uvvis spectroscopy
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UV/Vis-NIR absorption spectroscopy
The Electromagnetic Spectrum
Pro
tein
s
Bac
teria
Ani
mal
cel
ls
103 102 101 100 10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8 10-9 10-10 1011 10-12
Longer wavelength
Shorter wavelength
H2OViru
s
Tennis ballBuilding
Wavewlength(metres)
Generic name
Sources
Frequency (Hz)
Energy ofa photon
(ev)
FM
rad
io
10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 100 101 102 103 5 105 106
106 107 108 109 1010 1011 1012 1013 1014 1015 1016 1017 1018 10201019AM
rad
io
Mic
row
ave
oven
rada
r
Bod
y he
at
Ligh
t bul
b
X-r
ay m
achi
ne
radi
oact
ivity
Radio waves
Microwaves
Infrared ultraviolet
Soft x-rays
Hard x-rays
Gamma rays
IR Spectroscopy
UV/Vis Spectroscopy
UV/Vis-NIR spectroscopy
What region of the EM spectrum are we interested in?
UVC 210-280 nmUVB 280-320 nmUVA 320-400 nmVisible 400-700 nm
Far Red 700-1100 nmNear-Infra red 1100-2500 nm (9000 cm-1- 4000 cm-1)
(increasingly important for materials science)Mid Infrared 2500-6000 nm (4000 - 60 cm-1)
(unusual for electronic absorptions)
UV/Vis spectrum
300 400 500 600 700 8000.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Abs
Wavelength /nm
•Plot of the absorbance (unitless dimension) or molar absorptivity (M-1 cm-1) against wavelength•Expressed in nanometres (nm, 10-9 m)•In older books and papers Angstroms (Å, 10-10 m) are used•In the near infrared region (from 900-2500 nm) microns or micrometres (µm) are used
UV region visible region far red region
Wavelength vs wavenumber
300 400 500 600 700 8000.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Abs
Wavelength /nm
40000 35000 30000 25000 20000 150000.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Abs
wavenumber /cm-1
Electronic Absorption E = hν
λ
EM radiation has a magentic and an electronic vector
If electrons move from one orbital to another then there is a change in the charge distribution in a molecule and hence a change in the molecules electric dipole.
Selection rules
-Resonance condition-Change in principle quantum number-Change in orbital angular momentum-No change in spin: (2S+1) must not change-Laporte’s rule: transitions between orbitals of the same parity are forbidden
singlet triplet
singlet singlet
Electronic Absorption
• Total internal energy Sum of the electronic, vibrational and rotational energy
• Etotal = Eelec + Evib + Erot
• For an electronic transition:
∆Etotal= ∆Eelec+∆Evib+∆Erot
Molar Absorptivity
• Probability that a photon of light will be absorbed upon passing through an optically dilute solution
• Proportional to the square of the transition moment (change in electron density)
• Forbidden transitions
Chromophores & Conjugation
Chromophore – brings colourThe absorption of light is quantisedMixing of electronic, vibrational and rotational energy levels leads to broad spectra instead of lines
C = O C = C
Pigments & Dyes, I
• Retinal – cis/trans isomerisation is the first step in sensing light
• Chlorophylls absorb light for photosynthesis
• Types of transitionsn – π* (auxochromes have free electron pairs e.g C=ö: )
n – σ*σ –σ*π – π*
Vibrational progression
28000 26000 24000 22000 200000.0
0.2
0.4
0.6
0.8
1.0
Nor
mai
llise
d ab
sorp
tion
and
emis
sion
wavenumber /cm-1
9,10-di-phenyl-anthracene
Frank-Condon state
0�10�0
0�2
The Bougier-Beer-Lambert Law
An Empirical law – i.e. based on observation not theoretical
derivation
Transmittance
• The light passing through a sample• Solvents and optical material are
transparent (100% transmittance) only in certain regions of the EM spectrum
• Solvent cut-off (see table 7.6, Hesse):– Water – 190 – CH3CN – 190– Acetone -350
Transmittance
εεεε
T = I1/I0
T = 10-A
The Beer-Lambert-Bouguer Law(empirical law)
• The intensity of light passing through a sample decreases exponentially and the absorbance of light is proportional to the concentration of the chromophore
• The absorbance of light is proportional to the pathlength through which the light travels
A = εclA is absorbance, c is concentration (mol L-1), l is pathlength
(in cm) and ε is the molar absorptivity (L mol-1 cm-1)
N.B. Assumption that all particles behave independently
(no shadow effect)
Justification of BLB lawdI = - κ[J]I dl
where [J] is the molar concentration of the absorbing species, I is the incident intensity,
dI is the reduction in intensity of I, κ is the proportionality coefficient and dl is the
pathlength or thickness of the layer.
Rearranged the equation is
dI = - κ[J] dl
I
And for a series of pathlengths dl and integral is used:
I dI = - κ l [J] dl
I0 I 0
ln (I/I0) = - κ[J] l and since ln (I/I0) = ln 10 * log (I/I0)
Then - log (I/I0) = (1/2.303)κ[J] l = ε c l
This is describes the absorption at a single wavelength however sometimes the
absorption of an entire band is being treated and in this case the integrated
absorption coefficient is used which corresponds to the area uinder the absorption
band Α = ε (v) dv
∫∫
~~∫
Bicomponent systems
• Two species present in solution• Species behave independentlyA = A1 + A2 = ε1c1l + ε2c2l = (ε1c1 + ε2c2)l
pH dependence of fluorescein
Isosbestic pointNo change in abosrbance
Instrumentation
How do we record spectra?
Single beam diode array spectrometer
Halogen lamp for visible& deuterium lamp for UV
Diode array detector
grating
Single beam scanningspectrometer
From Halogen lamp for visible& deuterium lamp for UV
To sample and then PMT Detector
grating
Czerny-Turner monochromator
Dual beam scanningspectrometer
From Halogen lamp for visible& deuterium lamp for UV
Two PMT (190-1100 nm) or PbS (1100-2500 nm) Detectors
From: http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm
Wavelength vs wavenumber
300 400 500 600 700 8000.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Abs
Wavelength /nm
40000 35000 30000 25000 20000 150000.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Abs
wavenumber /cm-1
In converting from wavelength to wavenumber you have to correct for band pass. The slit width is constant in nm over the whole spectrum but varies in cm-1.
5 nm, 1112 cm-1 5 nm, 156 cm-1
Sample Handling• Spectroscopic grade solvents• Concentration should be known• Cuvette – generally quartz glass (QS), also OS or plastic
cuvettes can be used• Typically 1 cm pathlength cuvettes (cells) are used• Cleaning - hot nitric acid• Cleaning – do not use of strong hydroxide bases or
sources of fluoride as these etch the glass surface• UV/Vis light used to measure can also result in
photochemistry – do not expose the sample to light any more than is necessary.
Measuring spectra
Single Beam vs. Dual beam spectrophotometers Single beam (diode arrays) are fast but often less stableFirst measure a solution not containing the compound of interest
(Spectrum 1)Second measure a solution containing the compound of interest
(spectrum 2)Subtract the reference spectrum from the spectrum with the sample
Dual beam (PMT) are slower but very stableFirst measure a baseline using two solutions that do not contain the
compound of interest (baseline/background)Second measure a solution containing the compound of interest
together with a blank solution Subtract the reference spectrum from the spectrum with the sample
Measuring Molar Absorptivity
Measure absorbance over a range of concentrations
Use different pathlength cells 1 mm to 10 cm to increase dynamic range
Overlap data points i.e. 1 mM with a 1 mm pathlength cell and 0.1 mM with a 1 cm cell
Deviations from linearity
• Assumption of independently absorbing species– Inner filter effect
• Aggregation of samples at higher concentration– Scattering– Intermolecular interactions (H and J aggregates in perylenes)
• Photochemistry• Change in gas content in solution• Reaction with solvent
• Scatter – the wall!
Micellaneous topics
Photochromism
250 300 350 400
0.00
0.05
0.10
0.15
0.20
0.25
Abs
Wavelength /nm
S
S
∆
hv
R
R
Anti-foldedSyn-folded
S
S
R
R
Pigments and Dyes II
250 300 350 400 450 500
0.2
0.4
0.6
0.8
1.0
Abs
Wavelength /nm
S
hv
S
HH -H2
S
H H
hv
HH
-H2
H H
Solvatochroism
Interaction of a part of the chromophoric group, e.g. a ketone, or amine, result in a change in the energy of the frontier orbitals and hence a shift in the absorption spectrum
pH dependence of fluorescein
Isosbestic pointNo change in abosrbance
Computational methods
300 400 500 600 700 800 900
ZINDO/S 1 ZINDO/S H
21
300 400 500 600 700 800 900
TD-DFT 1 TD-DFT H
21
300 400 500 600 700 800 900
MeCN 1 MeCN H
21
300 400 500 600 700 800 900
H2O 1
H2O H
21
N
NNNN
N NNMe Me
Ru
RuN
N
NN
NN
N
N
N N
2+
__
= 2,2'-bipyridine
Predicted UV.Vis electronic
spectra is done on the basis of
an isolated molecule (in
vacuo)
However solvent is not
innocent in determining the
absorptivity of chromophores
Band GapsSemiconductor materials: TiO2 (titanium dioxide –
white paint, some suncreams, solar cells), ITO (Indium doped SnO2), NiO (nickel oxide)
VB (NiO)
CB (NiO)
hν
Self cleaning windows generate active oxygen and charges
Diffuse Reflectance
Uses an integrating sphere – used to look at rough surfacesAlso used for opaque solutions samples can be prepared as dilute‘solutions’ in KBr
Specular reflectance
Brewster’s AnglePolarisation angle (angle at which light is fully polarised)
Applications: Following Reactions
• Isosbestic points• Multiple reaction steps
250 300 350 400
0.00
0.05
0.10
0.15
0.20
0.25
Abs
Wavelength /nm
S
S
∆
hv
R
R
Anti-foldedSyn-folded
S
S
R
R
Applications: Analysis
• Structural assignments• Quantitative analysis
– HPLC– Following reactions
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