Uv Vis

Learning Outcomes
By the end of the topic students will be able to:

Explain the absorption process in the uv-vis region in
terms of its absorbing species and electronic transitions
Define the following terms as used in uv-vis
spectroscopy; chromophore, auxochrome, hypsochromic
shift and bathochromic shift
Explain the limitation to Beers law
Draw and explain the important components in uv-vis
spectrometer
Differentiate the difference between a single-beam and
double-beam uv-vis instrument and state the advantages
of each
Apply Ultraviolet-visible spectroscopy for quantitative and
qualitative analysis
Predict lmax for dienes & enones.
Ultraviolet-visible Spectroscopy
Absorption measurement based upon Uv-vis
application
Qualitative Analysis
Identification
determination
Quantitative Analysis
Amount
Organic &
Inorganic species
Review:
Review:
Qualitative Analysis
molar absorptivities (emax)
Beers Law
A= ebc
How Does Matter Absorb Ultraviolet-visible Radiation?
Electronic Transition
Electrons of a molecule may be raised to a higher
electron energy
E3
E2
E1
E0
An energy level diagaram showing 4
electronic levels and the possible
electronic transitions
What are the types of compounds that

absorbs in the ultraviolet-visible region?
Just any compound will absorb??
Molecular Absorption of Visible & Ultraviolet Radiation
Absorption by Organic Compounds
(not all, specific ones)
Absorption by Inorganic Compounds
Charge-transfer absorption
Absorption of uv-vis radiation results

from excitation of bonding electrons
3 types of electronic transitions
p, s, and n electrons
d & f electrons
Charge transfer electrons
Electrons that contribute to absorption by
organic molecule are
those that participate directly in bond
formation between atoms
Non bonding or unshared outer electrons (that
are largely localized about atoms such as
oxygen, halogens, sulfur & nitrogen
energetically favored electron promotion will be
from the
highest occupied molecular orbital (HOMO)
to the lowest unoccupied molecular orbital
(LUMO),
.
and the resulting species is called an excited
state
hn
Organic compounds that absorb in the uvvis region are called chromophores
Chromophores unsaturated organic
functional groups that absorb in the uv-vis
regions
Example of chromophores
d & f electrons absorption
3d & 4d electrons
1st and 2nd transition metal series
Absorption bands are broad
Strongly influenced by chemical
environmental factors
d & f electrons
absorption
4f & 5f electrons
Lanthanide and actinide
series
Narrow, well defined
characteristic absorption
peaks
Electronic Transitions
Review:
formaldehyde
Electronic
transitions
1) s
s*
2) n
s*
3) n
p*
4) p
p*
e
e
e
s
s*
Never observed in the ordinarily accessible

ultraviolet region
n
s*
saturated compounds containing atoms

with unshared electron pairs (non bonding
electrons)
l range: 150 250 nm
e range: 100 3000 Lcm-1mol-1
tend to shift to shorter l in polar solvents.
e.g. H2O , CH3CH2OH
Some examples of Absorption due to n
lmax (nm)
emax
H2O
167
1480
CH3OH
184
150
CH3Cl
173
200
CH3I
258
365
(CH3)2O
184
2520
CH3NH2
215
600
Compound
s*
n
p*
Unsaturated compounds containing atoms

with unshared electron pairs (non bonding
electrons)
Unsaturated functional group
to provide the p orbitals
l range: 200 700 nm

e range: 10 100 Lcm-1mol-1
Examples
n p*
Examples n
p*
n
p*
examples of inorganic species

Nitrate (313nm)
Nitrite (360, 280 nm)
p
p*
compounds with unsaturated functional

groups to provide the p orbitals
l range: 200 700 nm
e range: 1000 10,000 Lcm-1mol-1
(Note: conjugation of chromophores shifts absorption to longer l)
examples: p
p*
examples: p
p*
charge-transfer complex
charge-transfer complex generally contain an
electron donor & an electron acceptor group within
the complex.
absorption of radiation involves transition of an
electron from donor gp to an orbital that is largely
associated with the acceptor.
the excited state is the product of an internal
oxidation-reduction process.
Molar absorptivites of charge transfer peaks
are among the largest (e >10,000)
Organic & inorganic complexes
Examples:
Thiocyanate & phenolic complexes of iron (III)
Iron (II) complex of orthophenanthroline
Ferro-ferricyanide complex
Absorption
Mechanisms by which a metal complex can absorb
radiation
excitation of the metal ion
excitation of the ligand
charge transfer transition
Important terminologies:
Auxochrome:
does not itself absorb radiation but if present in a
molecule, it can enhance the absorption by a
chromophore or shift the l of absorption when
attached to a chromophore
e.g. hydroxyl groups, amino groups & halogens
hypsochromic shift:
Absorption maximum shifted to shorter l
bathochromic shift:
Absorption maximum shifted to longer l
Terminologies
Nature of Shift
Descriptive Term
To Longer l
Bathochromic
To Shorter l
Hypsochromic
To Greater Absorbance
Hyperchromic
To Lower Absorbance
Hypochromic
b is a constant, 1cm
Beers Law
A
x
x
x
A = ebc
y = mx
concentration
A ebc
absorbance
molar
absorptivity
(Lmol-1cm-1)
slope = e
concentration (mol/liter)
pathlength (cm)
Absorbance and concentration relationship
Limitation to the Applicability of Beers Law

Factors that cause the Beers law relationship to
depart from linearity:
failure to use monochromatic radiation

existence of stray radiation
experimental uncertainties in
measurement of low absorbances
molecular interaction at high absorbances
concentration dependent association or
dissociation reaction
(pg 338 - 343 ) Skoog, Holler & Crouch, 6th Edition
Limitation to the Applicability of Beers Law
Beers law
describes the absorbance behaviour only of dilute
solutions
solutions concentration < 0.01M

solution with concentration >0.01M;
the average distances between ions or molecules of
the absorbing species are diminished to the point
where each particle affects the charge distribution and
the extent of absorption of its neighbours.
Limitations to the Applicability of Beers Law
Basically Beers law is limited to:

apparent chemical deviation
apparent instrumental deviation

apparent deviation from stray radiation
*** Please read the details on pages I have mentioned in the earlier slides.
INSTRUMENTATION
What are the important components in
a Uv-vis spectrophometer?
1
Source lamp
2
l selector
3
Sample
holder
4
Detector
UV region:
Deuterium lamp;
H2 discharge
tube
Visible region:
Tungsten
lamp
Prism/
monochromator
Quartz /
fused silica
Prism/
monochromator
Glass/quartz
Phototube,
PM tube,
diode array
Phototube,
PM tube,
diode array
5
Signal
processor
and readout
The effect of the slit width on the spectra in uvvis spectrophotometry.

The slit width determines the spectral bandpass, the wavelength range of the light
passing through the sample.
The smaller the slit width, the more nearly monochromatic the light beam will be.
But if the slit width is too large, the polychromatic light effect will cause the spectral
peaks to be shorter and broader than they would be at narrower slit widths.
If you are trying to measure an accurate absorption spectrum, for example for use as a
reference spectrum for future measurements or for identification of that absorber, then
you should use a narrow slit.
However, the signal-to-noise ratio decreases as the slit width is reduced, so it is not
always practical to use the smallest slit width possible.
If the spectral bandpass is one tenth (1/10th) of the spectral width (full width at halfmaximum) of the narrowest band in the spectrum, then the maximum peak height error
caused by polychromaticity will be less than 1%.
Uv-visible instrument
1. Single beam
2. Double beam
Single beam
One radiation source

Filter/monochromator (l selector)
Cells
Detector
Readout device
Single beam instrument:
or
computer
Single beam
stabilized voltage supply to avoid errors
resulting from changes in the beam intensity
during the time required to make the 100%T
adjustment and determine %T for the analyte
Double beam Instrument
What is the difference?
Double beam Instrument
Double-beam Instruments
Advantage:
1.
Compensate for all but most short-term fluctuations in

the radiant output of the source as well as for drift in the
transducer and amplifier
2.
Compensate for wide variations in source intensity with l
3.
Continuous recording of transmittance or absorbance

spectra
1. Calibration Curve Method
2. Standard Addition Method
Calibration Curve Method
A general method for determining the
concentration of a substance in an
unknown sample by comparing the
unknown to a set of standard sample of
known concentration.
(Explain on whiteboard)
Standard Addition Method
Involves adding one or more increments of
a standard solution to sample aliquots of
the same size
Each solution is diluted to a fixed volume
before measuring its absorbance
Standard Addition
Assume several identical aliquots, Vx, of
the unknown solution with concentration,
cx, are transferred to volumetric flasks
having a volume Vt.
To each of these flasks is added a
variable volume, Vs (mL), of a standard
solution of the analyte having a known
concentration, cs.
Each solution is diluted to volume
Standard Addition
If Beers law is obeyed,
ebVs cs ebVx cx
A
Vt
Vt
where k is a constant equal to
eb
Vt
kVs cs kVx cx
Standard Addition
A plot of A as a function of Vs should yield a
straight line of
A mVs b
where the slope m and intercept b are:
m kcs
b kVx cx
Standard Addition (esp. multiple point std addition)
m kcs
b kVx cx
bcs
cx
mVx
m
Vs
Standard Addition
For a standard addition with only 2 increments of
sample
Standard Addition
sample
Absorbance of
diluted sample
Absorbance of
diluted sample
plus standard
solution.
A2
ebVx cx
A1
Vt
ebVx cx
Vt
ebVs cs
Vt
Eq.1
Eq.2
Standard Addition
sample:
solve 1st equation for eb & substitute into the

2nd equation and solve for cx:
A1csVs
cx
A2Vx A1Vx
A1csVs
cx
( A2 A1 )Vx
Example Standard Addition (single point

addition)
The single-point addition method was used in the
determination of phosphate by the molybdenum blue
method. A 5.00-mL urine sample was treated with
molybdenum blue reagents to produce a species
absorbing at 820 nm, after which the sample was
diluted to 100 mL. A 25.00-mL aliquots of this solution
gave an absorbance of 0.314 (solution 1). Addition of
1.00 mL of a solution containing 0.0500 mg of
phosphate to a second 25.0-mL aliquot gave an
absorbance of 0.734 (solution 2) Use these data to
calculate the concentration of phosphate in milligrams
per milliliter of the specimen.
Example Standard Addition (single point

addition)
A1csVs
(0.314)(0.0500mgPO43 / mL)(1.00mL)
cx
A2Vx A1Vx (0.734)(25.00mL) (0.314)(25.00mL)

3
3
4
cx 1.50 10 mgPO / mL
This is the concentration of the diluted sample. To obtain the concentration
of the original urine sample, we need to multiply by 100.00/5.00.
mg 100mL
[ PO ] 1.50 10
mL 5.00mL
3
4
= 3.00 x 10-2 mg/mL
Standard Addition Method

When do you use Std. Addition Method?
Standard addition technique needs to be

conducted if an analyte is in a complex matrix
(matrix effect).
Main reason - the matrix may contain other

components that interfere with the analyte signal
causing inaccuracy in the determination of sample
concentration
Absorbance Measurement
Measurements are usually made at the wavelength where
absorption is at its maximum.
Highest absorbance
highest
greater sensitivity.
The change in absorbance value per unit concentration is

greatest at this point.
Absorbance is nearly constant with wavelength where
absorption is at its maximum.
lmax
added conjugation in naphthalene, anthracene
and tetracene causes bathochromic shifts of
these absorption bands
Empirical Rules for Dienes, lmax

Homoannular
(cisoid)
Heteroannular
(transoid)
l = 253 nm
l = 214 nm
30
30
Alkyl substituents or ring residue
Exocyclic double bond
-OCOCH3
-OR
-Cl, -Br
-NR2
60
60
Parent
Increment for:
Double bond extending
conjugation
Polar groupings
pp 396
Empirical Rules for Dienes, lmax

Homoannular diene vs
cisoid or s-cis
Homoannular diene
Heteroannular diene
transoid or s-trans
Heteroannular diene
Example 1 ( lmax for dienes)
Transoid
CH3
H
C=C
CH3
C=C
CH3
214 nm
Alkyl groups: 3 x 5 =
15 nm
lmax 229 nm
H
Observed lmax 228 nm
Homoannular
CH3
CH
CH3
CH3
253 nm
Alkyl substituent:
5 nm
Ring residues: 3 x 5 =
Exocyclic double bond:
H3C
COOH
15 nm
5 nm
lmax 278 nm
Heteroannular
CH3
214 nm
Ring residues: 3 x 5 =
Exocyclic double bond:
15 nm
5 nm
lmax 234 nm
Empirical Rules for Enones
(pp 400)
Base values:
Six membered ring or acyclic parent enone
= 215 nm
Five membered ring parent enone
= 202 nm
Acyclic dienone
= 245 nm
Increments for:
Double-bond-extending conjugation
30
Alkyl group or ring residue
a 10
b 12
g and higher 18
Polar groupings:
Empirical Rules for Enones
(pp 400)
Polar groupings:
-OH
a 35 nm
b 30 nm
d 50 nm
-OCOCH3
a,b,d 6 nm
5 nm
Homocyclic diene component
39
Solvent correction
Variable
Example 1 ( lmax for enones)
O
CH3
C=C
CH3
CH3
CH3
Acyclic enone:
215 nm
a-CH3:
10 nm
b-CH3: 2 x 12 =
24 nm
249 nm
Observed:
249 nm
Example 2 (lmax for enones)

O
CH3
a
b
OCOCH3
Six membered enone:
215 nm
Double-bond-extending conjugation
30 nm
d-Ring residue:
18 nm
Homocyclic diene
39 nm
lmax 302 nm
Observed lmax : 300 nm
Example 3 ( lmax for enones)

CH3
O
a
Br
Five-membered enone:
202 nm
a-Br
b-Ring residue: 2 x12 =
25 nm
24 nm
5 nm
lmax 256 nm
Observed lmax : 251 nm
Other tables for lmax calculations/predictions

Empirical Rules for Unsaturated
Aldehydes
Empirical Rules for Unsaturated Acids
and Esters
Empirical Rules for Benzoyl
Derivatives

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Learning Outcomes

By the end of the topic students will be able to:

What are the types of compounds that

Absorption of uv-vis radiation results

3 types of electronic transitions

Never observed in the ordinarily accessible

saturated compounds containing atoms

Unsaturated compounds containing atoms

l range: 200 700 nm

examples of inorganic species

compounds with unsaturated functional

Absorbance and concentration relationship

Limitation to the Applicability of Beers Law

failure to use monochromatic radiation

Limitation to the Applicability of Beers Law

solutions concentration < 0.01M

Limitations to the Applicability of Beers Law

Basically Beers law is limited to:

apparent instrumental deviation

The effect of the slit width on the spectra in uvvis spectrophotometry.

One radiation source

Compensate for all but most short-term fluctuations in

Compensate for wide variations in source intensity with l

Continuous recording of transmittance or absorbance

solve 1st equation for eb & substitute into the

Example Standard Addition (single point

Example Standard Addition (single point

A2Vx A1Vx (0.734)(25.00mL) (0.314)(25.00mL)

= 3.00 x 10-2 mg/mL

Standard Addition Method

Standard addition technique needs to be

Main reason - the matrix may contain other

The change in absorbance value per unit concentration is

Empirical Rules for Dienes, lmax

Alkyl substituents or ring residue

Exocyclic double bond

Empirical Rules for Dienes, lmax

Example 1 ( lmax for dienes)

Example 2 ( lmax for dienes)

Example 3 ( lmax for dienes)

Empirical Rules for Enones

Five membered ring parent enone

Alkyl group or ring residue

Empirical Rules for Enones

Exocyclic double bond

Homocyclic diene component

Example 1 ( lmax for enones)

Example 2 (lmax for enones)

Six membered enone:

Example 3 ( lmax for enones)

Exocyclic double bond

Other tables for lmax calculations/predictions

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