Chromatographic

Techniques of Anaylsis

Dr. Anant R. Kapdi

Historical Significance
Liquid-liquid extractions were known for many years.
e.g. Fe/Mg solution in 6 M HCl where Fe forms FeClx salt is first
treated with ethyl acetate.
Mg which acts as an interferent because of higher affinity for water
goes into water phase, while Fe is extracted into ethyl acetate by
thoroughly shaking the mixture of two immiscible solvents.

Chromatography
It is the partitioning of components of a sample according to their
distribution coefficient between a mobile phase and a stationary phase.
Stationary phase is always a solid surface such as silica
Mobile phase represents the solvents.
Chromatography was first discovered by Mikhail Tswett.
Calcium carbonate used as the stationary phase for separating
coloured pigment from plant extract using pet. ether as mobile phase.

The word comes from Greek words Chromos which means colour and
graphy which means to write.

Classification of chromatographic separations

a)Physical state of mobile and stationary phase
Mobile phase:
i) liquid- Column chromatography, Thin layer chromatography,
High performance liquid chromatography
ii) Gas- Gas chromatography
Stationary phase:
i) solid- Column, TLC
ii) liquid film coated on solid surface- GC, HPLC
iii) Porous gel- Size exclusion chromatography
Method of contact between the mobile and stationary phase
Chemical or physical mechanism for separation
i) Adsorption chromatography: Column, GC
ii) Partition: HPLC

Thin Layer Chromatography (Adsorption chromatography)

Mobile Phase: liquid + Stationary phase: solid adsorbent (silica)

Sovents used:
DMF>MeOH>DCM>EtOAc>Et2O>Pet
ether>Hexane>pentane
Solvent system:
polar
EtOAc

non-polar solvent
Hexane or Pet. Ether

Et2O
MeOH

Pentane
Hexane

Gradient elution: Increase concentration of one solvent to elute very

closely places solutes.

Relation between distance

substance is given by

e.g.

travelled

by

solvent

and

the

TLC preparation process

Preparing the Chamber

Preparing Plates for Development
Developing the Plates
Identifying the Spots
Interpreting Data
http://www.chemilp.net/labTechniques/TLCVideo.htm

Stain development of TLC plates

UV-light: for most organic compounds
Iodine: for unsaturated compounds
Phosphomolybdic acid
Dinitrophenyl hydrazine: aldehydes and ketones
Vanillin: general organic compounds
KMnO4: sensitive groups to oxidation
Cerric ammonium molybdate

On a TLC plate Rf values obtained were 0.87, 0.54, 0.31, 0.15 for
a solvent system Hexane: EtOAc (90:10). Assign the Rf values to
the following compounds
a)4-methoxy Phenol
b) 2,4,6-trimethyl benzene
c)Nitrobenzene
d)p-Amino phenol

Column Chromatography
Column chromatography can be used on both a large and small scale. The
applications of this technique are wide reaching and cross many disciplines
including biology, biochemistry, microbiology and medicine.

Column chromatography is technique which works on the same principle

as TLC. However, unlike TLC where different compounds are separated
on a TLC plate, it is possible to collect these compounds separately
using column chromatography.

In TLC the direction of solvent flow is

down to up while reverse is the case
for Column. However, the theory
remains the same as TLC. Non-polar
component will elute first from the
column followed by the polar.

Process involved in the separation of components using Column

chromatography
1) Stationary phase: Silica or Alumina
2) Choice of solvent: The solvent system developed during TLC which
allowed best
separation of the components of the mixture
3) Apparatus: Glass column, beakers
4) Packing column:
method

a) Dry Packing

b) Slurry

5) Sample application

6) Gradient elution

General theory of Chromatographic

analysis

The retention time, tr, is the elapsed time

from the introduction of the solute to the
peak maximum.
The retention time also can be measured
indirectly as the volume of mobile phase
eluting between the solutes introduction
and the appearance of the solutes peak
maximum. This is known as the retention
volume, Vr.
Void time, tm

Chromatographic resolution

The goal of chromatography is to separate a sample into a series of

chromatographic peaks, each representing a single component of the sample.
Resolution is a quantitative measure of the degree of separation between two
chromatographic peaks, A and B,

In a chromatographic analysis of lemon oil a peak for limonene has a

retention time of 8.36 min with a baseline width of 0.96 min. gTerpinene elutes at 9.54 min, with a baseline width of 0.64 min. What
is the resolution between the two peaks?

Capacity factor
A solutes capacity factor can be determined from a
chromatogram by measuring the columns void time, tm, and
the solutes retention time, tr

adjusted retention time: The difference between a

solutes retention time and columns void time (tr).

In a chromatographic analysis of low-molecular-weight acids,

butyric acid elutes with a retention time of 7.63 min. The columns
void time is 0.31 min. Calculate the capacity factor for butyric acid.

Column Selectivity
The relative selectivity of a chromatographic column for
a pair of solutes is given by the selectivity factor, a,
which is defined as

In a chromatographic analysis of low-molecular-weight acids,

butyric acid elutes with a retention time of 7.63 min. The columns
void time is 0.31 min. The retention time for isobutyric acid is 5.98
min.
What is the selectivity factor for isobutyric acid and butyric acid?

Column efficiency
Band broadening: With time the width of the solute peak while
passing through the column starts broadening. This affects the
efficiency of the column. Broader the peaks efficiency of the column
to seprate the solute reduces.

Thereotical plates: Martin and Synge have proposed hypotehtical

plates that are nothing but cross-sections of the column where the
distribution of the solute between the stationary and mobile phase
takes place.

Thereotical plate could be calculated

as:-

N = number of thereotical plates, H= height of each thereotical plate,

L = length of the column
A chromatographic column having a length of 2 m has 20,000
thereotical plates. Calculate the height of each thereotical plate
In case of a chromatogram where the retention time and baseline
width of the solute is provided it is also possible to calculate the
thereotical plates using following calculations:-

tr= retention time, w = baseline width

A chromatographic analysis for the chlorinated pesticide Dieldrin gives
a peak with a retention time of 8.68 min and a baseline width of 0.29
min. How many theoretical plates are involved in this separation?
Given that the column used in this analysis is 2.0 meters long, what is
the height of a theoretical plate?

KB = Capacity factor, = column selectivity, R = resolution

GAS CHROMATOGRAPHY
Mobile Phase: Gas (carrier gas)
Stationary Phase:
solid support

solid

or

Gas-solid chromatography
chromatography

liquid coated on
Gas-liquid

Schematic of GC

a) Mobile Phase: also called as Carrier Gas as it helps in carrying the

solutes from the injector to the detector. Inertness with the solute is
an important criteria.
Argon
Helium
Nitrogen

Chromatographic columns
a) Packed columns

For GSC
Partitioning of solute between mobile phase and solid stationary
phase
Made: stainless steel, aluminium, glass, copper
Length: 2-7 m
Stationary phase: Diatomeceous earth
GLC
Partitioning of solute between mobile phase and liquid stationary
phase coated on a solid support
Made: Stainless steel, glass etc.
Solid support: To avoid the adsorption of solute molecules on
exposed packing material, which degrades the quality of the
separation, surface silanols are deactivated by silanizing with
dimethyldichlorosilane and washing with an alcohol (typically
methanol) before coating with stationary phase

b) Capillary columns: also known as open tubular columns

Made: fused silica coated on polymer support
Length: 100 m

Two types: 1) Wall coated open tubular columns (WCOT): stationary phase
attached to the inner wall of the column
2) Support coated open tubular columns (SCOT): liquid stationary phase
coated on a thin layer of solid support attached to the inner wall of the
column
Packed column
a) Length
2-7 m
b) Thereotical plates
less
c) Efficiency
Good
d) Pressure
Low
e) Amount analyzed
Large
f) Cost
Low ($200)

Capillary column
100 m
More
Very high
High
Small
Very High ($1000)

Detectors in GC:
a)Thermal conductivity detector: it is based on the thermal conductivity
of the mobile phase. Higher the capacity of the carrier gas to conduct
heat, better is the detection capactiy. Helium is used as carrier gas due
to its high thermal conductivity.
1. Universally applicable
2. non-destructible
3. poor detection limit
b) Flame Ionization detector: Combustion of organic compounds in H 2/air
leads to electrons and ions. Voltage of 300 V leads to the generation of
an analytical signal.
Good for detection of organic molecules.
Destructible
Excellent detection limit

Composition of Scotch Whiskey

1. Acetaldehyde (20 C)
2. Ethyl Acetate (77 C)
3. Methanol (65 C)
4. Ethanol (79 C)
5. n-Propanol (98 C)
6. Isobutanol (108 C)
7. Amyl Alcohol/Isoamyl Alcohol (138
C)
8. Acetic Acid (123 C)
Acetaldehyde and Ethylacetate have
less stronger H-bonding interaction
with the stationary phase so they
come out first, followed by MeOH,
EtOH etc. Amyl alcohol in comparison
to Acetic acid has higher boiling point
but acetic acid has a stronger HArrange the following compounds in the order of elution
bonding.
a)MeOH
b)4-Nitro benzoic acid
c)Benzaldehyde
d)4-Methoxy aniline

Hyphenated Techniques: Gas Chromatography-Mass

Spectrometry

http://www.shsu.edu/~chm_tgc/sound
s/flashfiles/GC-MS.swf

High Performance Liquid

Chromatography

HPLC Columns
Analytical Columns
Internal diameters: 2.1 mm to 4.6 mm
Length: 30-300 mm
Thereotical plates: 40-60,000
Packing material: porous silica
Types: a) Microcolumns that are very efficient with thereotical plates
ranging upto 2,50,000
b) Open tubular microcolumns: upto 1 million thereotical plates.
However, difficult to get less than 10 micron diameters.

Guard Columns: Degradation of

Analytical columns taking place due
to some solutes which bind strongly
with the column. Particulate matter
can also pass through the solution
injected. This is taken care by
introducing a shorter and cheaper
column just ahead of analytical
column.

Stationary phases: Generally the same as GC but depending on the

polarity the column could be classified as Normal or Reverse phase
column.

Normal Phase Chromatography:

If R above is a polar group then the column stationary phase is polar
in nature and in that case use of a normal polar or slightly polar
solvent.
General organic compounds can be separated using this type of
chromatography.
Less polar solute comes out first followed by the more polar (same
principle as TLC, Column)
Reversed phase:
R group in most cases is a non-polar group such as octyl or
octyldecyl hydrocarbon chain.
Highly polar solvent such as MeOH or buffered solution could be
used. However, the pH cannot be more than 7.5 due to the possibility

Mobile phase:

Preparative HPLC: In normal HPLC the solutes can be detected

and quantified. However, physical separation is not possible.
Preparative HPLC allows physical separation to give the
products as analytically pure samples.

Detectors:
a)Spectroscopic detectors: UV-Vis or Fluorescence detectors
1. Absorbance is detected as function of time
2. volumes are low around 1-10 L
3. Important criteria is the mobile phase should strongly absorb in
the given range
4. Very good detection limit upto ng

b) Electrochemical detectors:
1. Change in potential wrt reference electrode is used to detect
samples
2. Very good detection limit upto 10 pg
3. Volumes are high

Hyphenated Technique: Liquid Chromatography- Mass Spectrometry