Application of Molecular Absorption Spectroscopy
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Transcript of Application of Molecular Absorption Spectroscopy
APPLICATION OF MOLECULAR ULTRAVIOLET-
VISIBLE ABSORPTION SPECTROSCOPY
Introduction and Background
• Involves absorption of ultraviolet or visible radiation for qualitative and quantitative purposes.
• Most common analytical technique in the analytical laboratory
• Absorption commonly occurs with many– Organic molecules– Metals– Metal-organic complexes
ABSORBING SPECIES
The absorption of ultraviolet or visible radiation by an atomic or molecule can be considered to be a two step process :
M + hƲ M* M* M + heat
There are three types of electronic transitions :1.π, σ, and n electrons2. d and f electrons3.charge-transfer electrons
Ene
rgy
*
*
n
*
*
n
*
n
*
Antibonding
Antibonding
Nonbonding
Bonding
Bonding
Absorption by Organic Compounds
Many common organic compounds absorb in the UV region
Absorption by Inorganic Species
Many free metals and inorganic metal complexes absorb in the visible region of the spectrum
Absorption by Charge Transfer Complexes
• Many inorganic and organic complexes form charge transfer complexes
• A charge transfer complex consists of an electron donor group bonded to an electron acceptor group
• Charge transfer complexes exhibit broad band absorption in the visible region of the EMR spectrum
• Useful analytically because of the large molar absorption
What is Light?
• Light is a form of energy• Light travels through space at
extremely high velocities – The speed of light (c) ~ 3 x 1010
cm/sec or 186,000 miles per second
• Light is classified as electromagnetic radiation (EMR)
Characteristics of Light
• Light behaves like a wave.– That is, it can be modeled or characterized
with wave like properties.
• Light also behaves like a particle.
• Today, we envision light as a self-contained packet of energy, a photon, which has both wave and particle like properties.
The Electromagnetic Spectrum
EMR Wave Characteristics• Wavelength (l) is the distance from one wave
crest to the next.• Amplitude is the vertical distance from the
midline of a wave to the peak or trough.• Frequency (v) is the number of waves that pass
through a particular point in 1 second (Hz = 1 cycle/s)
Wave Properties of Electromagnetic Radiation
• EMR has both electric (E) and magnetic (H) components that propagate at right angles to each other.
Particle Properties of EMR
• The energy of a photon depends on its frequency (v)
Ephoton = hv
h = Planck’s constant
h = 6.63 x 10-27 erg sec or 6.63 x 10-34 Js
V = Wave Number (cm-1)l = Wave LengthC = 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 (Ergsec)
V =C
E = h = hC
C
= C =
Electromagnetic Radiation
How Light Interacts with Matter.
• Atoms are the basic blocks of matter.
• They consist of heavy particles (called protons and neutrons) in the nucleus, surrounded by lighter particles called electrons
How Light Interacts with Matter.
• An electron will interact with a photon.• An electron that absorbs a photon will
gain energy.• An electron that loses energy must
emit a photon.• The total energy (electron plus photon)
remains constant during this process.
Molecular Absorption
• More complex than atomic absorption because many more potential transitions exist– Electronic energy levels– Vibrational energy levels– Rotational energy levels
• Emolecule = Eelectronic + Evibrational + Erotational
Eelectronic > Evibrational > Erotational
• Result - complex spectra
Emission of EMR We distinguish several types of
emission1. Atomic2. X-Ray3. Fluorescence
Involves moleculesResonance and non-resonance modes
4. Phosphorescence• Non-radiative relaxation• Similar to fluorescence only relaxation
times are slower than fluorescence• Involves metastable intermediates
Energy Level Diagrams of Excitation and Emission
Auxokrom : gugus jenuh yang bila terikat pada kromofor mengubah panjang gelombang dan intensitas serapan maksimum. Ciri auxokrom adalah heteroatom yang langsung terikat pada kromofor, missal : -OCH3, -Cl, -OH dan NH2.Pergeseran batokromik : Pergeseran serapan kearah panjang gelombang Yang lebih panjang disebabkan substitusi atau pengaruh pelarut (pergeseran merah).Pergeseran hipsokromik : Pergeseran serapan kearah panjang gelombang yang lebih pendek disebabkan substitusi atau pengaruh pelarut (pergeseran biru).Efek Hiperkromik, kenaikan dalam intensitas serapan.Efek hipokromik, penurunan dalam intensitas serapan.
A few metal chlorides, which fluoresce strongly in the visible wavelengths,are the basis for almost all the colors in modern fireworks.
Barium chloride produces green; strontium chloride produces red; copper chloride produces blue
Single Beam Instruments
Double-Beam Instruments• A double beam instrument is one in
which the light source can be passed (simultaneously) through both a reference and a sample cell
• Purpose and Approach1. Adjust light output of the instrument
to 100% transmission (0 % absorbance)
2. Allows correction of the sample absorbance signal for non-analyte absorbance
Double-Beam Instruments
Reference and Sample Cell OptionsReference and Sample Cell OptionsReference Reference Reference Reference Sample or Standard Sample or StandardCellCell Cell Cell Cell CellSolution (pure HSolution (pure H
22O) Solution (pure HO) Solution (pure H22O) Solution (pure HO) Solution (pure H
22O)O)
ReagentsReagents Reagents Reagents Analyte or SampleAnalyte or Sample
Signal Due toReagents Only(Can be used to estimatereagent blank)
Signal Due toAnalyte Only
Example of UV-Visible Instrument
QUALITATIVE ANALYSIS• SOLVENTS
Transparant
Polar solvents [water, alcohols, esters,
ketones]
water 190 nm cyclohexane 210 nm ethanol 210 nm benzene 280 nm n-hexane 195 nm diethyl ether 210 nm
acetone 330 nm 1,4-dioxane 220 nm
• DETECTION OF FUNCTIONAL GROUPS
Spectroscopy Terms Describing Absorption (Beer’s Law)
• Consider a beam of light with an (initial) radiant intensity Po
• The light passes through a solution of concentration (c)
• The thickness of the solution is “b” cm.
• The intensity of the light after passage through the solution (where absorption occurs) is P
P0hv P
b
Co
nce
ntr
atio
n (
c)
We Define
• Transmittance (T) = P/P0 (units = %)
• Absorbance (A) (units = none)– A = log (P0/P)
– A = -log (T) = log (1/T)
– A = abc (or εbc) <--- Beer’s Law• a = absorptivity (L/g cm)• b = path length (cm)• c = concentration (g/L)• ε = molar absorptivity (L/mol cm)
– Used when concentration is in molar units
Beer’s Law
Major Point: There is a linear relationship
between absorbance and concentration (but not absorbance and transmission)
A = abc = εbc = log (Po/P) = log (1/T)
P0 = 10,000 P = 5,000
-b-
Example
A = -log T = -log (0.5) = 0.3010
P0 = 10,000 P = 2,500
25.010000
2500
0
P
PT
--2b--
Example
A = -log T = -log (0.25) = 0.6021
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0 1 2 3 4 5 6 7 8 9 10
Thickness, multiples of b
Ab
sorb
an
ce Absorption vs. Absorption vs.
TransmissionTransmission
0
0.2
0.4
0.6
0.8
1
1.2
0 1 2 3 4 5 6 7 8 9 10
Thickness, multiples of b
Tra
nsm
itta
nce
A = abc
T = 10-abc
Limitations to Beer’s Law
• Real– At high concentrations charge distribution effects
occur causing electrostatic interactions between absorbing species
• Chemical– Analyte dissociates/associates or reacts with
solvent
• Instrumental– ε = f(λ); most light sources are polychromatic
not monochromatic (small effect)– Stray light – comes from reflected radiation in
the monochromator reaching the exit slit.
Instrumental Limitations - ε = f(λ)
• How/Why does ε vary with λ?
• Consider a wavelength scan for a molecular compound at two different wavelength bands
• In reality, a monochromator can not isolate a single wavelength, but rather a small wavelength band
Instrumental Limitations – Stray Light
• Result – non-linear absorption (Analyte vs. Conc.) as a function of analyte concentration– Similar to
polychromatic light limitations
QUANTITATIVE ANALYSIS
• SELECTION OF WAVELENGTH• VARIABLES THAT INFLUENCE
ABSORBANCE• CLEANING AND HANDLING OF CELLS• DETERMINATION OF THE RELATIONSHIP
BETWEEN ABSORBANCE AND CONCENTRATION
• STANDARD ADDITION METHOD• ANALYSIS OF MIXTURES OF ABSORBING
SPECIES
SPEKTROFOTOMETRI DERIVATIF
• Mengalihbentuk data spektrum• Diperoleh dengan cara memplotkan turunan
pertama atau turunan lebih tinggi absorban terhadap panjang gelombang :
A = f(λ)
dA/d λ = f ‘(λ)
d2A/d λ2 = f “(λ)
dst
zero crossing
Zero order spectrum
A Gaussian absorption band and its first to fourth order derivatives
223 nm 266 nm
246 nm Thiamine
Riboflavine
Mixture of thamine and riboflavine
Thiamine
Riboflavine
223 nm
246 nm 266 nm
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
VITAMINS
Vitamin A H3C CH3
CH3
CH3
CH3
CH2OH
- Carotene
CH3
CH3
CH3 CH3 CH3
CH3 CH3 CH3
H3 C
CH3
H3 C CH3
CH3
CH3 CH3
H3 C CH3
CH3
CH3 CH3
CH2OH
Oxidation
C H
O
Retainal
Retinol (Vitamin A)
- 2H
Food
KOH (Alcoholic) Saponification
3 hrs. at room temperature
Ether for Extraction
Extract (vit.A and carotenoids)
Total Carotenoids only at 440 nm
Vitamin A + Carotenes Carr-Price Reagent Measure at 620 nm
Vitamin A and - Carotene Determination
-CAROTENE STANDARD ABSORBANCES AT 440 AND 620 nm
A a
t 440 n
m
an
d 6
20
nm
at 440 nm
at 620 nm
A
A
Use absorbances at 440 nm and then convert this to absorbance at 620 nm and subtract from the absorbance at 620 nm to determine the absorbance at 620 due to Vitamin A.
62 0
Carotenoid Absorbance at 440nm Vitamin A Absorbance at 620 nm
Carotenoid (g/ml) Vitamin A g/cuvette (sample)
Abs
orba
nce
at 4
40nm
Abs
orba
nce
at
n
mx
x
x
x
x
xx
x
x
x
x
THIAMIN DETERMINATION THIOCHROME (Fluorescent)
N
K3Fe(CN)6Oxidation
N
S
N
NH3C
CH2 CH3
CH2CH2OH
N
S
N
NH3C
CH2
NH2
CH3
CH2CH2OH
pyrimidine thiazole
Excite thiochrome at 365 nm and measure the absorbance at 435 nm
PHOTOMETRIC TITRATIONSPhotometric measurements can be employed to advantage in locating the equivalence of a titration, provide the analyte, the reagent, or the titration product absorbs radiation.
A photometric titration curve is a plot of absorbancecorrected for volume changes, as a function of the volume of titrant. If conditions are chosen properly,the curve will consist of two straight line regions with differing slopes, one occuring at the outset of the titration and the other located well beyond the equivalence region. The EP is taken as the intersection of extrapolated linear portions.
A(λ1) = aI(λ1) cI + aII(λ1) c II ……………………(1)A(λ2) = aI(λ2)cI + aII(λ2) cII ……………………… (2)Dimana :•nilai A(λ1 ) dan A(λ2) diperoleh dari pengukuran sample pada masing2 panjang gelombang maksimum.•aI(λ1) diperoleh dari harga tangens dari kurva baku komponen I pada λ1•aII(λ1) diperoleh dari harga tangens dari kurva baku komponen II pada λ1•aI(λ2) diperoleh dari harga tangens dari kurva baku komponen I pada λ2•aII(λ2) diperoleh dari harga tangens dari kurva baku komponen II pada λ2•CI dan CII adalah konsentrasi komponen I dan komponen II yang tidak diketahui