spectrometry
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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.
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.
Wave Number (cycles/cm)
X-Ray UV Visible IR Microwave
200nm 400nm 800nm
Wavelength (nm)
Spectral Distribution of Radiant Energy
V = Wave Number (cm-1)
λ = Wave Length
C = Velocity of Radiation (constant) = 3 x 1010 cm/sec.
υ = Frequency of Radiation (cycles/sec)
The energy of photon:
h (Planck's constant) = 6.62 x 10-27 (Erg×sec)
V = υC λ
=1
E = h = hC
λυ C
=λ
υ C = υλ
Electromagnetic Radiation
Visible
Ultra violet
Radio
Gamma ray
Hz
cmcm-1Kcal/mol eV
Type
Quantum Transition
Type
spectroscopy
Type
Radiation
Frequency
υ
Wavelength
λ
Wave
Number VEnergy
9.4 x 107 4.9 x 106 3.3 x 1010 3 x 10-11 1021
9.4 x 103 4.9 x 102 3.3 x 106 3 x 10-7 1017
9.4 x 101 4.9 x 100 3.3 x 104 3 x 10-5 1015
9.4 x 10-1 4.9 x 10-2 3.3 x 102 3 x 10-3 1013
9.4 x 10-3 4.9 x 10-4 3.3 x 100 3 x 10-1 1011
9.4 x 10-7 4.9 x 10-8 3.3 x 10-4 3 x 103 107
X-ray
Infrared
Micro-wave
Gamma ray emission
X-ray absorption, emission
UV absorption
IR absorption
Microwave absorption
Nuclear magnetic resonance
Nuclear
Electronic (inner shell)
Molecular vibration
Electronic (outer shell)
Molecular rotation
Magnetically induced spin states
Spectral Properties, Application and Interactions of Electromagnetic Radiation
Dispersion of Polymagnetic Light with a Prism
Polychromatic Ray
Infrared
RedOrange
Yellow
Green
Blue
Violet
Ultraviolet
monochromatic Ray
SLIT
PRISM
Polychromatic Ray Monochromatic Ray
Prism - Spray out the spectrum and choose the certain wavelength(λ) that you want by slit.
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.
Electronic Excitation
The absorption of light energy by organic compounds in the visible and ultraviolet region involves the promotion of electrons in σ, π, 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.
Electronic Molecular Energy Levels
The higher energy transitions (σ →σ*) occur a shorter wavelength and the low energy transitions (π→π*, n →π*) occur at longer wavelength.
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
••••
σ and σ* orbitals π and π* orbitals
Spectrophotometer
An instrument which can measure the absorbance of a sample at any wavelength.
Light Lens Slit Monochromator
Sample Detector Quantitative Analysis
Slits
Instrument to measures the intensity of fluorescent light emitted by a sample exposed to UV light under specific conditions.
Emit fluorescent lightas energy decreases
Ground state
Sample
90°C
DetectorUV Light Source
Monochromator Monochromator
Antibonding
Antibonding
Nonbonding
Bonding
BondingEnergy
σ
π
σ π
σ −>σ
π −>π
'
'
'
''
n->n
σ n->π'
Electron's molecular energy levels
Fluorometer
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
••••
σ and σ* orbitals π and π* orbitals
Beer – Lambert Law
Glass cell filled with concentration of solution (C)
IILight
0
As the cell thickness increases, the transmitted intensity of light of I decreases.
R- Transmittance
R = I0 - Original light intensity
I- Transmitted light intensity
% Transmittance = 100 x
Absorbance (A) = Log
= Log = 2 - Log%T
Log is proportional to C (concentration of solution) and is
also proportional to L (length of light path through the solution).
I
I0
I
I0
I0
I
1
T
I
I0
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
E =Absorbance x Liter
Moles x cm
UNITS
A = ECL
A = No unit (numerical number only)
E = Liter
Cm x Mole
Steps in Developing a Spectrometric Analytical Method
1. 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
Wavelength (nm)
Abs
orba
nce
0.0
2.0
200 250 300 350 400 450
Slope of Standard Curve =∆ A∆C
1 2 3 4 5
1.0
0.5
Concentration (mg/ml)
A a
t 280
nm
There is some A vs. C where graph is linear.
NEVER extrapolate beyond point known where becomes non-linear.
x
x
x
Spectrometric Analysis Using Standard Curve
1 2 3 4
0.4
0.8
1.2
A a
t 540
nm
Concentration (g/l) glucose
Avoid very high or low absorbencies when drawing a standard curve. The best results are obtained with 0.1 < A < 1. Plot the Absorbance vs. Concentration to get a straight line
Light Sources
UV Spectrophotometer
1. Hydrogen Gas Lamp
2. Mercury Lamp
Visible Spectrophotometer
1. Tungsten Lamp
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.
Chromophoric Structure
Group Structure nm
Carbonyl > C = O 280
Azo -N = N- 262
Nitro -N=O 270
Thioketone -C =S 330
Nitrite -NO2 230
Conjugated Diene -C=C-C=C- 233
Conjugated Triene -C=C-C=C-C=C- 268
Conjugated Tetraene -C=C-C=C-C=C-C=C- 315
Benzene 261
UV Spectrometer Application
Protein
Amino Acids (aromatic)
Pantothenic Acid
Glucose Determination
Enzyme Activity (Hexokinase)
Visible Spectrometer Application
Niacin
Pyridoxine
Vitamin B12
Metal Determination (Fe)
Fat-quality Determination (TBA)
Enzyme Activity (glucose oxidase)
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?
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
Spectroscopy Homework
1. 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 1.002.5 x 10-3 0.2 1.004.0 x 10-5 50 50002.0 x 10-4 150 [X](M) = Concentration in Mole/L
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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