Dr.

Muniyandi Sankaralingam
Assistant Professor
Department of Chemistry
National Institute of Technology Calicut
1
 Spectroscopy
Reference Books:
1. Fundamentals of Molecular Spectroscopy, 4th Edition, by
Colin N. Banwell and Elaine M. McCash
2. Introduction to Spectroscopy, 5th Edition, Donald L.
Pavia, Gary M. Lampman, George S. Kriz
3. Organic Spectroscopy By William Kemp
4. Spectroscopy of Organic Compounds By P. S. Kalsi
5. Elementary Organic Spectroscopy By Y. R. Sharma

2
 IR Range of EM Spectrum
Increasing frequency ()
γ-Ray X-Ray Ultraviolet Visible Infrared Microwave Radio
Increasing wavelength (m)

Near IR Middle IR Far IR

0.8 2.5 25 250

12500 4000 400 40

E = 1-10 kcal/mol

Bond Vibrations (most useful I.R. region) 3

4
D
E
T
E
C
T
O
R

5
 IR Spectroscopy is concerned with the study of absorption of
IR radiation, which causes vibrational transitions in the
molecule due to the change in the dipole moment. Hence IR
spectroscopy is also known as Vibrational spectroscopy.

 Used in structural elucidation to determine the functional

groups.

6
O

H
C
C
H
3
3

7
Theory of IR Spectra - Vibrational Motion in Covalent Bond

Normal Vibration Vibration having Absorbed Energy

Ground State Excited State

A bond will absorb radiation of a frequency similar to its

vibration(s)

8
 Types of Bond Motions

Symmetric

Stretching

Asymmetric

In Plane Bending

Bending

Out of Plane Bending

9
 Types of Stretching Motions

H H
Symmetric
C

H H

Asymmetric
C

10
 Types of Bending Motions

In Plane Bending Out of Plane

Scissoring Rocking Twisting Wagging

11
The energy of a diatomic molecule:
The two atoms in a molecule are settle at a mean inter-nuclear
distance such that these forces are just balanced and the total energy
of the whole system is minimum.

13
 Stretching IN/OUT Motions
Stretching-Out

δ-Cl Hδ+

Electric Field
Induced
Fluctuating Dipole
of Polar Molecule

Stretching-In
Induced Electric Field Interacts with the EM Radiation
14
 The vibrating bond will absorb EM radiation (in IR Range)
only when its frequency is same as the frequency of the
oscillating dipole.

Frequency
Absorbed

Electromagnetic Fluctuating Dipole of

Polar Molecule
Radiation Frequency 𝝑 IR spectra
Frequency 𝜗

Each Vibrational mode with change in dipole moment of the

system leads to absorption of frequency in IR range

15
 Criteria for IR Absorption

Dipole Moment of the bond must change during

Vibrational Motion

e.g. H2, N2, O2, Cl2 e.g. H2O, SO2, HCl

IR-Inactive IR-active

16
 Example CO2

No Change in Dipole
IR Inactive

Change in Dipole
IR Active

Change in Dipole
IR Active

Though CO2 is non polar it is IR active

17
 Simple Harmonic Oscillator (SHO)
Bond is elastic in nature!
Energy

Obey Hooke’s law

SHO

0 req
Internuclear distance
Morse Energy Diagram 18
For diatomic molecule behaving as SHO
Frequency of a vibrational stretching

Where, 𝑘 = Force constant

𝟏 𝒌 𝜗ҧ = Wavenumber
ഥ=
𝝑 𝑐 = Velocity
𝟐𝝅𝒄 𝝁
𝜇 = 𝑅𝑒𝑑𝑢𝑐𝑒𝑑 𝑚𝑎𝑠𝑠

𝑚1 𝑚2
𝜇 = 𝑅𝑒𝑑𝑢𝑐𝑒𝑑 𝑚𝑎𝑠𝑠 =
𝑚1 + 𝑚2

19
 𝟏 𝒌
ഥ=
𝝑 𝒄𝒎−𝟏
𝟐𝝅𝒄 𝝁

Two conclusions can be drawn from the above equation:-

ഥ∝ 𝒌
𝝑
Reveals 𝐬𝐭𝐫𝐨𝐧𝐠𝐞𝐫 𝐛𝐨𝐧𝐝𝐬 𝐚𝐛𝐬𝐨𝐫𝐛 𝐟𝐫𝐞𝐪𝐮𝐞𝐧𝐜𝐲 𝐢𝐧 𝐡𝐢𝐠𝐡 range

𝟏
ഥ∝
𝝑
𝝁
Reveals heavier atoms absorb frequency in low wavenumber

20
 Problems

𝟏 𝒌
ഥ=
𝝑 𝒄𝒎−𝟏
𝟐𝝅𝒄 𝝁

1. What is the order of decreasing vibrational frequency

for C — H & C — D?
Hint: μ = reduced mass (C-H, 0.92 & C-D, 1.71)

2. What is the correct increasing order of stretching

frequencies for C ≡ C, C = C and C — C?
Hint: μ = reduced mass 6 (same for C-C, C=C, C ≡ C)

21
 𝟏 𝒌
ഥ=
𝝑 𝒄𝒎−𝟏
𝟐𝝅𝒄 𝝁

Energy of Vibrational level is expressed as

1
𝐸𝑛 = 𝑛 + ℎ𝜗 𝐽𝑜𝑢𝑙𝑒𝑠
2
(Derived from schrödinger equation)

𝐸 1
𝜀𝑛 = = (𝑛 + )𝜗ഥ 𝑐𝑚−1
ℎ𝑐 2
Where, 𝒏 = 𝒗𝒊𝒃𝒓𝒂𝒕𝒊𝒐𝒏𝒂𝒍 𝒒𝒖𝒂𝒏𝒕𝒖𝒎 𝒏𝒖𝒎𝒃𝒆𝒓 = 𝟎, 𝟏, 𝟐, 𝟑, … …

22
 𝑬 𝟏
𝜺𝒏 = ഥ 𝒄𝒎−𝟏
= (𝒏 + )𝝑
𝒉𝒄 𝟐
𝟏𝟑
n=6 𝜺𝟕 = ഥ
𝝑 1
𝟏𝟏
𝟐
𝐴𝑡 𝑛 = 0, 𝜀𝑂 = 𝜗ҧ 𝑐𝑚−1
n=5 𝜺𝟓 =
𝟐
ഥ
𝝑 2
𝟗 Zero Point Energy
𝜺𝟒 = 𝝑 ഥ
n=4 𝟐 3
𝐴𝑡 𝑛 = 1, 𝜀1 = 𝜗ҧ 𝑐𝑚−1
Energy

𝟕
𝜺𝟑 = 𝝑 ഥ 2
n=3 𝟐
𝟓 5
𝜺𝟐 = 𝝑 ഥ
n=2 𝟐 𝐴𝑡 𝑛 = 2, 𝜀2 = 𝜗ҧ 𝑐𝑚−1
n=1 𝜺𝟏 = 𝝑
𝟑
ഥ 2
𝟐 Selection rule for a SHO
𝟏
n=0 𝜺𝒐 = 𝝑 ഥ
𝟐 undergoing vibrational changes

Internuclear distance
𝒏 = 𝟏
Energy levels of SHO are equally spaced
 A molecule can never have zero vibrational energy.
 The energy of the molecule corresponding to n = 0 level is known as
Zero Point Energy. 23
 Energy

Internuclear distance

1 1
𝜀𝑛→𝑛+1 = 𝑛+1+ 𝜗ҧ − 𝑛+ 𝜗ҧ 𝑐𝑚−1 = 𝜗ҧ 𝑐𝑚−1
2 2

24
 The Anharmonic Oscillator (AHO)
Real molecules do not obey laws of SHO for large amplitude of
vibrations. 𝟐
𝟏 𝟏
𝜺𝒏 = 𝒏 + 𝝎𝒆 − 𝒏 + 𝝎𝒆 𝝌𝒆 𝒄𝒎−𝟏
𝟐 𝟐
Where, 𝒏 = 𝟎, 𝟏, 𝟐, … . ; 𝝌𝒆 = 𝐀𝐧𝐡𝐚𝐫𝐦𝐨𝐧𝐢𝐜𝐢𝐭𝐲 𝐂𝐨𝐧𝐬𝐭𝐚𝐧𝐭
𝝎𝒆 = 𝐄𝐪𝐮𝐢𝐥𝐢𝐛𝐫𝐢𝐮𝐦 𝐎𝐬𝐜𝐢𝐥𝐥𝐚𝐭𝐢𝐨𝐧 𝐅𝐫𝐞𝐪𝐮𝐞𝐧𝐜𝐲 𝐢𝐧 𝒄𝒎−𝟏
Dissociation Energy

 With increasing n
value the energy
decreases means they
Energy

n=6
n=5
n=4
De are more closely each
n=3 others.
𝟓 𝟓
n=2 𝜺𝟐 = 𝟏 − 𝝌𝒆 𝝎𝒆
𝟐 𝟐  The selection rules
𝟑 𝟑
n=1 𝜺𝟏 =
𝟐
𝟏 − 𝝌𝒆 𝝎𝒆
𝟐
for anharmonic
n=0
𝜺𝒐 =
𝟏 𝟏
𝟏 − 𝝌𝒆 𝝎𝒆
oscillator
𝟐 𝟐 n = 1, 2, …. etc
Internuclear Distance 25
Comparison Between Harmonic and
Anharmonic Oscillator

26
 2
1 1
𝜀𝑛 = 𝑛 + 𝜔𝑒 − 𝑛 + 𝜔𝑒 𝜒𝑒 𝑐𝑚−1
2 2

1 1
𝜀𝑛 = 𝜔𝑒 𝑛+ {1 − 𝜒𝑒 𝑛 + } 𝑐𝑚−1
2 2

Where, 𝒏 = 𝟎, 𝟏, 𝟐, … . ;
𝝌𝒆 = 𝐀𝐧𝐡𝐚𝐫𝐦𝐨𝐧𝐢𝐜𝐢𝐭𝐲 𝐂𝐨𝐧𝐬𝐭𝐚𝐧𝐭
𝝎𝒆 = 𝐄𝐪𝐮𝐢𝐥𝐢𝐛𝐫𝐢𝐮𝐦 𝐎𝐬𝐜𝐢𝐥𝐥𝐚𝐭𝐢𝐨𝐧 𝐅𝐫𝐞𝐪𝐮𝐞𝐧𝐜𝐲 𝐢𝐧 𝒄𝒎−𝟏

Calculate the Zero Point Energy?

27
 Probable Transitions
 𝒏 = 𝟎  𝒏 = 𝟏, 𝒏 = 𝟏
𝑰𝒏𝒕𝒆𝒏𝒔𝒆 𝑨𝒃𝒔𝒐𝒓𝒑𝒕𝒊𝒐𝒏 𝑭𝒖𝒏𝒅𝒂𝒎𝒆𝒏𝒕𝒂𝒍 𝑨𝒃𝒔𝒐𝒓𝒑𝒕𝒊𝒐𝒏
𝚫𝜺 = 𝝎𝒆 (𝟏 − 𝟐𝝌𝒆 ) 𝒄𝒎−𝟏

1 1
𝜀𝑛 = 𝜔𝑒 𝑛+ {1 − 𝜒𝑒 𝑛 + } 𝑐𝑚−1
2 2

 𝒏 = 𝟎  𝒏 = 𝟐, 𝒏 = 𝟐
𝑾𝒊𝒕𝒉 𝑺𝒎𝒂𝒍𝒍 𝑰𝒏𝒕𝒆𝒏𝒔𝒊𝒕𝒚 𝑭𝒊𝒓𝒔𝒕 𝒐𝒗𝒆𝒓𝒕𝒐𝒏𝒆
𝚫𝜺 = 𝟐𝝎𝒆 (𝟏 − 𝟑𝝌𝒆 ) 𝒄𝒎−𝟏

 𝒏 = 𝟎  𝒏 = 𝟑, 𝒏 = 𝟑
N𝒆𝒈𝒍𝒊𝒈𝒊𝒃𝒍𝒆 𝒊𝒏𝒕𝒆𝒏𝒔𝒊𝒕𝒚 (𝑺𝒆𝒄𝒐𝒏𝒅 𝒐𝒗𝒆𝒓𝒕𝒐𝒏𝒆)
𝚫𝜺 = 𝟑𝝎𝒆 (𝟏 − 𝟒𝝌𝒆 ) 𝒄𝒎−𝟏
28
Q1. The fundamental and first overtone transitions of NO are
centred at 1876 cm-1 and 3724 cm-1, respectively.
Evaluate the equilibrium vibrational frequency (𝝎𝒆 ), the
anharmonicity constant (𝝌𝒆 ) and the zero point energy.
Ans: 𝚫𝜺 = 𝝎𝒆 𝟏 − 𝟐𝝌𝒆 𝒄𝒎−𝟏
𝚫𝜺 = 𝟐𝝎𝒆 (𝟏 − 𝟑𝝌𝒆 ) 𝒄𝒎−𝟏
~ 𝜔𝑒 =1903 cm-1, 𝜒 𝑒 =7.3 x 10-3 cm-1
zero point energy = 949 cm-1
Q2. The spectrum of HCl, shows a very intense absorption at
2886 cm-1 a weak one at 5668 cm-1 and a very weak one at
8347 cm-1. Calculate the equilibrium frequency (𝝎𝒆 ) and
anhormonicity constant (𝝌𝒆 ).

Ans. 𝝌𝒆 = 0.0174, we = 2990 cm-1

30
Harmonic oscillator Anharmonic oscillator

n=4
ℎ𝜗 E
n=3
E n=2

n=1 n=2
n=0 n=1 First Overtone
Fundamental
n=0

Bond distance Bond distance

Fundamental

First overtone Second overtone

Frequency (𝜗) Frequency (ϑ)

32
Degree of freedom: The number of directions in which atom can
move freely is defined as its degree of freedom.
A molecule composed of N atoms. The total number of
coordinates to specify the locations of N atoms is 3N (e.g. x,
y, z per atom). Says that the molecule has 3N degree of
freedom.
3N = degrees of freedom of translational + rotational +
vibrational motions
For linear or non-linear molecule – translational motions are 3
For linear molecules rotational motion = 2
For non-linear molecule rotational motion = 3

33
 The degree of freedom of vibrational motion

For linear molecule 3N-5

Non-linear molecule 3N-6
} Fundamental vibrations

No. of stretching motions of linear or non linear molecule is N-1

No. of bending motions for
linear molecules = 2N-4,
Non-linear molecules = 2N-5

34
Transmittance

Wavenumber (cm-1)

35
 IR band shapes come in various forms. Two of the most common
are narrow and broad. Narrow bands are thin and pointed, like a
dagger. Broad bands are wide and smoother.
 A typical example of a broad band is that displayed by O-H bonds,
such as those found in alcohols and carboxylic acids, as shown
below.
Transmittance

Wavenumber (cm-1)
36
Band Depends on Frequency
Position Reduced mass of Lighter atoms – higher
atoms frequency
Bond strength Strong – higher frequency
Strength Change in dipole Large dipole moment gives
moment strong absorption
Width Hydrogen bonding Strong H-bond gives wide
peak

37
 Not all covalent bonds display bands in the IR spectrum. Only
polar bonds do so. These are referred to as IR active.
 Strongly polar bonds such as carbonyl groups (C=O) produce
strong bands.
 Medium polarity bonds and asymmetric bonds produce
medium bands.
 Weakly polar bond and symmetric bonds produce weak or non
observable bands.

Symmetric bond that has identical or nearly identical groups on

each end will not absorb in the infrared
38
• Stronger bonds have a larger force constant K and vibrate at higher
frequencies than weaker bonds.

• Bonds between atoms of higher masses (larger reduced mass) vibrate

at lower frequencies than bonds between lighter atoms.

μ – less μ – more
39
• Bending motions occur at lower energy (lower frequency) than the
typical stretching motions because of the lower value for the bending
force constant K.

• Hybridization affects the force constant K, also sp>sp2>sp3

40
 Equations to Remember
For, SHO 𝟏 𝒌
ഥ=
𝝑 𝒄𝒎−𝟏
𝟐𝝅𝒄 𝝁

𝑬 𝟏
𝜺𝒏 = ഥ 𝒄𝒎−𝟏
= (𝒏 + )𝝑
𝒉𝒄 𝟐
For, Anharmonicity
2
1 1
𝜀𝑛 = 𝑛 + 𝜔𝑒 − 𝑛 + 𝜔𝑒 𝜒𝑒 𝑐𝑚−1
2 2

1 1
𝜀𝑛 = 𝜔𝑒 𝑛+ {1 − 𝜒𝑒 𝑛 + } 𝑐𝑚−1
2 2
41
Group Frequencies

42
 Infrared Spectroscopy Table

Bond Class of compound Range (cm-1)

C-C Alkane 1200 - 700

C=C Alkene 1680 - 1620
C≡C Alkyne 2150 - 2130

C-H Alkane (C-sp3) 2965 - 2850

C-H Aromatic (C-sp2) > 3000

C-H C-sp > 3150

43
 Infrared Spectroscopy Table
Bond Class of compound Range (cm-1)

Aldehyde 1740 – 1720

Ketone 1725 - 1705

Carboxylic acid 1725 - 1700

Amide 1700 - 1630 44

 Infrared Spectroscopy Table

Bond Class of compound Range (cm-1)

O-H Alcohol (monomer) 3650 - 3590

O-H…O Alcohol (H-bonded) 3420 – 3200

O-H Carboxylic acid 3300 - 3250

(H-bonded)

C-O Alcohols, Esters, 1300 - 1000

Acids and Ethers

45
 Infrared Spectroscopy Table

Bond Class of compound Range (cm-1)

N-H Amine/Amide 3500 (approx.)

CN Nitrile 2260 - 2240

C-X X = Chloride 800 - 600

X = Bromide 600 - 500

X = Iodide 500 (approx.)

46
 IR Absorption Full Range

4000 cm-1 2700 cm-1 2000 cm-1 1600 cm-1 400 cm-1

47
 Looking at a Spectrum

Fingerprint region: complex and

difficult to interpret reliably.

Focus your analysis on this region

(Functional group region). This is where
most stretching frequencies appear.

 Fingerprint region is rich in Bending Vibration of C-C, C-H, C-N, C-O

etc.

48
Hydrocarbons

49
Note the strong bands in the 3000-2850 cm-1 region due to C-H stretch.
The C-H scissoring (1470 cm-1), methyl rock (1383 cm-1), and long-chain
methyl rock (728 cm-1) are noted on this spectrum.
50
51
3000 1000

52
53
 Carboxylic Acid

3000 1000

54
 Amines

z
z

3000 1000

55
 Amines

3000 1000

56
Factors Influencing the Vibrational Frequency of C=O Bond

Inductive Effect
 Addition of +I groups result in weakening of CO bond and the 𝜗𝑐𝑜
value decreases.

1750 cm-1 1745 cm-1 1715 cm-1

Reason: Decrease in Bond Order and hence the Bond Strength

57
Factors Influencing the Vibrational Frequency of C=O Bond

 Introduction of –I groups results in increase in bond order of

CO and hence the absorption frequency (𝜗𝑐𝑜 ) increases.

1750 cm-1

1715 cm-1 1725 cm-1 1740 cm-1 1750 cm-1

58
 Mesomeric Effect
 Conjugation lowers the 𝝑𝒄𝒐 value by weakening the C-O
bond.

 Mode of conjugation enhances, the 𝝑𝒄𝒐 value further

decreases.
No conjugation Conjugation Extension of conjugation

1715 cm-1
1706 cm-1
1693 cm-1
59
• Resonance also affects the strength and length of a bond and
hence its force constant K will change.
• Thus, whereas a normal ketone (acetone) has its C=O stretching
vibration at 1715 cm−1; a ketone that is conjugated with a C=C
double bond absorbs at a lower frequency, near 1675 to 1680 cm−1

• That is because resonance lengthens the C=O bond distance and

gives it more single-bond character.
No two molecules will give exactly the same IR
spectrum (except enantiomers).

60
 Problems
1. In the IR spectra, Benzamide and Phenyl acetate showed
𝝑𝒄𝒐 stretching frequency at 1663 cm-1 and 1730 cm-1,
respectively. Explain the reason for difference in their peak
position.

2. Arrange the following compounds according to their 𝝑𝒄𝒐

absorption frequency: 1677 cm-1, 1700 cm-1, 1770 cm-1.
H3C C O H3C C O H3C C O

O
NO2 CH3

1770 cm-1 1677 cm-1

1700 cm-1
61
 Hydrogen Bonding

 H-bonding brings downward frequency shifts.

 Stronger the H-bonding, greater is the absorption shift

towards lower frequency.

 Inter-molecular H-bonding gives rise to broad band and

strong peak.

 Intra-molecular H-bonding gives rise to sharp and defined

peak.

62
 Intermolecular Hydrogen bonding

Neat/concentrated Diluted with CCl4 Very diluted, with CCl4

Effect of dilution on Hydrogen bonding - effect on O-H

stretching
Intermolecular hydrogen bonding weaken the O-H bond,
thereby shifting the band to lower frequency.
63
 Intramolecular H-bonding

Intramolecular-H bonding shifts the OH band to lower

frequency
“Does not depend on the concentration”

64
Intermolecular Hydrogen bonding

3000 1000

65
Hydrogen bonding

1058 cm-1

1109 cm-1

66
Hydrogen bonding

1220 cm-1

67
 Explain why,

1. In dilute solution, amines show stretching

frequency at 3650 cm-1, while a broad band is
appeared at 3300 cm-1 in condense phase spectra.
Explain why?

68
 Explain why,

2. The spectrum of glycol in CCl4 shows two peak at

3644 and 3612 cm-1.
In tra m o le c u la r H -b o n d in g
H H
O O
H O H H
H
H H H H
H O
H
S yn A n ti

No Intermolecular H-bonding, 3644 cm-1 Intermolecular H-bonding, 3612 cm-1

69
70
71
Problems

72
Ans:

73
74
Ans: Double bond equivalents/Degree of unsaturation can be
calculated using the following equation.

75
76
Ans:

77
78
Ans:

79
80
81
82
 Answer all questions

1. a) Define zero point energy. (2 marks)

Define with plot
b) The fundamental vibrational frequency of 1H19F is 8.67 1013 Hz. Calculate the fundamental
vibrational frequency (in Hz) of 2D19F on the assumption that the force constants of both the
bonds are equal. (2 marks)

(0.5 mark)

(calculation of µ= 0.5)

ν DCl = νHCl / (µDCl/µHCl)1/2 = 8.67 1013 Hz/(3.1411x10-27 kg /1.61418x10 -27 kg)1/2 (0.5 mark)
= 6.215x1013 Hz (0.5 mark) 83
 = 273 nm.nm.
= 273 (1(1mark)
mark)

4. The –OH stretching frequencies -1 -1

4. The –OH stretching frequenciesininthe
thefollowing
following substituted
substituted phenols are observed
phenols are observed atat3608
3608cm cm
-1 -1 -1
(broad)
(broad) A, A,
for for at at
3605
3605cmcm (broad)
(broad)and
and3643 cm-1(sharp)
3643cm for B
(sharp) for B and at 3643 cm-1-1 (sharp)
3643 cm forC.C.
(sharp)for
What is the
What reason
is the forfor
reason this shift
this ofof–OH
shift –OHfrequencies
frequenciesfor
forA,
A,BB and
and C? (3(3marks)
marks)

OH
OH OH
OH OH

AA BB C
C
Answer:
Answer:
Compound A: As the0 30-butyl group is in the para position, -OH can participate in intermolecular H-bonding
Compound A: As the 3 -butyl group is in-1 the para position, -OH can participate in intermolecular H-bonding
hence broader spectra around 3608 cm is observed. (1 mark)
hence broader spectra around 3608 cm-1 is -1 observed. (1 mark)
Compound B: broader peak (3605 cm -1 ) is due to the intermolecular H-bonding while sharper band (3643
Compound B: broader peak (3605 cm ) is due to the intermolecular H-bonding while sharper band (3643
cm-1) is due to the free –OH group, steric hindrance offered by the 300-butyl group at ortho position prevents
-1
cm some
) is due to theoffree
fraction –OH group,
molecules steric hindrance
from undergoing offered H-bonding.
intermolecular by the 3 -butyl group at ortho position(1prevents
mark)
some fraction of molecules from undergoing intermolecular
0 H-bonding.
Compound C: steric hindrance offered by the 3 -butyl groups on either sides (ortho position) of –OH group (1 mark)
Compound C: steric hindrance
prevent association through intermolecular 30-butyl groups
offered by the H-bonding, henceononly
either
onesides
sharp(ortho
peak position) –OHtogroup
(3643 cm-1of) due the
-1
prevent association
presence free –OHthrough
group. intermolecular H-bonding, hence only one sharp peak (3643 cm ) due to the
(1 mark)
presence free –OH group. (1 mark)

5. Two components A and B are separated by elution with hexane from a column packed with 84silica
5. Two components A and B are separated by elution with hexane from a column packed with silica
Thank you

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