Molecular Spectroscopy with

UV-Vis Spectrophometry
Ultra Violet (UV)
• Three regions of UV

• UV-A:
• long-wave UV, near-ultraviolet, black light, or Wood’s light
• between 320 and 400 nm
• UV-B:
• medium-wave UV
• between 280 and 320 nm
• UV-C:
• short-wave, far ultraviolet, or germicidal UV
• between 180 and 280 nm
• ( untuk desinfectan)

Visible : 380 – 800 nm

Sumber cahaya

3
Interaksi UV-Vis dengan materi
• Transisi elektronik
• Materi menyerap UV-Vis untuk keperluan transisi elektronik
• Transisi yang terjadi tergantung struktur elektronik senyawa
• Gugus kromofor : extensively conjugated pi-electrons

4
 UV & Visible Spectrophotometry
• Observations

Incident light, I0 Emergent light, I

(UV or visible)
ultraviolet visible infra-red

200 - 400 400 - 800 800 - 15

C
nm nm nm nm nm mm
b

When a light of intensity I0 goes through a liquid of concentration C & layer thickness b
• The emergent light, I, has less intensity than the incident light I0
• scattering, reflection
• absorption by liquid
• There are different levels of reduction in light intensity at different wavelength
• detect by eye - colour change
• detect by instrument
 The method used to measure UV & visible light absorption is called spectrophotometry (colourimetry refers to the
measurement of absorption of light in visible region only)
UV & Visible Spectrophotometry

Incident light Emergent light

I0 I

• Theory of light absorption

Quantitative observation C

• The thicker the cuvette

- more diminishing of light in intensity b

• Higher concentration the liquid

- the less the emergent light intensity
UV & Visible Spectrophotometry
These observations are summarised by Beer’s Law:
Successive increments in the number of identical absorbing molecules in the path of a
beam of monochromatic radiation absorb equal fraction of the radiation power travel
through them
light fraction
absorbed of light
b

x
dI dI
s
2
  k ' I   k' Ncs 2
dx  acdx
I0
I Ncs dx I
I b dI b Ib
s
number of   ac  dx  ln  acb
I0 I 0 I0
molecules Absorbance
N-Avogadro number
dx I0
 log  abc  A
I
 Transmitansi (T) dan Absorbansi (A)

Besarnya I
dipengaruhi oleh :
 Konsentrasi
 Panjang lintasan
 Watak bahan

I I
T   %T   100  A   log T
Io Io

8
Hukum Beer-Lambert

 Banyaknya intensitas cahaya/GEM yang terserap (Absorbansi) berbanding

lurus terhadap konsentrasi senyawa

 Semakin besar konsentrasi semakin besar absorbansi

 Jika konsentrasi suatu senyawa diketahui (standard) diukur absorbansinya,

maka hasil absorbansinya dapat digunakan untuk menentukan konsentrasi
senyawa lain.

9
Hukum Beer-Lambert
Transmitansi (T) dan Warna

 Color is an important property of a substance.

 The color of matter is related to its absorptivity or reflectivity.
 The human eye sees the complementary color to that which is absorbed
11
Relationship between light absorption and color
Absorbed vs. Observed

R
O RV

V
Y

YG BV

G B
BG

Color absorbed → Complementary color

observed
Spektrofotometer dapat berfungsi
untuk identifikasi senyawa

ANALISIS KUALITATIF
Fotometer ( trace pada 1 panjang gelombang

Pendekatan analisis Kuantitatif

Perbandingan
Absorbansi standar
Standard adisi
Kurva kalibrasi
standar
monokromatik

15
Atomic Spectra & Molecular Spectra
• Comparison of atomic and molecular spectra

Atomic spectra Molecular spectra

Adsorption spectra Yes Yes

Emission spectra Yes Yes

Energy required for excitation high low

Change of energy level related change of e-’s orbital change of vibration states
to
Spectral region UV mainly visible

Relative complexity of spectra simple complex

• Quantum mechanics is the basis of atomic & molecular spectra

• The transitional, rotational and vibrational modes of motion of objects of atomic / molecular level
are well-explained.
Beer’s Law: A = abc
The amount of light absorbed (A) by a sample is dependent on the path length (b), concentration of the
sample (c) and a proportionality constant (a – molar absorptivity)

Amount of light absorbed is dependent on frequency ()

Increasing Fe+2 concentration 

Absorbance is directly proportional to concentration Fe +2

Molecular Spectra

Absorption Emission

Intensity

40 50 60 70
0 0 l (nm) 0 0

Bands vs. Lines

Beer’s Law: A = abc

Transmittance (T) = I/Io %Transmittance = %T = 100T

Absorbance (A) = log10 Io/I

No light absorbed- % transmittance is 100%  absorbance is 0

All light absorbed- % transmittance is 0%  absorbance is infinite

Relationship Described in Terms of Beer’s Law

A = Absorbance = abc = -log(%T/100)

a = molar absorptivity: constant for a compound at a given frequency (n) or

wavelength (l)  units of L mol-1 cm-1

b = path length: cell distance in cm

c = concentration: sample concentration in moles per liter.

Therefore, by measuring absorbance or percent transmittance at a given frequency

can get information related to the amount of sample (c) present with an identified a
and l.
Note: law does not hold at
high concentrations, when A
>1
UV & Visible Spectrophotometry
• Terms, units and symbols for use with Beer’s Law

Name alternative name symbol definition unit

Path length - b (or l) - cm

Liquid concentration - c - mol / L

Transmittance Transmission T I / I0 -

Percent transmittance - T% 100x I / I0 %

AbsorbanceOptical density, A log(I / I0) -
extinction
Absorptivity Extinction coeff., a (or e, k) A/(bc) [bc]-1
absorbance index
Molar absorptivity Molar extinction coeff., a A/(bc)
molar absorbancy index [or aM AM/(bc’) ] M-molar weight
c’ -gram/L
UV & Visible Spectrophotometry
• Use of Beer’s Law
 Beer’s law can be applied to the absorption of UV, visible, infra-red & microwave
 The limitations of the Beer’s Law
• Effect of solvent - Solvents may absorb light to a various extent,
e.g. the following solvents absorb more than 50% of the UV light going through them
180-195nm sulphuric acid (96%), water, acetonitrile
200-210nm cyclopentane, n-hexane, glycerol, methanol, ethanol
210-220nm n-butyl alcohol, isopropyl alcohol, cyclohexane, ethyl ether
245-260nm chloroform, ethyl acetate, methyl formate
265-275nm carbon tetrachloride, dimethyl sulphoxide/formamide, acetic acid
280-290nm benzene, toluene, m-xylene
300-400nm pyridine, acetone, carbon disulphide
• Effect of temperature
• Varying temperature may cause change of concentration of a solute because of
• thermal expansion of solution
• changing of equilibrium composition if solution is in equilibrium
UV & Visible Spectrophotometry
• What occur to a molecule when absorbing UV-visible photon?
• A UV-visible photon (ca. 200-700nm) promotes a bonding or non-bonding electron
into antibonding orbital - the so called electronic transition * Antibonding
• Bonding e-’s appear in s & p molecular Antibonding
*
orbitals; non-bonding in n
• Antibonding orbitals correspond to the

n *

n *
 *

*
non-bonding
bonding ones

Energy
n
• e-’s transition can occur between various Bonding

states; in general, the energy of e-’s 

transition increases in the following order:
(n®p*) < (n®s*) < (p ®p*) < (s ®s*)


• Molecules which can be analysed by UV-visible absorption

• Chromophores
functional groups each of which absorbs a characteristic UV or visible radiation.
UV & Visible Spectrophotometry
• The functional groups & the wavelength of UV-visible absorption
Group Example lmax, nm Group Example lmax, nm
C=C 1-octane 180 arene benzene 260
naphthalene 280
C=O methanol 290 phenenthrene 350
propanone 280 anthracene 375
ethanoic acid 210 pentacene 575
ethyl ethanoate 210
ethanamide220 conjugated 1,3-butadiene 220
1,3,5-hexatriene 250
C-X methanol 180 2-propenal 320
trimethylamine 200 b-carotene (11 C=C) 480
chloromethane 170
bromomethane 210 each additional C=C +30
iodomethane 260
 UV & Visible Spectrophotometry
• Instrumentation

UV visible
Light source Hydrogen discharge lamp Tungsten-halogen lamp
Cuvette QUARTZ glass
Detectors photomultiplier photomultiplier
UV & Visible Spectrophotometry
• Applications

a) Analysis of unknowns using Beer’s Law calibration curve

b) Absorbance vs. time graphs for kinetics
c) Single-point calibration for an equilibrium constant determination
d) Spectrophotometric titrations – a way to follow a reaction if at least
one substance is colored – sudden or sharp change in absorbance at
equivalence point
Lambert’s Law of Absorption for Thin Film
Lambert described how intensity changes with
distance in an absorbing medium of thin film.
• The intensity I0 if a beam of light decreases exponentially as it passes though a uniform
absorbing medium with the linear decay constant α.
Restatement: In a uniform absorbing medium, the intensity of a beam of light decreases
by the same proportion for equal path lengths traveled.
• The linear decay constant α is a characteristic of the medium. It has units of reciprocal
length. α is the path length over which the intensity is attenuated to 1/e.
l
α
I  I 0 e  l The distance traveled through the medium is
called the path length.
I0 I dI
I ( x )  I 0 e  x
d I   I d x   I
dx
I(x)
 x I  x
I  I 0e e
x
I0
Simple Method of Measuring the Band Gap
Energy Value of TiO2 in the Powder Form
using a UV/Vis/NIRm Spectrometer

PerkinElmer, Inc.
Shelton, CT USA
Transmittance and Pathlength
For cells with same pathlength: constant T
Double pathlength: square T

#1 #2
2
I0 I2 I2 I1 I 2  I1 
    
I0 I 0 I1  I 0 

I1 I2
Ttotal  T1  T2  T1 
2
I0 I1
T and Pathlength
For b cells (b pathlengths = 1cm)  Tb

#1 #b
b
I0 Ib I b  I1  I1 Ib
     
… I0  I0  I0 I b-1

I1 Ib

Ttotal  T1   T1    Tb
I0 I b-1 b

b in power
Transmittance and Concentration
1.0 M reference solution gives

I0 I1 I2 b
… T   I1 cm 
 I 
I1 I2  0 
I0 I1

2.0 M: Double concentration,c→“Double pathlength,

b”…
bc
 I1 cm, 1M 
T  T1 
bc
   bc in power
 I0 
pathlength and
concentration
Molar Extinction Coefficient, 

T: concentration c & pathlength b

What about: molecular identity?
 Probability of absorption
– Specific to molecule
– Function of l

lmaxMost efficient absorption

– Peak of absorption curve
– “Best l” for experiment
b, c, and 
 I1 cm, 1M 
ε   log  
bc  I0 
 I1 cm, 1M 
T  T1   I1 cm, 1M 
bc
      10 ε
 I0   I0 
bc in power e in power
pathlength and concentration molar extinction coeff.

bc
 I1 cm, 1M 
Therefore: T     10   10
-ε bc -εcb

 I0 
 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.
Chemical Limitations
A reaction is occurring as you record Absorbance measurements

Cr2O72- + H2O 2H+ + CrO42-

CrO42-

Cr2O72-
A550 A446

300 400 500 concentration concentration

wavelength
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
Larger the Bandwidth – larger deviation band
 Solution too Concentrated
• Refractive index changes with larger concentrations

A  e en n
n2 + 2