lecture 4 chromatography & tlc

1
Chromatography
Pham Van Hung, PhD
What on Earth did scientist do before Chromatography?
- Extraction
is based on the difference in solubility material
is grounded, placed with a solvent which
dissolves soluble compounds. A second
extract solvent . The mixture is placed in a
separatory funnel
- Crystallization
also based on the difference of solubility. The
solubility is solved in a fixed volume of solvent.
The purified compound crystallizes as solution
cools, evaporates or diffuses
- Distillilation
separates components based on their volatility
typically via vaporization-condensation
method
- Filtration
separate components of a mixture based on
their particle size. Used most often to
separate a liquid from a solid
What entices the scientists to Chromatography?
Just like the previous techniques,
chromatography is a way to separate two
components based on a specific
characteristic
What makes chromatography so useful:
The results are reproducible with better
accuracy than the before mentioned

separation techniques
Chromatography can separate more
complex mixtures than the previous
techniques
Chromatography is less time consuming and
cheaper
Brief History of Chromatography
 1903 – Tswett, a Russian botanist coined the
term chromatography. He passed plant
tissue extracts through a chalk column to
separate pigments by differential adsorption
chromatography.
 1915 R.M Willstatter, German Chemist win
Nobel Prize for similar experiment.
 1922 L.S Palmer, American scientist used
Tswett’s techniques on various natural
products.
 1931 Richard Kuhn used chromatography to
separate isomers oh polyene pigments; this
is the first known acceptance of
chromatographic methods.
History of the Main techniques
 1938 Thin Layer chromatography by
Russian scientist N.A Izamailov and M.S
Shraiber
 1941 Liquid-Liquid partition
chromatography developed by Archer
John, Porter Martin and Richard Laurence
Millington Synge
 1944 Paper Chromatography one of the

most important methods in the
development of biotechnology
 1945 Gas Chromatography 1
st
analytical
gas-solid (adsorption) chromatography
developed by Fritz Prior
 1950 Gas Liquid Chromatography by
Martin and Anthony James; Martin won the
Nobel Prize in 1952
British chemist Archer John Porter Martin, co-
recipient, with Richard L. M. Synge, of the 1952
Nobel Prize in chemistry, "for their invention of
partition chromatography."
History of the Main Techniques
 1966 HPLC named by Csaba
Horvath, but didn’t become a
popular method until 1970s
 1950s Ion-Exchange
chromatography declassified this
technique
 1970s Ion Chromatography was
developed by Hamish Small and
co-workers at the Dow Chemical
company
 1930s Affinity Chromatography
was developed for the study of
enzymes and other proteins
2
Chromatography

A technique exploiting the interaction of the
components of a mixture with a stationary phase
and a mobile phase (solvent) in order to separate
the components.
Components are separated by different levels of
adsorption to the stationary phase and solubility
in the the mobile phase.
Principles of Chromatography
 Chromatography is used when there is a difference in the retention times of
different components
 Two types of phases
1) Stationary phase
2) mobile phases
 Properties of Chromatographic Properties
1) immiscible stationary and mobile phases
2) an arrangement where a mixture is deposited at one end of the
stationary phase
3) flow of the mobile phase towards the other end of the stationary
phase
4) different rates of partitioning for each component
5) means for visualizing the separation of each component
Column Chromatography
Gas Liquid Chromatography (GLC)
Gas Liquid Chromatography (GLC)
High Performance Liquid Chromatography (HPLC)
High Performance Liquid Chromatography (HPLC)
Paper Chromatography and Thin Layer Chromatography (TLC)
Types of Chromatography
Chromatography
 There are two basic types of

chromatography
 Gas
 Liquid
 Liquid includes TLC and high performance
liquid chromatography (HPLC)
Thin-layer chromatography
 TLC is a form of liquid chromatography
consisting of:
 A mobile phase (developing solvent) and
 A stationary phase (a plate or strip coated with a
form of silica gel)
 Analysis is performed on a flat surface under
atmospheric pressure and room temperature
Principles of TLC
 TLC is one of the simplest, fastest, easiest
and least expensive of several
chromatographic techniques used in
qualitative and quantitative analysis to
separate organic compounds
 Michael Tswett is credited as being the father
of liquid chromatography. Tswett developed
his ideas in the early 1900’s.
3
TLC
 The two most common classes of TLC are:
 Normal phase
 Reversed phase
Normal Phase
 Normal phase is the terminology used when
the stationary phase is polar; for example

silica gel, and the mobile phase is an organic
solvent or a mixture of organic solvents which
is less polar than the stationary phase.
Reversed Phase
 Reversed phase is the terminology used
when the stationary phase is a silica bonded
with an organic substrate such as a long
chain aliphatic acid like C-18 and the mobile
phase is a mixture of water and organic
solvent which is more polar than the
stationary phase.
Adsorbents for TLC
 Silica gel
 Silica gel-F (Fluorescing indicator added)
 Magnesium Silicate (Florisil)
 Polyamides
 Starch
 Alumina
Steps in TLC Analysis
 The following are the important components
of a typical TLC system:
 Apparatus (developing chamber)
 Stationary phase layer and mobile phase
 Application of sample
 Development of the plate
 Detection of analyte
Thin Layer (and Paper) Chromatography
TLC plates are inert supports (glass, plastic, aluminium)
with a thin veneer of chromatographic media (silica,etc…)
 Apply a concentrated drop of sample (•)

with a capillary or dropping tube to
bottom of plate (origin pencil line)
•
•
•
•
• Stand plate in a sealed vessel.
• carefully add solvent
(keep solvent level below sample).
• Allow solvent to adsorb up the plate,
drawing the sample with it.
4
Thin Layer (and Paper) Chromatography
•
•
•
•
The ratio of distance travelled by the component (from
origin) compared
with the distance travelled by the
solvent front (from origin) is called the R
f
value.
Solvent front
Solvent front
x
a
b
c
R

f
of = a/x
R
f
of = b/x
R
f
of = c/x
R
f
of = a/x
R
f
of = b/x
R
f
of = c/x
Thin Layer and Paper Chromatography
A solution of a mixture is applied as a spot/band at the
bottom of the plate and allowed to travel with the solvent
up the plate.
A B C A+B+C
standards
Mixed
standards
Unknown +
standards
•
•
•

•
•
•
•
•
•
•
•
•
A+B+C
?
?
standards
Mixed
standards
Unknown +
standards
A B C A+B+CABC A+B+CA+B+CABC
Column Chromatography
 Similar to thin layer
chromatography
 Stationary phase =
silica gel on support
 Mobile phase = liquid solvent
 In column chromatography,
this stationary phase is
packed into a vertical glass
column.
 Mobile phase moves down
the column as a result of

gravity.
Column Chromatography
 Blue compound = more polar
 Adsorb more to the
silica gel
 Elutes slower
 Yellow compound =
less polar
 Spends much of its
time in the mobile
phase
 Elutes faster
 Example of column chromatography separation:
HPLC Introduction:
 HPLC = improved form of column chromatography
 Instead of the mobile phase moving through the
column as a result of gravity, it is forced through the
column under high pressure.
 Typical operating pressures: 500-6000psi
 To get improved separation – smaller sized packing
material is required (<10µm).
 Smaller packing = greater resistance to flow
 Low flow rate = solute diffusion
 Higher pressures needed to generate the needed solvent
flow
 Gravity is too slow- high pressure greatly speeds up the
procedure.
1903: Russian botanist Mikhail Tswett
 Separated plant pigments through column adsorption
chromatography

 Packed open glass column with particles
 Calcium carbonate and alumina
 Poured sample into column, along with pure
solvent
 As the sample moved down the vertical column,
different colored bands could be seen.
 Bands correlated to the sample components.
 Coined the term chromatography from the Latin word
meaning “color writing”.
HPLC History
5
Early 1950s: First appearance of GC
 Almost immediately became popular.
 Work began on improving LC
1964: J. Calvin Giddings
 Published a paper entitled “Comparison of the Theoretical
Limit of Separating Ability in Gas and Liquid Chromatography”
in the journal Analytical Chemistry.
 Outlined ways to improve LC: smaller packing size, increased
pressure
 In theory, he demonstrated how LC could actually be more
efficient than GC.
 Increased number of theoretical plates
HPLC History
HPLC History
1966: Horváth
 Built the first HPLC instrument and gave it its name
 HPLC = High Pressure Liquid Chromatography.
1970’s: HPLC became popular with an increase in
technology

 Improved columns and detectors
 Production of small silica packing material
 By 1972 particle sizes less than 10µm were introduced
 This allowed for more precise and rapid separations.
 As new technology continued to develop, HPLC became more efficient.
 HPLC = High Performance Liquid Chromatography
Overview of the HPLC Process
 Mobile phase pumped through column at high pressure.
 Sample is injected into the system.
 Separation occurs as the mobile phase and sample are pumped
through the column.
 Each sample component will elute from the column, one at a time,
and will be detected by one of several possible detector types.
 The response of the detector to each component eluted will be
displayed on a chart or computer screen.
 Known as a chromatogram.
 Each compound eluted will show up as a peak on this chromatogram.
 Data processing equipment are used to analyze the data generated.
Diagram of HPLC Apparatus:
1.
The end!
Happy time now?