is the molar extinction coefficient of
compound Y?   4. Calculate the molar
extinction coefficient E at 351 nm for
aquocobalamin in 0.1 M phosphate buffer. pH
7.0 from the following data which were obtained
in 1 Cm cell. Solution C x 105 M I0 I
A 2.0 100 30
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Spectroscopy Homework

• A substance absorbs at 600 nm and 4000 nm. What
  type of energy transition most likely accounts
  for each of these absorption processes?
• 2. Complete the following table.
•  X(M) Absorbance Transmittance()
  E(L/mole-cm) L(cm)
•   30 2000 1.00
• 0.5 2500 1.00
• 2.5 x 10-3 0.2 1.00
• 4.0 x 10-5 50 5000
• 2.0 x 10-4 150
•   X(M) Concentration in Mole/L
•  

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3. The molar absorptivity of a pigment
(molecular weight 300) is 30,000 at 550 nm. What
is the absorptivity in L/g-cm.    4. The iron
complex of o-phenanthroline (Molecular weight
236) has molar absorptivity of 10,000 at 525 nm.
If the absorbance of 0.01 is the lowest
detectable signal, what concentration in part per
million can be detected in a 1-cm cell?
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