This document provides an overview of chromatography techniques. It begins with a brief history of chromatography from 1903 to 1965. It then discusses the importance of chromatography and its applications in various fields. The document defines chromatography and describes various techniques including thin layer chromatography, gas chromatography, high performance liquid chromatography, and ion exchange chromatography. It explains the theoretical bases and factors affecting chromatographic separation. Finally, it discusses terminology, applications, and detection methods in chromatography.
2. HISTORY
Chromatography
(from Greek: chromatos -- color , "graphein" -- writing)
• 1903 Tswett - plant pigments separated on chalk columns
• 1931 Lederer & Kuhn - LC of carotenoids
• 1938 TLC and ion exchange
• 1950 Reverse phase LC
• 1954 Martin & Synge (Nobel Prize)
• 1959 Gel permeation
• 1965 instrumental LC (Waters)
3. Importance
Chromatography has application in every branch of
the physical and biological sciences
12 Nobel prizes were awarded between 1937 and
1972 alone for work in which chromatography
played a vital role
5. Chromatography is a physical method of separation in which the
components to be separated are distributed between two phases,
one of which is stationary while the other moves in a definite
direction
Definition
Stationary phase
Mobile phase
Solid
Liquid (supported on a solid or gel)
Liquid
Gas
6. Theoretical bases of chromatography
Distribution equilibrium:
-Substances are eluted from the chromatographic column in
inverse order of their distribution coefficients with respect to
the stationary phase.
-The distribution of solutes between two phases is governed by an
equilibrium constant known as distribution coefficient, K (or
partition coefficient in certain types of chromatography).
7. Larger K value
K = Cstationary / Cmobile
Cstationary = concentration of solute in stationary phase
Cmobile = concentration of solute in mobile phase
More time is spent in the stationary phase
Smaller K value
The solute will be eluted very fast with the mobile phase
8. 1-Velocity of the mobile phase
2-The ratio of volume of the stationary phase to the volume of
the mobile phase
3-The value of the distribution coefficient (it is characteristic for
each component)
Rate of travel
Factors limiting rate of travel:
Determination of the rate of travel:
1- By measuring the distance travelled by each solute after a
fixed time (planar chromatography)
2- By measuring the time interval at which each component
appears after a fixed distance (column chromatography)
10. Development
It is the process by which chromatographic
separation takes place by allowing the mobile phase
to pass through the stationary phase
Elution
Removal of adsorbed solutes from stationary phase
using Eluent
Eluate
Fraction of the mobile phase that comes out the
column containing the solutes
11. Effluent Any solvent that passes out the column
Retention
volume
It is the volume of the mobile phase that comes
out the column before elution of any solutes
Retention
time (Rt)
Denotes the time needed before any solute comes
out of the column
13. A- According to
the separation theory
B-According to the method of
holding the stationary
phase
C- According to the
development procedure
D- According to the purpose
of use
Classification of
chromatographic
techniques
14. A- According to the mechanism of separation
(separation theory)
Adsorption Partition
Ion exchange
Molecular exclusion
( Gel permeation , Gel filtration)
Affinity chromatography
18. Adsorption chromatography
Adsorption is a surface phenomenon
adsorptive
(solute)
Sorbent (St.phase)
In adsorption, the
concentration at the
interface solid stationary
phase/ mobile phase is
higher than in the
surrounding medium
More polar molecules, will
be more strongly adsorbed
(eluted more slowly)
22. Alumina
Commercial alumina is available
as: neutral, basic or acid alumina
Used for separation of sterols
and vitamins
Not used for separation of
phenolics or carboxylic acids
23. Silica gel or Silicic acid
The adsorptive properties depend on the hydroxyl
groups attached to silicon atoms which interact with
polar or unsaturated molecules by hydrogen bonding
Not heated silica (not activated) have variable
amounts of physically adsorbed water. It acts by
partition
Heating silica removes this
water without loss of surface
hydroxyls. This treatment
gives adsorbent of maximum
activity
Used for column and TLC
24. The Ideal adsorbent must fulfill the following
requirements:
1- Selection of the
adsorbent
(stationary phase)
Inert to the
solutes
Colourless
Suitable
particle size
Insoluble in
the mobile
phase
25. Fine particle size slowing rate of elution
Suitable particle
size
great surface
area
efficient
adsorption
26. Strong eluents (more polar solvents)
decrease adsorption while weak eluents
increase it.
Elution of the column can be carried out
using solvents of increasing polarity
Mobile phase:
i-Selection of the solvent:
27. A B CMobile phase A:
is a solvent (not adsorbed)
Mobile phase B:
is a developer more affinity to
the adsorbent than the solvent
but less than the solutes (it
allows the solutes to move)
Mobile phase C:
Eluent
It has more eluting power than the
developer and can take solutes out
of the surface of the adsorbent.
D
Mobile phase D:
Displacer
It is the strongest mobile phase and
is more strongly adsorbed than
solutes.
28. Factors affecting column efficiency
and chromatographic separation
• Decreasing the particle size of the adsorbent
results in good separation, but very small
particles will offer considerable resistance to
flow
1-Particle size of the
supporting medium
• The column efficiency is improved as the
length /width ratio of the column is increased
2-Column dimensions
• Irregular packing results in uneven and
irregular movement of solvent front and less
uniform zone formation
3-Uniformity of
packing of the column
29. • As temperature is increased, the elution
speeds up as the adsorption is generally
reduced at higher temperatures
4-Column
temperature
• Uniform and low flow rate will result in a
satisfactory separation and more uniform
zone formation than a fast flow rate
5-Solvent flow rate
• It is not recommended to interrupt the
experiment and continue later
6-Constancy of flow
30. • The flow rate is inversely proportional to
the viscosity, low viscosity solvents result
in high efficiency separation
7-Selection of
solvents
• Highly concentrated solution results in
rapid movement through the column
8-Concentration
32. 1. Wet packing
The adsorbent + the first solvent to be used in the
separation =(slurry), is gradually added to the column.
The packing is allowed to settle between additions
The suspending liquid is allowed to flow out slowly (the
liquid level in the column is maintained above the
packing at all times)
Advantages: Homogenous columns
Disadvantages: Air bubbles formation and uneven
separation of zones
33. 2. Dry packing
The adsorbent is added in portions with vibrations between
additions. The process is repeated until the column is
adequately filled
The column is then washed carefully with the first solvent
34. iv.Detection of the sample components
On-column detection
Examination of the separated
zones on the column in either
• visible light (for colored
substances)
• or in UV light (for
fluorescent compounds)
Out-of column detection
Collecting the eluate in fractions
from the whole column, then
testing each of the eluted
fractions for the presence of the
components.
This can be carried out by using
• Thin layer (TLC)
• or paper chromatography
(PC).
• The process is called
monitoring the fractions
35. Applications
Isolation and purification of:
• vitamins and hormones
• alkaloids of Cinchona, Ergot, Opium and Nux- vomica
• cardiac glycosides from Digitalis
• anthraquinones from Senna species
Purification of tincture of alkaloids from pigments before
determination of their alkaloidal content
37. Partition chromatography
The separation of the components of a mixture is dependent
on differences in the partition coefficients of the
components between two immiscible liquids (or liquid and
gas)
The liquid stationary phase is adsorbed on an
inert support, which may be either
packed in a chromatographic tube (column
partition chromatography)
or layered on a glass plate (TLC) or in the
form of sheets of paper (PC)
Inert
support
Liquid st.
phase
38. stationary phase may be
Liquid
mobile
phase
Liquid / Liquid
Chromatography
(LLC) which flows
through the
stationary phase in a
counter-current
manner
liquid Gas
Gas
mobile
phase
Gas / Liquid
Chromatography
(GLC) which flows
through the
stationary phase in a
counter-current
manner
39. Paper chromatography
Stationary phase: is a specified type
of paper formed of cellulose
Mechanism of separation:
partition chromatography.
Applications: It is the method of
choice for the fractionation of
some groups of substances, e.g.
flavonoids, sugar samples.
41. G X L U
After drying and spraying
Retardation factor: It is the
ratio between the distance
traveled by solute to that
traveled by the solvent (Rf).
It has values less than one.
42. General techniques for paper
chromatography
i. Ascending technique
ii. Descending techniques
43. • The samples are applied at a small
distance from the bottom edge of the
paper.
• The mobile phase is poured in the
bottom of the chromatographic jar and
is allowed to flow upward.
Advantages:
Any well-closed chamber e.g. test tubes,
cylinders of flasks may be used as
chromatographic jars.
Disadvantages:
The upward flow of the solvent is against
gravity and becomes very slow after a
certain height is reached.
i. Ascending development (ascending chromatography)
44. ii. Descending technique
The paper is held with its upper end in the solvent trough and passes
upward and over an, which suspends it away from the edges of the
trough. The other longer end hangs free in the jar. The starting line is
situated at 1 to 1.5 cm below the glass rod
The solvent is allowed to flow down the paper till its front becomes
near the lower end
Advantages:
There is no gravity resistance to flow, as it is downward
45. Thin layer chromatography
It is a type of planar
chromatography (such as
paper chromatography)
A thin layer of finely divided
adsorbent supported on a
glass, plastic or aluminium
sheet is used
Separation of
plant pigments
46. Applications
Mechanism of separation
Separation of lipophilic compounds
e.g. terpenoids,steroids and alkaloids
It is based on the two mechanisms of
chromatographic separation: adsorption and
partition
47. 1-Inexpensive
( c.f. instrumental
analysis).
4-Flexibility of choice of
mobile, stationary phases,
spray reagents (conc. and
drastic reagents can be
used)2-Requires little
training or
knowledge of
chromatography
3-Easy scale-up to
preparative mode
with quick isolation
of milligrams to
gram amount
5-A large number of samples
may be analyzed or
separated simultaneously
Advantages of TLC
48. Spot detection (location or visualization)
1-Physical methods
visualization of the spots in
the visible or UV light
2-Chemical methods
spraying with certain reagents
coloured or fluorescent spots
49. Iodine solution or vapor
or modified Dragendroff's
reagent for Alkaloids
Aluminum chloride
for Flavonoids
Antimony trichloride
in CHCl3 for Steroids
Alkali for
anthraquionone
derivatives
Ninhydrin for
Amino acids
Ferric chloride
for Phenols
Spray reagents
50. Gel Chromatography
This type of chromatography lacks
an attractive interaction between
the stationary phase and solute.
It separates the molecules according
to their size.
The molecules are eluted in the
order of decreasing particle size
smaller molecules enter into the gel
pores, pass slowly.
larger molecules pass through the
column at a faster rate than the
smaller ones.
Mechanism of separation
51. Cross linked dextrans “Sephadex”
Cross linked polyacrylamide
Cross linked agarose (Sepharose) characterized by
very large pores used to separate very large
molecules
Mobile phase : A Buffer
Column packing (stationary phase):
Spherical porous beads of definite size
52. This technique is limited to natural products
that carry charge on their functional groups
Separation is achieved by differences in affinity between :
• ionic components of the mixture and
• the stationary phase(the bond strength is different)
Ion exchange chromatography
Mechanism of separation
Applications
Stationary Phase: Ion Exchange Resins
Mobile Phase: An aqueous buffer of certain ionic
strength
54. According to the nature
of the solid support
According to the nature
of the charged groups
Polystyrene
Cation exchangeAnion exchange
weak strong weak strong
3ry amines
Quaternary
ammonium
salts
Carboxylic
acids
Sulfonic
acid resin
Properties of ion exchangers
( Resins )
Cellulose
Dextrans
56. Gas chromatography is a chromatographic technique that
can be used to separate volatile organic compounds
Gas chromatography
GC instruments
57. 1. The injection port
2. Columns
Packed columns Capillary columns
61. Example of GC chromatogram
Chromtogram: It Is a plot of detector signal (absorbance, fluorescence,
refractive index etc… versus retention time of solutes in a chromatographic
column. The area under the peak is related to solute concentration i.e.
essential for quantitative analysis).
62. High performance liquid chromatography (HPLC(
HPLC is widely used separation technique for analysis and
isolation of bioactive natural products.
Applications
65. Stationary phases
• Normal-phase partition chromatography
• Reverse-phase partition chromatography
• Ordinary Stationary phase as silica or
alumina
66. Different modes of mobile phase run
• Constant mobile phase
composition
• The strength (polarity) of the
mobile phase is increased in
increments by raising the
amount of organic solvent
fraction.
68. Advantages of HPLC
The versatility of stationary phases made
it popular method for bioassay-guide
isolation
Run samples, print out chromatograms
and spectra automatically
Different mode of mobile phase run
(gradient, isocratic)
Can range-up to preparative scale
69. It can be used for the majority of natural compounds that are
soluble in organic solvents and can be adapted to ion exchange
for isolation of highly polar compounds
Excellent separating power
Reproducibility of results
Advantages of HPLC (cont.)
70. It is expensive
( preparative one is more expensive)
Preparative column have short life
Needs high purity solvents (HPLC grade)
Disadvantages of HPLC
71. Example of HPLC application for separation of
natural products: