Gas chromatography and high performance liquid chromatography are analytical techniques used to separate mixtures. Mikhail Tswett invented chromatography in 1901 to separate plant pigments using a glass column packed with calcium carbonate. Liquid chromatography was later refined using smaller packing materials and pumps to deliver solvents at high pressures, allowing for faster separations and the development of high performance liquid chromatography. Chromatography works by separating components in a mixture based on how they interact differently with a stationary and mobile phase as they flow through a column.

1. Gas Chromatography
&
High Performance Liquid Chromatography

2. Invention of Chromatography
Mikhail Tswett
Russian Botanist
(1872-1919)
Mikhail Tswett invented
chromatography in 1901
during his research on
plant pigments.
He used the technique to
separate various plant
pigments such as
chlorophylls, xanthophylls
and carotenoids.

3. Original Chromatography Experiment
Later
Start: A glass
column is filled
with powdered
limestone
(CaCO3).
End: A series of
colored bands is
seen to form,
corresponding to
the different
pigments in the
original plant
extract. These
bands were later
determined to be
chlorophylls,
xanthophylls and
carotenoids.
An EtOH extract
of leaf pigments
is applied to the
top of the column.
EtOH is used to
flush the pigments
down the column.

4. Comparing Chromatography to
the Flow of a River...
Base
Water flow
Light leaf
Heavy stone

5. 5
Interaction Between Solutes, Stationary
Phase, and Mobile Phase
• Differences in the interactions between the solutes and
stationary and mobile phases enable separation.
Solute
Stationary
phase
Mobile phase
Degree of adsorption,
solubility, ionicity, etc.

6. How Does Chromatography Work?
In all chromatographic separations, the sample is
transported in a mobile phase. The mobile phase can be a
gas, a liquid, or a supercritical fluid.
The mobile phase is then forced through a stationary phase
held in a column or on a solid surface. The stationary
phase needs to be something that does not react with the
mobile phase or the sample.
The sample then has the opportunity to interact with the
stationary phase as it moves past it. Samples that interact
greatly, then appear to move more slowly. Samples that
interact weakly, then appear to move more quickly.
Because of this difference in rates, the samples can then be
separated into their components.

7. Chromatography is based on a physical equilibrium
that results when a solute is transferred between the mobile
and a stationary phase.
A
A
A
A
A
A
A
A
A
A
A
A
K = distribution coefficient
or partition ratio
K =
CS
CM
Where CS is the molar
concentration of the solute in
the stationary phase and CM is
the molar concentration in the
mobile phase.Cross Section of Equilibrium in a column.
“A” are adsorbed to the stationary phase.
“A” are traveling in the mobile phase.

8. Elution : always (100%) dilution
What is Chromatography?
s a m p le
in
e lu e n t
in
C a C O 3
( a d s o r p t io n )
c o lu m n
e lu a n t
o u t
d e t e c t o r
c h r o m a t o g r a m
( m a s s s p e c t. IR
s p e c t . e t c )

9. Chromatography:(Greek = chroma “color” and
graphein “writing” ) Tswett named this new technique
chromatography based on the fact that it separated the
components of a solution by color.
Common Types of Chromatography
Tswett’s technique is based on Liquid Chromatography.
There are now several common chromatographic
methods. These include:
Paper Chromatography
Thin Layer Chromatography (TLC)
Liquid Chromatography (LC)
High Pressure Liquid Chromatography (HPLC)
Ion Chromatography
Gas Chromatography (GC)

10. Three States of Matter and
Chromatography Types
Mobile phase
Gas Liquid Solid
Stationary
phase
Gas
Liquid
Solid
GasGas
chromatographychromatography
LiquidLiquid
chromatographychromatography

11. Classification based on Mobile
Phase
Gas ChromatographyGas Chromatography
Gas - solidGas - solid Gas - liquidGas - liquid
Stationary Phase

12. Classification based on Mobile
Phase
Liquid chromatography (LC)
Column
(gravity flow)
High performance
(pressure flow)
Thin layer
(adsorption)

13. polar s.p.
Adsorption and Partition Chromatography
for GC & LC for GC

14. Ion Exchange and Gel Permeation Chromatography
resin-SO3
-
gel filtration
resin-N(CH3)3
+
by size

15. Paper and Thin Layer Chromatography
Later
The solvent moves up paper by capillary action,
carrying mixture components at different rates.
solvent
solvent
front

16. • The retention factor, or Rf, is defined as the
distance traveled by the compound divided by the
distance traveled by the solvent
For example, if a compound travels 2.1 cm and the
solvent front travels 2.8 cm, the Rf
is 0.75:
Retention factor, Rf

17. Gas chromatography is a technique used for separation of volatile
substances, or substances that can be made volatile, from one
another in a gaseous mixture at high temperatures. A sample
containing the materials to be separated is injected into the gas
chromatograph. A mobile phase (carrier gas) moves through a
column that contains a wall coated or granular solid coated
stationary phase. As the carrier gas flows through the column,
the components of the sample come in contact with the
stationary phase. The different components of the sample have
different affinities for the stationary phase, which results in
differential migration of solutes, thus leading to separation.

18. Flow
As a material travels through the column, it assumes a
Gaussian concentration profile as it distributes between the
stationary packing phase and the flowing mobile gas or
liquid carrier phase.
In a chromatography column, flowing gas or liquid
continuously replaces saturated mobile phase and results
in movement of A through the column.
Column is packed
with particulate
stationary phase.

19.  Good for volatile samples (up to about 250 o
C)
 0.1-1.0 microliter of liquid or 1-10 ml vapor
 Can detect <1 ppm with certain detectors
 Can be easily automated for injection and data analysis
Gas Chromatography

20. Components of a Gas Chromatograph
Gas Supply: (usually N2 or He)
Sample Injector: (syringe / septum)
Column: 1/8” or 1/4” x 6-50’ tubing packed with
small uniform size, inert support coated with
thin film of nonvolatile liquid
Detector: TC - thermal conductivity
FID - flame ionization detector

21. Schematic of a Commercial Gas Chromatograph

22. A carrier gas should have the following properties:
1. Highly pure (> 99.9%)
2. Inert so that no reaction with stationary phase or
instrumental components can take place, especially at high
temperatures.
3. A higher density (larger viscosity) carrier gas is preferred.
4. Compatible with the detector since some detectors require
the use of a specific carrier gas.
5. A cheap and available carrier gas is an advantage.

23. 23
Injectors
Septum type injectors are the most common. These are composed of
a glass tube where vaporization of the sample takes place. The
sample is introduced into the injector through a self-sealing
silicone rubber septum. The carrier gas flows through the injector
carrying vaporized solutes. The temperature of the injector should
be adjusted so that flash vaporization of all solutes occurs. If the
temperature of the injector is not high enough (at least 50 degrees
above highest boiling component), band broadening will take
place.

24. 24
Column Configurations and Ovens
The column in chromatography is undoubtedly the heart of the
technique. A column can either be a packed or open tubular.
Traditionally, packed columns were most common but fast
developments in open tubular techniques and reported
advantages in terms of efficiency and speed may make open
tubular columns the best choice in the near future. Packed
columns are relatively short (~2meters) while open tubular
columns may be as long as 30-100 meters.

25. Column Ovens
• Column temperature is an important variable that
must be controlled to a few tenths of a degree for
precise work. Thus, the column is ordinarily housed in
a thermostated oven. The optimum column
temperature depends upon the boiling point of the
sample and the degree of separation required.
• Roughly, a temperature equal to or slightly above the
average boiling point of a sample results in a
reasonable elution time (2 to 30 min). For samples
with a broad boiling range, it is often desirable to
employ temperature programming, whereby the
column temperature is increased either continuously
or in steps as the separation proceeds.

26. 26
Detection Systems
Several detectors are available for use in GC. Each
detector has its own characteristics and features as
well as drawbacks. Properties of an ideal detector
include:
1. High sensitivity
2. Minimum drift
3. Wide dynamic range
4. Operational temperatures up to 400o
C
5. Fast response time
6. Same response factor for all solutes
7. Good reliability
8. Nondestructive
9. Responds to all solutes (universal)

27. 27
a. Thermal Conductivity Detector (TCD)
This is a nondestructive detector which
is used for the separation and
collection of solutes to further
perform some other experiments on
each purely separated component.
The heart of the detector is a heated
filament which is cooled by helium
carrier gas. Any solute passes across
the filament will not cool it as much
as helium does because helium has
the highest thermal conductivity.
This results in an increase in the
temperature of the filament which is
related to concentration. The
detector is simple, nondestructive,
and universal but is not very
sensitive and is flow rate sensitive.
Heated wire

28. TCD characteristics include:
1. Rugged
2. Wide dynamic range (105
)
3. Nondestructive
4. Insensitive (10-8
g/s)
5. Flow rate sensitive

29. 29
Flame Ionization Detector (FID)
This is one of the most
sensitive and reliable
destructive detectors.
Separate two gas
cylinders, one for fuel and
the other for O2 or air are
used in the ignition of the
flame of the FID. The fuel
is usually hydrogen gas.
The flow rate of air and
hydrogen should be
carefully adjusted in
order to successfully
ignite the flame.

30. 30
Characteristics of FID
• Rugged
• Sensitive (10-13
g/s)
• Wide dynamic range (107
)
• Signal depends on number of carbon atoms in
organic analytes which is referred to as mass
sensitive rather than concentration sensitive
• Weakly sensitive to carbonyl, amine, alcohol, amine
groups
• Not sensitive to non-combustibles – H2O, CO2, SO2,
NOx
• Destructive

31. 31
Electron Capture Detector (ECD)
This detector exhibits high intensity for halogen
containing compounds and thus has found wide
applications in the detection of pesticides and
polychlorinated biphenyls. The mechanism of sensing
relies on the fact that electronegative atoms, like
halogens, will capture electrons from a β emitter
(usually 63
Ni). In absence of halogenated compounds, a
high current signal will be recorded due to high
ionization of the carrier gas, which is N2, while in
presence of halogenated compounds the signal will
decrease due to lower nitrogen ionization.

32. 32
Characteristics of ECD
Simple and reliable
Sensitive (10-15
g/s) to electronegative groups
(halogens)
Largely non-destructive
Insensitive to amines, alcohols and
hydrocarbons
Limited dynamic range (102
)
Mass sensitive detector

33. How do we describe a chromatogram?
1) Chromatogram :
A graph showing the detectors response as a function of
elution time :
band’s shapes, position, resolution.
2) For individual band :
a) Retention time (tr) :
The time needed after injection for an individual solute to
reach detector.
b) An ideal chromatographic peak
⇒ Gaussian shape.
w½ = 2.35σ, w = 4σ

34. How do we describe a chromatogram?

35. Theoretical plates (N): (from distillation)
the more plates on a column, the more
equilibration steps, and the better the
separation.
Number of plates on column :
N = 5.55(tr/w½)2
Plate height : H = L/N
The smaller plate height
⇒ narrower peaks ⇒ better separation
How do we describe a chromatogram?

36. Why do bands spread ?
1) Why broadening?
a) diffusion
b) slow equilibration of solute between the
m.p and s.p.
c) irregular flow paths.

37. Why do bands spread ?
2) Longitudinal
diffusion :
the faster the flow
⇒ the less a band
spends in column.
⇒the less time for
diffusion.
⇒ broadening
u
1
∝

38. 3) solute requires time to equilibrate between
phases.
(s.p.↔m.p.) with temp.
broadening ∝ u
Can’t equilibrate rapidly enough.
Why do bands spread ?
m.p.
s.p.

39.  Substances that vaporize below 300°C can be measured
quantitatively.
 In assuring the quality of products in the chemical industry
 Measuring toxic substances in soil, air or water
 Gas Chromatography is used extensively in forensic science.
 Since the samples have to be volatile, human breathe,
blood, saliva and other secretions containing large amounts of
organic volatiles can be easily analyzed using GC.
 In food industry
Applications of Gas Chromatography

40. Advantages of Gas Chromatography
• Requires only very small samples with little preparation
• Good at separating complex mixtures into components
• Results are rapidly obtained (1 to 100 minutes)
• Very high precision
• Only instrument with the sensitivity to detect volatile
organic mixtures of low concentrations
• Equipment is not very complex (sophisticated oven)

41. From Liquid Chromatography to High Performance
Liquid Chromatography
• Higher degree of separation!
→ Refinement of packing material (3 to 10 µm)
• Reduction of analysis time!
→ Delivery of eluent by pump
→ Demand for special equipment that can
withstand high pressures
The arrival of high performance liquid chromatography!

42. HPLC Separation Modes
• Adsorption (liquid-solid) chromatography
• Partition (liquid-liquid) chromatography
– Normal phase partition chromatography
– Reversed phase partition chromatography
• Ion exchange chromatography
• Size exclusion chromatography

43. Pump
Sample injection unit
(injector)
Column
Column oven
(thermostatic column
chamber)
Detector
Eluent
(mobile phase)
Drain
Data processor
Degasser
Flow Channel Diagram for High Performance
Liquid Chromatograph

44. Advantages of High Performance Liquid
Chromatography
• High separation capacity, enabling the batch
analysis of multiple components
• Superior quantitative capability and reproducibility
• Moderate analytical conditions
– Unlike GC, the sample does not need to be vaporized.
• Generally high sensitivity
• Low sample consumption
• Easy preparative separation and purification of
samples

45. • Biogenic substances
– Sugars, lipids, nucleic
acids, amino acids,
proteins, peptides,
steroids, amines, etc.
• Medical products
– Drugs, antibiotics, etc.
• Food products
– Vitamins, food
additives, sugars,
organic acids, amino
acids, etc.
• Environmental samples
– Inorganic ions
– Hazardous organic
substances, etc.
Applications of High Performance Liquid
Chromatography
• Organic industrial products
– Synthetic polymers,
additives, surfactants, etc.