Download Ultraviolet/Visible Absorption Spectroscopy

Ultraviolet-Visible
Absorption Spectroscopy
Electronic Excitation by UV/Vis Spectroscopy :
X-ray:
core electron
excitation
UV:valance
electronic
excitation
IR:
molecular
vibrations
Radio waves:
Nuclear spin states
(in a magnetic field)
Rays
γ rays
X rays
Far UV
Near UV
Visible
Near IR
2.5µm
Mid IR
Far IR
400µm
Micro waves
Radiowaves
Frequency
Wavelength
1020 -1016
1016 -1015
1015 -7.5X1014
7.5X1014 4.0 X1014
4.0 X1014 -1.2 X1014
0.01-10nm
10-50nm
50-200nm
200-400nm
400-800nm
0.8-
1.2 X1014 - 6 X1012
6 X1012 -1011
2.5-25µm
25-
1011 -108
108 - 105
400-25cm
25cm-1000m
Spectroscopic Techniques
UV-vis
UV-vis region
bonding electrons
Atomic Absorption
UV-vis region
atomic transitions (val. e-)
FT-IR
IR/Microwave
vibrations, rotations
Raman
IR/UV
vibrations
FT-NMR
Radio waves
nuclear spin states
X-Ray Spectroscopy
X-rays
inner electrons, elemental
X-ray Crystallography
X-rays
3-D structure
Ultraviolet and visible spectroscopy
It is used to measure the multiple bonds or
atomic conjugation within the molecule.
The UV-Visible region is subdivided as
below
– Vacuum UV: 100-200 nm
– Near UV: 200 to 400 nm
– Visible region: 400 to 800 nm
Vacuum UV is so named because
molecule of air absorb radiation in these
region. The radiation is assessable only in
special vacuum equipments.
Spectroscopy:
Synonyms: Spectrometry or spectrophotometry
Spectroscopy is made up of
Spectrum + Skopien → Given by Isaac Newton
He did simple experiment
V I BG YOR
Sunlight
According to Newton experiment, Spectrum is a band of color
or pattern of colors or arrangement or array of colors.
But extended definition of spectrum is, Separation of wave or
pattern of waves or arrangement /array of wave.
Today, Spectrum is defined as arrangement of array or pattern
of anything.
Skopein: Evaluation/examination
Spectroscopy: Evaluation/ Examination of spectrum
Metry: Measurement
Spectrometry: Measurement of spectrum
Photo: EMR (Electro magnetic radiation)
Spectrophotometry: Measurement of EMR spectrum
Fundamental principle of spectroscopy:
Drug + EMR → Drug* + EMR*
We measure difference between EMR and EMR*
EMR:
EMR is a radiant energy, that is transmitted through space in
normal velocity
Radiant energy has wave nature and being associated with electric
as well as magnetic field so called EMR
All EMR propagated through space with same speed (3X 1010
cm/sec) called speed of light
EMR is alternating electric field and associated magnetic field in
space
The two components oscillate in planes perpendicular to each
other and perpendicular to the direction of propagation of radiation
Crest
Electrical component
Direction of propagation of radiation
Trough Magnetic Component
EMR is vibration of wave produced by oscillating or
vibrating of electron in particular direction.
EMR are produced by periodic motion of charge
particles like electrons
Characteristics of EMR:
Wavelength
Frequency
Velocity
Wave number
Wavelength (λ) is defined as the distance between adjacent
peaks (or crest/troughs). Designated in meters, centimeters or
nanometers (10-9 meters).
Energy α 1/λ max
Frequency (µ) is the number of wave cycles passes a given
point in 1 sec. Expressed as cycles per second, or hertz (Hz).
µ α 1/λ, , µ = c/λ
Velocity (c) The distance traveled by wave in 1 sec.
c= λ x µ, c= 3x 1010 cm/sec
Wavenumber (σ) – the number of waves spread in length of 1
centimeter.
σ = 1/ λ
•
Energy α Frequency (µ)
E= h µ, E = hC/ λ
H= plank const= 6.626 x 10 -27 erg/sec
Spectroscopy: it is a branch of science deals with
interaction of EMR with matter
Spectroscopy can be divided in,
Study at molecular/atomic level
1)
Atomic spec e.g.: AAS, flame photometry
Change in E at atomic level
Deals with interaction of EMR with atoms which are in
their lowest energy state, i.e., ground state
2)
Molecular spec e.g.: UV, IR, fluorimetry
Change in E at molecular level
Transition between rotational and vibrational E levels in
addition to electronic transition
Study based on absorption/emission
1)
Absorption Spe
e.g.: UV, IR, X-ray, ESR, NMR
2)
Emission Spe
e.g.: flame photometry, fluorimetry
Study at electric/ magnetic level
•
Electric Spe
e.g.: UV, Colorimetry, fluorimetry (without magnetic field)
2)
Magnetic Spe
e.g.: NMR, ESR
UV Visible Spectroscopy also known as Molecular
absorption/ electronic absorption spectroscopy
Importance of UV spectra:
Simple in operation
High sensitivity
Speedy Analysis
Qualitative and quantitative application
Limitation: Non Selective
Molecular absorption spectrum/ UV Visible spectrum
is band spectrum
Atomic spectrum involves line spectrum
Molecular absorption spectrum/ UV
Visible spectrum is band spectrum
Why we get ∩ (band) and not ∏ on x-axis
even though conc. is same
B’se all the time energy absorbed is not
the same…
A molecular energy state is the sum of
an electronic, vibrational, rotational, and
translational component
E = E electronic + E vibrational
+E rotational +E translational
Electronic E level: Molecule possess an electronic
configuration & the electronic energy depends on
the electronic state of the molecule
Vibrational E level: The parts of molecule, i.e., atoms
or groups of atoms may move with respect to each
other. This motion is called vibration and associated
energy is called vibrational E level
Rotational E level: The molecule may rotate about an
axis & such rotation is characterized by the
rotational E level
Translational E level: The molecule as a whole may
move & this is called as transition & the associated E
is called translational E level
Eele, Ev, Er are the quantized/internal energy.
In order to absorption to occur, the E difference
between two e levels must be equal to conc. of
photons absorbs.
Means E2-E1= hv
The e- are mainly involved in absorbing E, so called electronic
absorption spectroscopy
Both UV and Visible Spe. Only the valance e- absorbs E,
thereby, the molecule undergoes transition fro GS to ES
The intensity of absorption depends on the conc. And path
length given by Beer-Lambert's law.
For Absorption in UV – visible intensity of absorption depends
on the conc., and hence given by Beer’s law
While in IR absorption Spe., it concerns with jumping
of e- from one vibrational E level to other vibrational E
level so called Vibrational Abn. Spe./ IR Abn. Spe.
For IR abn. Rotational spectrum is not important
analytically.
e- jump from one E level to another ----- Transition
If a molecule passes from one of its allowed E level to
a lower one, some of E must be released, which may
be lose as radiation.----Emission of radiation
If a molecule passes from one of it’s allowed E level to
higher one, some E must be absorbed, which may be
absorbed as radiation -------Absorption of radiation
Valance e- : The e- which are required for bond formation
present in outer most orbital.
Types of valance e- : 1) σ 2) π 3) n
1)
σ e- :
Present in saturated hydrocarbon
e.g.: paraffin ( C-C, C-H)
Highly stable
Required higher E for excitation and λ req. for excitation
is very low
Such e- do not absorbs near UV but absorbs vacuum UV
radiation < 200 nm
Hence the comp. ctg σ bond doesn't absorb in UV region
------transparent in UV range -----so used as a solvent.
For e.g.: Hexane
2) π e-:
Present in unsaturated hydrocarbon
e.g.: Double or triple bond…>C=C<,-C≡C- (alkenes,
alkynes, aromatic compd., and carbonyl compds
such as aldehydes and ketones, cyanides, azo
compds etc.
Relatively unstable and highly reactive
Required less E for excitation
3) n e- : non bonding eThese e- are not involved in bond formation
e.g.: S,O,N & halogen (X), such n e- are excited by
UV radiation
Principle of UV radiation
Any molecule has either n, σ, π or a combination of
these e-.
These bonding (σ and π) and non bonding (n) eabsorbs characteristic radiation
undergoes transition from GS to ES
By the characteristics absorption peaks, the nature of epresent, the mol. Stru. can be elucidated
Electronic Transition & Excitation process
C
LUMO
B
A
HOMO
A: Bonding Molecular Orbital: if e- are in these region: bond
formation takes place, which is highly stable
B: Non Bonding Molecular Orbital: bond formation doesn't
take place. Atomic orbital are themselves non bonding
molecular orbital prior to bond formation
C: Anti bonding molecular orbital: here, if e- are present in
these region, highly unstable---so no bond formation
σ, π (bonding) and n (non-bonding) electrons
The E required for excitation for different
transition are,
n→π* < π→π* < n→σ* < σ→σ*
n→π* ----required lowest E
σ→σ* ----required highest E
Electronic Transition
σ→σ* transition:
Required highest E
Absorption in ~150 nm
E.g. Hydrocarbons
Methane: λmax = 125 nm (High E compare to ethane, Lower λ)
Ethane: λmax = 135 nm (less E compare to methane, Higher λ)
B’se strength of C-C bond is < C-H bonds..
Propane: maxi abs. At 135 nm…
most of spectrophotometer doesn’t shows abs. < 180-200 nm,
σ→σ* not observed
Exception: Cyclopropane: shows abs. at 190 nm
In far UV (Vacuum) region, Oxygen present in
the air absorbs strongly, so to study σ→σ*
air must be evacuated from the instrument,
specially in case of Saturated HC
Since UV operated above 200nm, Saturated
hydrocarbon used as a solvent (non polar) as
it doesn’t give solvent peak
n→σ* transition:
Saturated compd ctg. atoms with unshared pair of e- (O,N,S/ X/ non
bonding e-)
Majority of comps in this class doesn’t shows abs. in near UV region
Transition in region of 150-250 nm, with most abs peak < 200 nm
Most commonly used solvent: Alcohols and ethers (abs < 185nm)
E.g.: Alkyl Halides
The E req. for n→σ* transition ↓es with ↑es in size of halogen atom/ ↓es in
the electro negativity of atom. (F, Cl, Br, I)
E.g.: Methyl chloride (λmax=173) and methyl iodide (λmax=259)
B’se of > electro negativity of chlorine than iodine, the n e- on chlorine
atom are comparatively difficult to excite, while n e- on iodine atoms
are loosely bound
Magnitude of molar extinction coefficient (εmax) for a particular
absorption αnal prob. of particular electronic transition.
εmax for CH3I= 400, εmax for CH3Cl= 200
n→σ* transitions are sensitive to H-bonding
For E.g.: alcohols and amines forms H-bonding with solvent
molecule (due to n bonding e-)
So, greater E req. for excitation and hence, H-bonding shifts
abs. towards shorter λ.
Amines abs at higher λ as compared to alcohols
B’se n bonding e- on N atoms in amine are loosely bound as
compared to o atoms in alcohol (B'se higher electro negativity
of O than N)
E.g.: Tri methyl amine: in aq. solution doesn't shows abs due
to n→σ*
B'se protonated amine does not contain any n bonding e-
Absorption Spe. Of org. compd. are based upon,
n→π* & π→π*
B’se E req. for these processes brings Abs peaks in
to spectral region (200-700nm).
Both
transitions
unsaturated
orbital.
req.
functional
the
group
presence
to
of
an
provide
π
π→π* transition:
In simple alkenes, several transitions are available.
abs. band between 170-190 nm in un conjugated
alkenes
E.g.: Ethylene in vapor phase absorbs at 165 nm, &
gives second band at 193 nm due to π→π*,
The intensities of olefinic double bonds is
independent of solvents due to non polar nature of
double bond
Band in π→π* transition also called as K-band.
K-band is obtained in the spectra of conjugated π
systems.
e.g. Butadiene, Mesityl oxide
K-band is also known as E-band (Ethylinic) & B band
(Benzenoid)
n→π* transition:
required lowest E (longer λ)
The peak due to this transition also called as
R-band ( longer λ)
Peak seen due to: n bonding e- is present in
compound ctg = or ≡.
e.g. aldehyde, ketone and nitro compd.
n→π*
Blue shift observed with an increase in solvent
polarity ( due to salvation and H-bonding to the lone
pair, large shift of 30 nm)
n→π* transition is characterized by taking spectrum
in acid solution.
E.g.: Pyridine: bands due to n→π* in pyridine
disappear in acid solution b’se of the formation of
bond between acidic proton and n eC6H5N: + H+ → C6H5NH+
Peak appear
Peak disappear
Characteristic difference between n→π* & π→π* is
found with effect of solvent
Blue shift observed with an increase in solvent
polarity in n→π*
Red shift observed with an increase in solvent
polarity in π →π*
135 nm
C C
165 nm
C C
H
C O
C O
n183 nm
weak
150 nm
n188 nm
n279 nm
weak
180 nm
A
279 nm

Visible Spectroscopy
Apparent color of the solution is always complementary
of the color absorbed
Beer’s Law:
When a monochromatic radiation passed
thro’ a transparent medium, a rate of
decrease in intensity of radiation with
concentration of medium is proportional to
the intensity of the incident light.
Intensity
of
transmission
radiation
decreases as the conc. of absorbing subs
increases arithmetically
The wavelength and amount of light that a compound
absorbs depends on its molecular structure and the
concentration of the compound used.
I0
logA

abc
I
where
I = intensity of monochromatic light transmitted
through the solution
I0 = intensity of light transmitted through the blank
A = absorbance
b = path length (usually in cm)
a = Absorptivity, a constant for a given solution
and a given wavelength
C = concentration in g/100 ml
Deviation from Beer’s Law
Positive deviation: when a small
change in conc. produce greater
change in absorbance.
Negative deviation: when a large
change in conc. produce small change
in absorbance.
The law strictly followed for dilute
system, as conc. increases causes
deviation
Environmental Deviation: Temp, solvent
Instrumental Deviation: Relative Conc. error, Stray
radiation, stability of radiation source, wavelength
selector, slit control (SSW), electronics and reliability
of optical parts
Chemical Deviation: change in chemical equilibrium
and change in pH, ionization, presence of complexing
agent, competitive metal ion reactions and conc.
Dependence (hydrolysis, association, polymerization,
ionization, H-bonding).
Refractive index of the sample
Non monochromacity of radiation
Beer’s law is not applicable to suspension, coagulated
particle system, impurity that fluorescence / absorbed
Methods to find Conc. By Beer’s law
1) Using Standard equation (using standard A(1%, 1 cm) and ε
A= ε b c
The absorbance of the solution, A, is defined as
A= - log T, A= -log [I/Io], T= -log [I/Io]
%T, of the solution is expressed as,
A = 2.00 – log (% T) = ε b c
ε = A(1%, 1 cm) x Mol weight
10
A(1%, 1 cm)/a (Spe. absorptivity) is given than conc. Is in
g/100ml
ε is given than conc. Is in mol/lit
2) Calibration Method)
(by serial dilution method)
3) Using Formula method
Au / As = Cu / Cs
Different terminologies used in UV-Visible spectroscopy
•
Chromophore: A chromophore is the part of a molecule responsible for its color.
The color arises when a molecule absorbs certain wavelengths of visible light
and transmits or reflects others.
•
Auxochrome: An auxochrome is a group of atoms attached to a chromophore
which modifies the ability of that chromophore to absorb light. (hydroxyl, amino,
nitro group)
•
Bathochromic shift: It is a change of spectral band position in the absorption,
reflectance, transmittance, or emission spectrum of a molecule to a longer
wavelength
•
Hypsochromic shift: It is a change of spectral band position in the absorption,
reflectance, transmittance, or emission spectrum of a molecule to a shorter
wavelength.
•
Hyperchromic shift: Increase in absorbance
•
Hypochromic shift: Decrease in absorbace
Important Terms in UV Spe.
Chromophore
1) Dependant
2) Independent
Auxochrome
1) Bathochromic gr.
2) Hypsochromic gr.
Bathochromic shift (Red shift)
Hypsochromic shift
(Blue shift)
Hyperchromic shift
Hypochromic shift
End absorption
Chromophore
Chromo (color) + Phore (producing)
It is covalently unsaturated group responsible for
electronic absorption
Minimum requirement for absorption in Uv: Minimum
two conjugated double bonds
C=C-C=C
C=C-C=O
C=C-C=N
C=C-C=S
Independant: single chromophore is sufficient to
impart color to compd.
e.g: -N=N-, -NO-, o & p quinoid gr.
Dependent: More than one chromophore is required
to produced color in chromogen
>C=O, C=C
e.g.: CH3COCH3
CH3COCOCH3
CH3COCOCOCH3
Colorless (acetone)
Yellow (Diacetyl keton)
Orange (Tri keto pentane)
Structures having same chromophoric group shows
same absorption in UV and can’t analyzed by UV, so
UV is non selective method
Auxochrome
Auxo: Auxiliary
Auxochrome is a functional group having non
bonding electrons which itself has no absorption but
when attached to chromophoric group, enhance the
absorption properties of chromophoric groups
An auxochrome contains unshared pair of electrons
e.g.: NH3, SH, RNH2, OH
Benzene
Nitro benzene
P-nitro aniline
-no chrom. group
-NO2 chromophore
-NO2 and -NH2 (auxo.)
-colorless
-pale yellow
- dark yellow
Auxochrome: two types
1) Bathochromic groups: deepen the color of chromogen,
shift to longer λ, e.g.: 1°, 2°, 3° amine
2) Hypsochromic groups: lighten the color of chromogen ,
Hyperchromic
Different Shifts

Hypsochromic
Bathochromic
Hypochromic
Terminology
Bathochromic shift: (Red shift) shift of lambda
max to longer side or less energy is called
bathochromic shift or read shift. This is due to
substitution or solvent effect.
Hypsochromic shift: (Blue shift) shift of lambda
max to shorter side and higher energy is called
hypsochromic or blue shift. e.g. solvent effect.
Hyperchromic effect: an increase in absorption
intensity
Hypochromic effect: an decrease in absorption
intensity
End Absorption
It is special phenomenon of increases in
absorption intensity as the λ decreases
towards 200nm (End of UV range), which is
due to n→σ*
Significance:
A
200nm
λ
400nm
End
absorption
is
possible due to only
compd having n electrons
& having σ bonds. e.g. n
electrons present in most
of the solvent like water
and alcohol.
N electrons have higher E
and lower λ.
absorption spectra
1. λmax: Position of Spectra
2. Intensity: Amt of radiation
Factors affecting λmax/ Position of Spectra
Two types
1.Internal
(Structural)
A) Substitution
B) Unsaturation
C) Geometry
D) Resonance
2.External
(Non Structural)
A) Solvent
B) pH
C) Effect of metal ion
D) Molecular aggregation/ Charge
transfer complex
E) Temperature
Factors affecting absorption intensity
(εmax/A(1%,1cm)
A) Resonance
B) Intensity of incident radiation
C) Conc.
D) Thickness
E) Some fundamental factors like SSW &
stray light