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SPECTROSCOPY

-branch of science in which electromagnetic radiation


of particular wavelength or range of wavelength is
used for qualitative and quantitative analysis of
matter.
UV-VIS Absorption
Spectroscopy
IR Absorption Spectroscopy

hc
hv E = =
Interactions of Light with Matter
Reflection
Transmission
Absorption
I
o
= I
r
+ I
t
+ I
a
I
o
= original intensity
I
r
= reflected intensity
I
o
= transmitted intensity
I
o
= absorbed intensity
Interactions of Light with Matter
Absorption
At certain wavelengths, a normally transmitting medium will absorb light
photons energy is converted into an atomic or molecular process
Usually, the wavelength(s) of the light absorbed is/are characteristic of the
absorbing species, or chromophore.
This is where the true power of spectroscopy lies, in that it imparts the ability to
differentiate between different forms of matter because each has a unique
spectrum of absorbed light.
It is important to know the appropriate material as sample holder, window
or lens for various types of spectroscopy
Beer-Lambert Law: cl
I
I
o
o = ln
abc A=
ATOMIC SPECTRA
1) When irradiated, the outer electrons of
many atoms will absorb photons and be
accelerated to higher energy levels
2) The excited electron has a tendency to
return to its normal state, and in so doing
emits a photon
3) The emitted photon possesses a definite
amount of energy dictated by the spacing
of the energy levels
4) With a highly energetic source of
excitation, many electrons can be excited
to varying degrees, resulting in emission
of photons with multiple discrete
wavelengths
Single-Beam Instrument
Double-Beam Instrument
Molecular Absorption
Absorption spectra for polyatomic substances are more
complex than the atomic spectra, because the number of
energy states is enormous.
The energy associated with the bands of a molecule is made up
of three components.
E = E
electronic
+ E
vibrational
+ E
rotational
Energy
electronic vibrational rotational
100 kJ/mol
1 kJ/mol
0.01 kJ/mol
UV-Vis IR
FIR, wave
Absorption of Organic Compounds
All organic compounds are capable of absorbing
electromagnetic radation
Transitions involving electrons forming single bonds
occurs in vacuum UV region (< 185 nm) ---
experimentally challenging!
Important transitions in UV-Vis: n t* and t t*
(unsaturated functional group)
Chromophores : (color-bearing) functional groups
capable of absorbing UV-Vis radiation (e.g., N O
2
, N=O
(nitroso), -N=N
-
(azo), -C=O)
Auxochromes : functional groups that can intensify the
color of a molecule. They generally do not absorb
significantly in the 200-800 nm region, but will affect the
spectrumof the chromophore to which it is attached.
The most important auxochromic groups are OH, NH
2
,
CH
3
and NO
2
.
The actual effect of an auxochrome on a chromophore
depends on the polarity of the auxochrome, e.g. groups
like -CH
3
, -CH
2
CH
3
, and -Cl have very little effect, usually
a small red shift of 5-10nm. Other groups such as -NH
2
and -NO
2
are very popular and completely alter the
spectra of chromophores.
Absorption of Inorganic Compounds
involves transitions between filled and unfilled d-orbitals with
energies that depend on
(1) Position of the element in the periodic table
(2) Oxidation state of metal
(3) Nature of ligands bonded to the transition metal
Generally weakly intense because they are Laporte forbidden
(symmetry forbiden) but weakly allowed due to vibronic
coupling
d d Transitions
Absorption of Inorganic Compounds
Charge-transfer complexes:
important for quantitative analysis
large molar absorptivities (c > 10,000) = | sensitivity
consist of an e-donor group bonded to an e-acceptor
pi accepting ligands:
CO, NO, CN
-
, N
2
, bipy,
phen, RNC, C
5
H
5
-
,
C=C double bonds,
C=C triple bonds
pi donor ligands: F
-
,
Cl
-
, Br-, I
-
, H
2
O, OH
-
,
RS
-
, S
2
-
, NCS
-
, NCO
-
From this spectra of an octahedral Chromium complex, we see that the d-d
transitions are far weaker than the LMCT. Since Chlorine is a pi donor ligand in
this example, we can label the CT band as LMCT since we know the electron is
transitioning from a MO of ligand character to a MO of metal character. The
Laporte forbidden (symmetry forbidden) d-d transitions are shown as less intense
since they are only allowed via vibronic coupling.
Absorption Spectroscopy
Terms employed in Absorption Spectroscopy
1. Transmittance, T
Consider the figure below which depicts a beam of parallel radiation before and
after a layer of solution having a thickness of b cm and a concentration c of an
absorbing species.
P
o
P
b
absorbing solution
of concentration c
Attenuation of a beam of radiation by an absorbing solution
As a consequence of interactions between the photons and absorbing particles, the
power of the beam is attenuated from Po to P.
The transmittance, T of the solution is the fraction of the incident
radiation transmitted by the solution.
T = P
Po
Transmittance is often expressed as a percentage or
%T = P x 100
Po
2. Absorbance, A
The absorbance A of a solution is defined by the equation
A = -log T = log Po
P
Absorbance is directly proportional to the path length b
through the solution and the concentration c of the absorbing
species.
A = a b c
a is a proportionality constant called absorptivity
When concentration is in M and cell length is in cm, the
absorptivity is called molar absorptivity, c.
A = c b c
where c has the units L mol
-1
cm
-1
.
The equations:
A = abc or A = cbc
are statements of Beer-Lambert-Bouger Law or simply known as Beers Law.
E
1
E
2
E
o
Resonance
fluorescence
excitation
Fluorescence Spectroscopy
The energy released when the molecule goes back to the ground state (i.e.
relaxes) can be in the form of radiation.
with specific wavelength
I = kC
Quantitative Application
I = fluorescence intensity
C = concentration
Standard Calibration Method
A series of standard solutions of the analyte is prepared and the absorbance of each
solution is measured.
Absorbance is plotted against concentration and the concentration of the analyte in
the sample is determined using the resulting regression equation.
The standards should approximate the overall composition of the actual samples
and should cover a reasonable concentration range of the analyte.
Standard Addition Method
Standard addition method is often helpful in counteracting matrix
effects (e.g. effect of interfering substances).
One method involves adding one or more increments of a standard
solution to sample aliquots of the same size.
Each solution is then diluted to a fixed volume before measuring its
absorbance.
Assume that several identical aliquots Vx of the unknown solution with a
concentration Cx are transferred to flasks of volume Vt.
To each flask is added a variable volume, Vs, of standard solution
having a known concentration Cs.
If Beers Law is followed, the absorbance is described by
As = cbVxCx + cbVsCs
Vt Vt
A plot of As as a function of Vs is a straight line of the form
As = mVs + b
where slope m and intercept b are given by
m = cbCs
Vt
and b = cbVxCx
Vt
A least-squares analysis is used to determine m and b.
Cx is obtained from the ratio of m and b and the known values of Cx
and Vx.
b = cbVxCx/Vt = VxCx
m cbCs/Vt Cs
In this problem,
Cs = 11.1 ppm
Vx = 10.00 mL
Vt = 50.00 mL
The equation of the line is
A = 0.03820 Vs + 0.2412
Thus,
Cx = 0.2412 x 11.1
0.0382 x 10.00
= 7.01 ppm
Example:
Ten-milliliter aliquots of a natural water sample were pipetted into 50.00 mL
volumetric flasks. Exactly 0.00, 5.00, 10.00, 15.00, and 20.00 mL of a standard
solution containing 11.1 ppm of Fe
+3
were added to each, followed by an excess of
thiocyanate ion to give the red complex Fe(SCN)
2+
. After dilution to volume,
absorbances were found to be 0.240, 0.437, 0.621, 0.809, and 1.009, respectively.
What was the concentration of Fe
+3
in the water sample?
Nickel forms a complex with PAN that can be represented as NiL2. A series of
solution of known nickel concentration were treated with an excess of PAN
and had the following absorbances:
Example:
Conc (ppm) 0.20 0.35 0.50 0.65 0.80 0.95
A at 575 nm 0.172 0.300 0.438 0.557 0.694 0.830
a.Plot a versus conc (ppm)
b.Determine the molar absorptivity if cell width is 1.00 cm
c.Determine %T of each solution and plot %T vs. conc.
d.Determine the concentration of an unknown solution that had an
absorbance of 0.417
Solution:
a)
m = b = 0.8745
b (intercept) = -0.0043
b. A = bc = m/1.0 = 0.8745 ppm
-1
cm
-1
c. A = -log T T = antilog (-A)
A 0.172 0.300 0.438 0.557 0.694 0.830
%T 67.3 50.1 36.5 27.7 20.2 14.8

d) Based on (a), equation of line is:
A = 0.8745 (conc) 0.0043
Conc. (ppm) = A + 0.0043 = 0.417 + 0.0043 = 0.48 ppm
0.8745 0.8745
The key fact that permits analysis of mixtures is that at each
wavelength, the absorbance of a solution (containing species M, N, O,)
is the sum of the absorbances of each species:
Analysis of Mixtures
M
N
A

A =
M
b [M] +
N
b [N] +
O
b [O] + .
Case A: Individual Spectra
are Well-resolved
Case B: Individual Spectra
Overlap Significantly
M
N

A
A: Individual Spectra are Well-resolved
For a mixture of two components whose individual spectra
do not overlap very much the absorbance at wavelengths
and are given by:
A = x b [X] + yb [Y] at
A = x b [X] + yb [Y] at
The absorptivities of X and Y at each wavelength must be
measured in separate experiments.
The two equations are solved for the two unknowns [X] and [Y].
Alternative to the common approach of solving simultaneous
equation is the use of Cramers Rule:
A y b x b A
[X] = A y b and [Y] = x b A
x b y b x b y b
x b y b x b y b
The previous equation is a determinant. It is a shorthand way of
writing (a x d) (b x c).
The absorbance data of conjugate base-acid pair at given
concentrations are given below: (cell width = 1.50cm).
Example
Conc.(M) A at 430nm A at 600nm
HIn 8.00 x 10
-5
0.965 0.148
In 7.50 x 10
-5
0.087 0.783
a) Calculate the molar absorpitivity if HIn and In- at each wavelength.
b) A solution containing both HIn and In- gave an absorbance of 0.625 at
430 nm and 0.243 at 600 nm when measured in 1.00-cm cells.
Calculate
molar concentration of each species in the solution.
Solution
a.) HIn at 430 nm: = A/bc = 0.96/(1.5)(8 x 10
-5
)
= 8042
at 600 nm: = A/bc = 0.148/(1.5)(8 x 10
-5
) = 1233
In at 430 nm: = 0.087/(1.5)(7.50 x 10
-5
) = 773
at 600 nm: = 0.783/(1.5)(7.50 x 10
-5
) = 6960
b.) A430 = 430 b [HIn] + 430 b [In]
A600 = 600 b [HIn] + 600 b [In]
0.625 = (8042)(1)[HIn] + 773(1)[In]
0.243 = (1233)(1)[HIn] + 6960(1)[In]
Using Cramers rule:
0.625 773
[HIn] = 0.243 6960 = (0.625)(6960) - (773)(0.243) = 4162.2 = 7.56 x 10
-5
8042 773 (8042)(6960) - (773)(1233) 5.5 x 10
7
1233 6960
8042 0.625
[In
-
] = 1233 0.243 = (8042)(0.243) - (0.625)(1233) = 1183.6 = 2.15 x 10
-5
8042 773 (8042)(6960) - (773)(1233) 5.5 x 10
7
1233 6960
B: Individual Spectra Overlap (Graphical Method)
The absorbance of the mixture (Am) at any chosen is
Am = x b [X] + y b [Y]
If a standard solution of species X with concentration [X]s is
prepared, its absorbance will be:
Axs = x b [X]s or x = Axs
b [X]s
Similarly for species Y:
AYs = x b [Y]s or y = Ays
b [Y]s
Substituting the expressions to the Am equation:
Am = Axs b [X] + Ays [Y] b [Y]
b [X]s b [Y]s
Dividing both sides by Axs:
Am = [Y] Ays + [X] (linear equation)
Axs [Y]s Axs [X]s
y = m x + b
A graph of Am/Axs versus Ays/Axs (all of which can be measured)
at various wavelengths has a slope of [Y]/[Y]s and an intercept
of [X]/[X]s.
Am = (0.607) Ays + 0.499
Axs Axs
Slope = 0.607 = [Y]/[Y]s
[Y] = [V
v
] = (0.607)(1.89 mM) = 1.15 mM
y-intercept = 0.499 = [X]/[X]s
[X] = [Ti
IV
] = (0.499)(1.32 mM) = 0.659 mM
METHOD OF CONTINUOUS VARIATION
Allows identification of complex
P + nX PXn
Principle: Maximum absorbance is reached at the composition
corresponding to the stoichiometry of the complex
Procedure: Aliquots of equimolar solution of P and X are mixed followed by
dilution to a constant V, such that the total concentration of P + X remains
constant. As are recorded for each mixture. Corrected A is plotted vs mole
fraction of X.
A
corr
= A
mix
A
p
- A
x
For the complex, PaXb, maximum absorbance occurs at:
mol fraction of X = b/(b+a)
Ex: PX2, a = 1 b = 2 ; Xx = 2/(2+1) = 0.667
PX , a = 1 b = 1 ; Xx = 1/(1+1) = 0.500
P3X , a = 3 b = 1 ; Xx = 1/(1+3) = 0.250
MOLE-RATIO METHOD
One reactant is held constant while the other is varied.
MOLE-RATIO METHOD
One reactant is held constant while the other is varied.
THE SLOPE-RATIO METHOD
useful for weak complexes
For the complex: aP + bX P
a
X
b
Total molar concentration for the reactants:
Cp = [P] + a [PaXb]
Cx = [X] + b [PaXb]
If [X] is constant and in excess, equilibrium shifts to the right; [P] << [PaXb]
Cp = a[PaXb] , [PaXb] = Cp/a
If Beers law is obeyed:
A
1
= b [P
a
X
b
]
A
1
= b C
p
/a
If [P] is constant and in excess, [X] << [PaXb]
Cx = b [PaXb], [PaXb] = Cx/b
Plot of A vs varying Cp
A
2
= b [P
a
X
b
]
A
2
= b C
x
/a
The ratio of the slopes:
b/a = b therefore, ratio of P:X is known
b/b a
Plot of A vs varying Cx
A
Volume
T > P > 0
S = 0
A
Volume
P > T > 0
S = 0
A
Volume
S > T > 0
P = 0
PHOTOMETRIC TITRATIONS
A
Volume
S = sample; T = titrant; P = product
S = P = 0
T > 0
A
Volume
S = T = 0
P > 0
A
Volume
P = T = 0
S > 0
The IR Region
Just below red in the visible region.
Wavelengths usually 2.5-25 m.
More common units are wavenumbers, or cm
-1
,
the reciprocal of the wavelength in centimeters.
Wavenumbers are proportional to frequency
and energy.
Molecular Vibrations
Covalent bonds vibrate at only certain
allowable frequencies.
) /( ) ( 2
1
Y X Y X
M M M M
f
c +
=
t
v
Hookes Law
where v = vibrational frequency (cm
-1
)
c = velocity of light (cm/s)
f = force constant of bond (N/m)

v
f
x
12
10 3 . 5

=
where (reduced mass, kg) = (MX x MY)/(MX + MY)
f = 500 N/m for single bond
f = 1000 N/m for double bond
f = 1500 N/m for triple bond
Calculate the approximate wavenumber and wavelength of the
fundamental absorption peak due to the stretching vibration of a
carbonyl group C=0.
Example
The mass of the carbon atom on kilograms is given by
Solution
mC = 12 x 10
-3
kg/mol x 1 atom = 2.0 x 10
-26
kg
6.0 x 10 atoms/mol
Similarly, for oxygen
mO = 16 x 10
-3
kg/mol x 1 atom = 2.7 x 10
-26
kg
6.0 x 10 atoms/mol
and the reduced mass is given by
= (2.0 x 10
-26
kg) x (2.7 x 10
-26
kg) = 1.1 x 10
-26
kg
(2.0+2.7) x 10
-26
kg
The force constant for the typical double bond is about 1 x 10 N/m.
Substituting this value and
v = 5.3 x 10
-12
s/cm x sqrt[(1 x 10 N/m)/1.1 x 10
-26
kg
v = 1600 cm
-1
The carbonyl stretching band is found experimentally to be in the
region of 1600 to 1800 cm
-1
.
Vibrational Modes
Symmetrical Stretching Asymmetrical Stretching
Stretching vibrations
Molecular Absorptions Leading to Vibrations
Summary of IR
Absorptions
Vibrational Modes
Symmetrical Stretching Asymmetrical Stretching
Stretching vibrations
Bending vibrations
Scissoring Twisting Rocking Wagging
Stretching Frequencies
Frequency decreases with increasing
atomic mass.
Frequency increases with increasing bond
energy.
Fingerprint of Molecule
Whole-molecule vibrations and bending
vibrations are also quantized.
No two molecules will give exactly the same
IR spectrum (except enantiomers).
Simple stretching: 1600-3500 cm
-1
.
Complex vibrations: 600-1400 cm
-1
, called
the fingerprint region.
An IR Spectrum
stretching
bending
group frequency region
fingerprint region
IR-Active and Inactive
Only vibrations that result in a rhythmical change in
dipole moment of the molecule are observed in the IR
A polar bond is usually IR-active.
A nonpolar bond in a symmetrical molecule will absorb
weakly or not at all.