Spectroscopic ANALYSIS
Part 5 – Spectroscopic Analysis
using UV-Visible Absorption
Chulalongkorn University, Bangkok, Thailand January 2012
Dr Ron Beckett
Water Studies Centre & School of Chemistry
Monash University, Melbourne, Australia
Email: Ron.Beckett@monash.edu
Water
Studies
Centre
1
UV-Visible Absorption Spectroscopy
Absorption of UV and visible light by a molecule causes
electronic excitation
Electronic
excitation
1018
-rays
X-rays
1016
UV
1014
Rotation
1012
Infrared
108
Radio
1020
Vibration
Visible
Cosmic
rays
Bond breaking
and ionization
Microwave
Visible Spectrum
400
500
600
700
2
UV-Visible spectral
peaks result from
electronic-vibrational
transitions
Case (b) in the
diagram is most
common which gives
the typical symmetric
peak shape
3
Molecular Orbitals
• Bonding in organic molecules is based on overlap
between s and p atomic orbitals.
• This can give rise to bonding s and p molecular
orbitals, nonbonding n molecular orbitals
antibonding s* and p* molecular orbitals
Two p atomic orbitals
overlapping to give a
s and a s*
molecular orbital
4
Molecular Orbitals
Two p atomic orbitals overlapping to
give a bonding p molecular orbital and
a nonbonding p* molecular orbital
A
A
+
p*
B
A
px
B
px
B
p
5
Molecular Orbitals and Electronic Jumps
s * (antibonding)
p * (antibonding)
n s*
p p*
s s*
n p*
n (non-bonding)
p (bonding)
s (bonding)
Electronic energy levels of polyatomic molecules6
Peak Position and the Type of Electronic Jump
Conjugated
p bonds
7
Peak Position for Molecules containing
Double and Triple Bonds
8
Effect of Conjugation on Peak Position
The greater the number of conjugated double
bonds the lower the energy jump and higher the
wavelength of the UV-visible peak
p p*
9
Effect of Conjugation on Peak Position
Highly conjugated molecules may be coloured if the
absorption peak moves into the visible region
10
Question Time !
Fanta
has red and green colours !
Will red light pass through each of these
solutions or will it be absorbed ?
(a)
(b)
(c)
(d)11
Question Time !
Fanta has red and green colours !
Will green light pass through each of these
solutions or will it be absorbed ?
(a)
(b)
(c)
(d)12
Complementary Colours
When white light is absorbed by a chromophore,
the eye detects the colours that are not
absorbed. This is called the complementary
colour to the colour absorbed.
VIBGYOR
ofmaximum
absorption
380-440
440-500
500-580
580-680
680-780
Colour Absorbed Colour Observed
violet-blue
blue-green
green- yellow
orange-red
purple
green- yellow
orange-red
violet-blue
blue-green
green
13
Colorimetric Analysis
Used for determination of the concentration
of analytes in solution when:
1. The analyte is a coloured compound
2. The analyte produces a coloured species
when a suitable reagent is added
14
Colorimetric Analysis
Determination of concentration depends on detection
of change in colour intensity (absorption) at a
particular wavelength.
Photometric measurement
(a) visual comparison using colour standards
Po
P
Eye
15
Colorimetric Analysis
(b) Colorimeter/Photometer
• Filters used to select a wavelength range
• Detection with photosensing device
Filter
wheel
P
o
P
Photodetector
16
Spectrophotometric Analysis
(c) Spectrophotometer
– Spectral bandwidth ≤ 1 nm, i.e very monochromatic light.
– can operate in both the visible and UV ranges
– Colorimetry and spectrophotometry provide sensitive
methods of analysis, i.e. ppm to ppb ranges.
Po
P
Monochromator
Photodetector
Prism or Grating
Phototube, photomultiplier
17
or photodiode
Single Beam Colorimeter
•
Single beam spectrometer
18
Quantifying Light Absorption
b
Incident beam
Pa(solute)
Transmitted beam
PI
Reflected
beam
P
Pr
Pa(solvent)
Absorbing solution
of concentration,c.
Incident Light Intensity (PI)
(sometimes Ii is used)
PI = Pr + Pa(solvent) + Pa(solute) + P
Pa(solvent) & Pa(solute) are absorbed light intensities
Pr ≈ 4% for air-glass interface
19
Quantifying Light Absorption
Intensity lost due to reflection and solvent absorption are
removed by measuring the transmitted intensity of a blank
containing only solvent
b
Incident beam
Pa(solvent)
PI
Reflected
beam
Transmitted beam
P0
Pr
Absorbing solvent
Transmitted Light (P0)
PI = Pr + Pa(solvent) + P0
P0 = PI - Pr - Pa(solvent)
20
Quantifying Light Absorption
Absorbance is defined as
P0
A = log
P
Transmittance defined as
P
T =
P0
Thus
A = log (1/T) = log(100/%T)
21
Relationship between
Absorbance and Concentration
Beer-Lambert Law
A=elc
Where:
• l is the path length in cm
• c is the concentration in mol/L
• eis the molar absorptivity
22
Applications of the Beer-Lambert Law
Analysis of a single analyte
1. Measure absorbance of a series of standard solutions
2. Plot a standard curve (should be a straight line ?)
3. Measure absorbance of unknown samples
4. Use standard curve to measure concentrations
Assumptions
– At fixed  and l, e is
constant for a given
solute
A4
A3
Ax
A=elc
A2
A1
– the chemical matrix
of the standards is the
same as the sample.
A0
C0
C
1
C
2
C
3
Cx
C
4
23
Concentration
Applications of the Beer-Lambert Law
Standard Addition Method
Used for samples with
complex matrix &
chemical interferences.
1. Measure A of sample
2. Repeat with known
additions of standard
to the sample.
Sample plus
standard additions
Sample
0
CAdd
Concentration of
Standard added (mL)
Sample
Concentration
24
Limitations of the Beer-Lambert Law
Concentration effects
– B-L law applies to dilute solutions (negligible interaction
between solute ions).
– Higher concentrations of analyte (i.e. > 10-2 M) or high
electrolyte concentrations, may produce molecular/ionic
interactions which result in reduced light absorption at
some wavelengths.
Adherence to B-L law
Deviation from B-L law
(loss of sensitivity)
Concentration
25
Experimental Considerations
Wavelength selection
• Choose  where A is large to obtain best sensitivity.
• Choose  where dA/d  = 0 or is small.
1
2
2
3
3
4
Wavelength
26
Experimental Considerations
Choice of reagents for colorimetric analysis
– Should be stable and pure
– Should not absorb at  of measurement
– Should react rapidly with analyte to give a stable
coloured compound (chromophore).
– Absorptivity, e, should not be sensitive to minor
changes in pH, Temp., electrolyte changes, etc.
– Should be selective for the analyte of interest.
27