Chemistry 264

SPECTROSCOPIC
ANALYSIS
Dr Megan Matthews
What is this?
Key Questions

Reactant A + Reactant B Product C

How do we know what was made?

Did we make Product C?
Did we make anything else besides Product C?
Does Product C have the correct stereochemistry?

Solution: Instrumental Structure Determination

UV-Vis Spectroscopy – ultraviolet light promotes electrons to higher
energy levels
IR Spectroscopy – Infrared radiation causes molecular vibrations
Mass Spectrometry – Electron bombardment forms molecule
fragments
NMR Spectroscopy – Radio frequency radiation causes excitation of
the nucleus
Spectroscopy interaction of matter w light
parts
measuring

change that tapface

molesales generates response

Change to molecule
 Contents
01 UV Spectroscopy
The electromagnetic spectrum
02 Infrared Spectroscopy
Process of IR Absorption
IR Spectrometer
Process of UV/Vis Absorption
Interpretation of IR Spectra
Substituent Effects

Beer-Lambert Law

Solvent Effects

Spectrophotometric Titrations
 Electromagnetic Spectrum source
wavelength peaktopeak

it.iespectrum
Mia s
L
High
frequency
pmipan
p

Short
wavelength I
misfire waves

199
travel
 Important Equations
Speed of light in Frequency (s-1)
vacuum: Light behaves like a wave

3 x108 m/s 𝒄 = 𝒗𝝀 Wavelength (m)

constant
Energy of the Frequency (s-1) Light behaves like a
quality photon (Joules) 𝑬 = 𝒉𝒗 particle

Planck’s
constant:
6.63 x10-34 Js
E hot Wahhmber
𝒉𝒄 Wavenumber (m ) -1

com aim 𝑬 = = 𝒉𝒄𝒗 Light behaves like a wave

12
𝝀
wavenumber
NOTE: energy is inversely proportional to wavelength
Class Example
How much energy in kJ is carried by one mole of photons of red light with a wavelength of 650 nm?

1663
E 519 J photon avogadro's no
10231
3.06 10 5 photon 1398
4 131,6
i E 184115 Mol

Calculate the frequency and energy of visible light with wavelength of 562 nm.

E
3.54101
16.63
1534 v
3.54 10 19 v 5.3 1014m.se
E nu 6.63 10 34115.33 1014 C un u
3.53 10 19 I photon

3.53 10 19 6.0271023 2131151ms notnecessary 5.33 1014Hz

Process of UV/Vis Absorption

higherenergystate

TheHigher M + h → M*
energy's
Process of UV/Vis Absorption
stateemission
must doing a
hort called
exaff.it
Excited 2

Excited 1

E1
E2
S
Ground
 Process of UV/Vis Absorption

Incident Emerging For

Radiation Radiation Absorption:
P0 P P0 > P

supplyPs P released
nm
Olour wavelength
violet 3 use absorption
blue 3 45485
spestrum

955 595 55798 absorpiphege

range 590 620

red 620 750 http://www2.chemistry.msu.edu
 Atomic vs Molecular Absorption
MEI
bonds
moretransition
Atoms absorption byatoms and Molecules than atoms
molecules
absorptionby
atomicorbitalsform

Electronic
MEdY
Transitions
9 8hpElectronic
t
> Vibrational > Rotational

Handy onlywithbonds

Atomic Orbitals Molecular

combintations
sp.YEY.EE
https://brainbrooder.com/lesson/33/atomic-orbitals

8e9eTtohtotatsaifai
https://www.expii.com/t/molecular-orbital-theory-overview-application-8363

Spectrum overlappingregion
are where bonds
form
Harris, Quantitative Chemical Analysis, 2010
https://webbtelescope.org/contents/media/images/
Emission spectroscopy
Light emitted during transition from excited state to ground state

R3

El
I

associated vibrational
energy
En

Eel Eub Erot

uvYvis mitrowave
in
 Electronic Energy Levels & Transitions
node Zero opportunity Molecular Orbital Diagram
of finding an electron Petenergy
Bonding orbitals from
Bonding orbitals have
constructive interference lower energy than Antibonding MO
antibonding orbitals
𝛔∗
AO AO
Antibonding orbitals from

Energy
destructive interference
1s 1s

Bonding MO

𝛔
e
Hrgy
p orbitals can combine
head-on or side-on to H H2
form p or p
bonding/antibonding
orbitals

https://www.expii.com/t/molecular-orbital-theory-overview-application-8363
 Electronic Energy Levels & Transitions
highestenergylevel HOMO
When radiation in UV-Visible range lowest energy level LUMO
is absorbed, electrons transition
from
bondingorbital
Highest Occupied Molecular Orbital
(HOMO) to
Lowest Unoccupied Molecular Orbital
(LUMO)
antibonding orbital
Molecules that absorb strongly in
UV/Vis region are called
NB chromophores

things that have colour

moreconjugated
molecule lower HOMO LUMOgap
lessenergy required to cause parothe
electron transitions Beta promoted from HOMO LUMO
 Substituent Effects on UV maxima
Hyperchromic

HOMO Tea88
Hypsochromic Bathochromic
1. Bathochromic shift
(Red shift)
higherwavelength shifting
to shifting towards
absobing lwenggyab.sorb Hypochromic higher wavelength
Auxochromes:
2. Hypsochromic shift lower -Not chromophores!

Absorbance
(Blue shift) causedmolecule wavelength -Functional groups
to absorb lower which cause red
3. Hyperchromic shift –
increased intensity
wavelength Absorbmoreenfffff.sn shifts
HOMO LUMOgap -Examples: -OH, -
increased OR, -NH2, -SH
4. Hypochromic shift –
decreased intensity
Patient's
hostage's uueb
 (nm) Laminin
gp se

to here for Al
 present
unerring
chromophore

mmmm
I

o
auxochrome chromophore
auxochrome
N N
Nass

doublebond
auxochrome don'thave
chromophore

molecule that absorb strongly

in visible region
chromophore
most common electronic transition from alkynes
to
alkenes
I A
Practice Problem
Predict which molecule would absorb at longer wavelength

Éwev engffer Homo LumsGAP

aye more conjugation

longer conjugation
u
pathway

0
A B
In the beginning there
was colorimetry…
Nessler’s Test for Ammonia

precipitateforms NH3

Colorimetry is an example of spectroscopic analysis – sample absorbs

visible light
Using the intensity of the colour, it is possible to determine the concentration
of the compound

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 NB
Beer-Lambert Law
passesthrough
samplein cuvette
comesout
Incident Radiation Meffin
P 0
Pathlength P
Emerging
Radiation
b
from light source
P
collected by light
detector
P is called irradiance:
Amount of energy
provided by a beam of Monochromator Cuvette
light in a second per unit
area (unit W/m2)

𝑷𝟎
T (Transmittance): 𝑨 = log = − log 𝑻
𝑷 A (Absorbance): 𝑷
Fraction of incident
𝑻= inverse of what
light which passes 𝑷𝟎 𝑨 = 𝜺𝒃𝒄 Beer’s Law
was transmitted
through sample
Molar extinction coefficient/ Molar
absorptivity (M-1cm-1) → how much light a
substance absorbs at a specific wavelength
 Practice Problem
1000 0.003
Calculate the absorbance and transmittance of a 3 mM solution of a sample with
molar absorptivity of 300 M-1cm-1 in a cell which has a 2 cm pathlength

A EDC
300 M em 2 cm X 0.003M
1.80
A
Pf logt logt
A log 10109T 10
A
1.80
T 10
T D 016 1.611
The reference
What happens to light besides absorption by
analyte?
• Reflection at cuvette interface
• Scattering (dust, undissolved particles)
• Absorption (cuvette, solvent)

Solution
Reference cuvette containing only solvent – baseline/blank
measurement

𝑷𝟎 𝑷𝒔𝒐𝒍𝒗𝒆𝒏𝒕
𝑨 = log = log
𝑷 𝑷𝒔𝒂𝒎𝒑𝒍𝒆

https://sbtinstruments.com/od600-principles-and-considerations
 When does Beer’s Law not work?

Chemical
High Multiple Instrumental
reactivity/
Concentration wavelengths considerations
equilibrium
In concentrated Molar absorptivity is Stray light reduces
If analyte dissociates
solutions, analyte different at each apparent absorption
based on chemical
molecules affect one wavelength Different
equilibrium,
another’s absorption Monochromatic light reference/sample cells
undissociated vs
due to proximity. required result in different
dissociated will have
Require dilute solutions pathlengths
different absorptivities
<0.01M
Photo-induced
structural changes
 Solvent Effects on UV maxima
Solvent should not absorb in the region where the
analyte absorbs

Ethanol

Absorbance
Isooctane

 (nm)
Journal of the American Chemical Society 1948, 70, 10, 3283-3292

Polar solvents can hydrogen bond with the analyte

– interaction causes loss of fine structure

Pavia, Introduction to Spectroscopy, 2009

 Solvent Effects on UV maxima
Solvents can cause stabilization of ground or excited states

stabilites higher excitedstate

 max = 180 nm  17 9 5
Benzene  max
en b ever 
0
Hexane: 180 nm
Absorbance

n
Methanol: 190 nm
n

 
he
f level Hexane Methanol

 (nm)

Comparisons of UV spectra should use the same solvent

Summary
In organic molecules most
transitions in UV/Vis range  anti-bonding
(200 – 700 nm) are n→* or
Excited
→* states

The molecule must have  anti-bonding

unsaturation to provide
 electrons!
willnearlyalways

Energy
have doubtfond
Transition which requires
lowest energy is usually n non-bonding
the most important
 bonding
UV transitions
 bonding
Energy required below 190 nm
The colour wheel
don't need to know

A molecule will have a

colour if it absorbs light of a
complementary colour

Complementary colours
appear on opposite sides
of the colour wheel

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 me un
Quantitative Linear relationship between absorption and
concentration
UV/Vis 𝑨 = 𝜺𝒃𝒄

Spectroscopy Methods to determine concentration

from absoption

É
c
1. Calculation using known 
2. Calibration curve

https://anvajo.com/inspiration/how-are-calibration-curves-created-and-what-information-can-they-provide/

gefffd.gsbrbanievalues

Increasing amount of toluene

edtodetermine standards
 Practice Problem
A solution prepared by dissolving 4.75 mg of KMnO4 in hexane and diluting to 300 mL
pathlength
has a maximum absorbance of 0.26 at 525 nm in a 1 cm cuvette. Determine the
concentration of a KMnO4 solution which has an absorbance of 0.52 at 525 nm in the
same cell.
ÉbÉ Éb's
A 1 11711 10 4
My 4 0.26
E 2600M t.cm
0.9348

a oops I m
sample
A Ebc
0.52 12600M am firm C
1
C 2 10 4M
0.2 MM
 1206 mins
Spectrophotometric Track the changes in absorbance during a titration to
determine equivalence point – intersection of lines of
Titrations different gradients
wherelinesintersect endpoint
A = T = 0 P >0 P = T = 0 A >0 P = A = 0 T >0

1981 9 9
productone

Absorbance
In'sS T > P > 0 A =0 P > T > 0 A =0 A > T > 0 P =0
productnot
absorbing

M. Tesfa, et al. MethodsX 9 (2022) 101721

endpoint

A T P
Pconstant ft 9 Titrant volume added
yqepqndmi.iofgtadded
stan pp
After A of fan
no longer
𝑨′ = 𝑨
𝒕𝒐𝒕𝒂𝒍 𝒗𝒐𝒍𝒖𝒎𝒆
𝒊𝒏𝒊𝒕𝒊𝒂𝒍 𝒗𝒐𝒍𝒖𝒎𝒆
A be made
https://chem.libretexts.org/Courses/Providence_College/CHM_331_Advanced_Analytical_Chemistry_1
Analysis of Mixtures Absorbance of a solution is the sum of the absorbances of
all components in the solution at a specific wavelength

If
saffonance
É E Ez b Cit 2
𝑨 = 𝜺𝒃𝒄 𝑨 = 𝜺𝑿 𝒃𝒄𝑿 + 𝜺𝒀 𝒃𝒄𝒀 …

Case I: X and Y have significant overlap of absorption bands

Measure absorbance of standards of individual

components at different wavelengths X
𝑨𝑿𝒔 𝑨𝒀𝒔
𝜺𝑿 = 𝜺𝒀 =
𝒃𝒄𝑿𝒔 𝒃𝒄𝒀𝒔
𝑨 = 𝜺𝑿 𝒃𝒄𝑿 + 𝜺𝒀 𝒃𝒄𝒀 we Y
Use least squares method to get best “guess” of cX
and cY

Case II: X and Y have little overlap in selected regions

Measure at ’ and ’’

onlyneedtoknow
secondcase
𝑨′ = 𝜺′𝑿 𝒃𝒄𝑿 + 𝜺′𝒀 𝒃𝒄𝒀 @’
𝑨′′ = 𝜺′′𝑿 𝒃𝒄𝑿 + 𝜺′′𝒀 𝒃𝒄𝒀 @’’

Solve simultaneous equations for concentrations

Harris, Quantitative Chemical Analysis, 2010

 don'tneed toknowthisjustifying
info makesurewavelengthremainsstable
Analysis of Mixtures Isosbestic points occur when starting material and product
have the same molar absorptivity at a specific wavelength

X→Y

In 1 : 1 reaction, cX + cY = c constant throughout reaction

𝑨 = 𝒃(𝜺𝑿 𝒄𝑿 + 𝜺𝒀 𝒄𝒀 )
Molar absorptivity is the same at this point, 𝜺𝑿 = 𝜺𝒀 = 𝜺

𝑨 = 𝒃𝜺(𝒄𝑿 + 𝒄𝒀 )

X converted to Y:
-Both absorb light
-Absorption spectrum of X
Absorbance of total reaction mixture at this wavelength
crosses spectrum of Y remains constant, i.e. independent of progress of reaction

Harris, Quantitative Chemical Analysis, 2010

 91 84 ION
like Practice Problem
A mixture of compounds X and Y in a 1.000 cm cuvette had an absorbance of 0.852 at 265 nm and 0.511 at
305 nm respectively. Find the concentration of X and Y in the mixture given the information below:

 (M-1 cm-1)
 (nm) X Y A E bix Elybey 265hm
265 16 400 3 870
305 3990 6 420 A E Xbox yboy 305 hm

A4265 1164007111ex 13870111 cy

A265 Axzos 852

É I
139907111Cx 6420711 Cy cosmos
A305 Axsos Ayzos 0.511
3870 y 16400
Cx 10.852

0.511 13990 h 1642s ey sub min

2
8 4
1
0.511 139901 1640Cy
387

0.30371 5415445 4m
 Practice Problem
You are asked to determine the hardness of a water sample from the Eerste River. You decide to perform an
EDTA titration of CaCO3 . A 200 mL sample of river water was titrated with 0.010 M EDTA solution.
Using the titration curve provided, determine:
i) The volume of EDTA required to reach the endpoint
ii) The amount of CaCO3 in the water sample in mg/L

CaCO3 + EDTA4- (aq) → Ca(EDTA)2- (aq) + CO32- (aq) Molar Mass CaCO3 = 100.1 g/mol

8736
0.00461 1.989 y
4
Other 1.989 1.357 0.18736
0.00461
0.004746 2.17636
458.6µLEDTA

is volumesoonlysolve for x
 Practice Problem
You are asked to determine the hardness of a water sample from the Eerste River. You decide to perform an
EDTA titration of CaCO3 . A 200 mL sample of river water was titrated with 0.010 M EDTA solution.
Using the titration curve provided, determine:
i) The volume of EDTA required to reach the endpoint
ii) The amount of CaCO3 in the water sample in mg/L

CaCO3 + EDTA4- (aq) → Ca(EDTA)2- (aq) + CO32- (aq) Molar Mass CaCO3 = 100.1 g/mol

Means MEDIA endpoint nesta WE 6 1061

near Mm 1,9Y 4m59

6m01 100.1gimo 4.59 104g

Mcacss 4.59 10

5 to convert to 1000misample
4.59 104g 1000mg 0.46mg in 20mi sample
2.3my I
 Contents
01 UV Spectroscopy
The electromagnetic spectrum
02 Infrared Spectroscopy
Process of IR Absorption

Modes of Vibration
Process of UV/Vis Absorption
IR Spectrometer
Substituent Effects
Interpretation of IR Spectra
Beer-Lambert Law

Solvent Effects

Spectrophotometric Titrations
 Electromagnetic Spectrum

High
frequency

Short
wavelength

𝒉𝒄 Wavenumber (m )
-1

𝑬= = 𝒉𝒄𝒗
𝝀
𝟏
𝟏
𝒗(𝒄𝒎 ) =
𝝀 (𝒄𝒎)
 Practice Problem
Calculate the wavenumber (cm-1) as well as the frequency (Hz) of a beam of infrared radiation with a
wavelength of 7 m

1m 1429 am
7 4 m
m

3 4.3 10 Hz
v 38fm's
 Process of IR absorption
Energy absorption → gain in energy →vibration of bonds in molecule
runiaufentityhfihf.ie
imme
noq.me Infrared Spectrum
Excited 2

Excited 1 Absorption of
E2 infrared is also
E1 quantized

Ground

Bonds are not static!

If infrared energy
matches frequency
of bond vibration,
vibration amplitude
is increased.
Ahluwalia, Instrumental Methods of Chemical Analysis, 2023
 Process of IR absorption
Not all bonds can absorb infrared energy Homonuclear diatomics do not absorb IR

H2, N2, O2
Infrared absorption
requires a changing
dipole moment

The dipole moment

depends on the molecule But symmetry can be deceiving!
orientation and changes
O=C=O
as the molecule
contracts/stretches

When IR energy matches

frequency at which dipole
moment is changing, IR
radiation can be absorbed
Modes of Vibration – Fundamental
Absorptions
most important nextimportant
Stretching Bending
C–H O
C H

Asymmetric stretching
vibrations occur at higher
wavenumbers

Stretching vibrations occur

at higher wavenumbers
than bending vibrations Pavia, Introduction to Spectroscopy, 2009
Modes of Vibration – Complex Bands
Why is this spectrum so messy? iiiiin.itiiaiumoInotsame
Fundamental Absorptions Complex Absorptions
Eel
Excitation from ground Excitation from ground
Ep Erstate to lowest energy state to higher energy
excited state excited states/ band
coupling
ibsketon
roauratdoublefsh.fi
1. Overtones – ground state to higher energy excited
states. Needs more energy, occurs less frequently
n
know
to
2. Combination/Difference Bands – Sum or difference
of two interacting vibration bands
needtoknow
don't
3. Fermi Resonance – combination of fundamental
vibration with complex vibration
Ahluwalia, Instrumental Methods of Chemical Analysis, 2023
Modes of Vibration – Trends bondieigiiitesied.sine9 m i gi
C = ne
alkynesian
Stronger bonds vibrate at C≡C C C-C
higher wavenumbers
than weaker bonds
I 2150 cm-1 1650 cm-1 1200 cm-1

C-H C-C C -O C -Cl C -I

Bonds between atoms of
higher masses vibrate at 3000 cm-1 1200 cm-1 1100 cm-1 750 cm-1 500 cm-1
lower wavenumbers Increasing mass of atom attached to carbon

Hybridization of bonds ≡ C-H = C-H – C-H

increases observed
wavenumber 3300 cm-1 3100 cm-1 2900 cm-1
sp>sp2>sp3

Ketone C=O stretch 1715 cm-1

Resonance decreases the
Conjugated ketone C=O stretch 1675 cm-1
observed wavenumber

Pavia, Introduction to Spectroscopy, 2009

 Interferometry

IR spectrometer
Deflected 90°

• IR radiation of all wavelengths is split into two beams Undeflected

• Perpendicular beam is returned by fixed mirror
• Undeflected beam is returned by moving mirror
• Return pathlength of two beams will be different → constructive +
destructive interferences
• Sample simultaneously absorbs all wavelengths for IR active
bonds
• Interferogram contains information about which wavelengths
were absorbed relative of reference beam.

Radiation Source
Hot wire made from inert solid – heated metals, ceramic rods (silicon
carbide/ Nernst filament)

Absorption Cells
Pavia, Introduction to Spectroscopy, 2009 Made from rock salt or KBr – conventional glass/quartz would absorb
infrared!

Detector
Converts thermal signature to electrical signal which is interpreted by
computer
 Interpretation of IR spectra
What to look out for:
1. Position of the bands (in
wavenumbers) line
carbonyl closertobase
it
2. Intensities of the bands (weak/
medium/ strong)

3. Shapes of the bands (broad or

sharp)

broadshape

sharp

Pavia, Introduction to Spectroscopy, 2009

Interpretation of IR spectra
Do not try to assign every peak!
Look for key bands that guide structure

fgueantjfpy.im Is C=O present?

(S, 1820 – 1660 cm-1) Is C-H present?
(S, 3000 cm-1)
Is O-H present? With 1460 & 1375 cm-1
(B, 3400 – 3300 cm-1) -Hydrocarbon
Is O-H present? YES NO With C-O @ 1300 –
(B, 3400 – 2400 cm-1) 1000 cm-1 NO
-Carboxylic Acid -Alcohol/Phenol
Is C≡N present?
NO
NO (M, 2250 cm-1)
-Nitrile
Is N-H present? If W @ 2150 cm-1
Is N-H present?
(M, 3400 cm-1) With C-H @ 3300 cm-1
(M, 3400 cm-1)
-Amine -Acetylene
-Amide

NO
NO NO

Is C-O present? Is C-O present? Is C=C present?

(S, 1300 – 1000 cm-1) (S, 1300 – 1000 cm-1) (w, 1650 cm-1)
NO
-Ester -Ether With C-H > 3000 cm-1
-Alkene
If M to S in 1600 – 1450 cm-1
NO With C-H > 3000 cm-1
-Aromatic
Are there 2x w bands NO
@ 2580 & 2750 cm-1? Ketone
-Aldehyde C-H

NBfunctionalgrp toknow N
 grp s

off I doublebond I ring

aturation

Practice Problem
Suggest a possible structure which matches the IR spectrum below. There may be more than one correct
answer.

alkyne carbonyl

150

abond
3 0 d.su 2137221 3 2 doublebonds

H CEC ee
carbons halogens

nitrogen hydrogens 3 chloropropyne

 Practice Problem
Suggest a possible structure which matches the IR spectrum below. There may be more than one correct

Ñ
answer.
2 1 0 OR I C C bothofthesearepossible

a
Ñ
D

d 0.4 216 2 0 10 2
2
 C=O Base Values

Wavenumber (cm-1)
Ester Aldehyde Ketone Carboxylic Acid Amide important values
1735 1725 1715 1710 1690 toknow

Conjugation decreases observed wavenumber:

Delocalisation of  electrons across C=O and C=C
which increases single bond character

Halogen in -position increases observed

wavenumber:
Electron withdrawing effect removes electrons
from C=O leading to bond shortening
 Practice Problem
Suggest a possible structure which matches the IR spectrum below. There may be more than one correct
answer.

II
cC Pursued
ring
RI

d0.4 2 22
14

y
d0.4 of 4 mostlikelyaromaticgroup
R2
 Aromatic out-of-plane bends
intensities

equal

µ
2x bands approx. equal intensity

Single band approx. 800 cm-1

Pavia, Introduction to Spectroscopy, 2009