High Performance Liquid Chromatography

1. Introduction to Chromatography & High Performance
Liquid Chromatography
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2. Chromatography: Chromatography is a laboratory
technique for the separation of a mixture. The mixture is
dissolved in a fluid (gas, solvent, water...) called the mobile
phase, which carries it through a system( a column , a
capillary tube, a plate or a sheet) on which is fixed a material
called the stationary phase.

3. Starting material Reaction mixture
Is there only
one
product?
How do I
know !!!
After some
time
In a reaction,
• May form different byproduct.
• Starting material may left (not 100% conversion).
• Different isomer may form.

4. Processes of separation
1. Extraction (depending upon
solubility in different solvent)
2. By Crystallization method
3. Chromatography: a) Gas chromatography
b) Liquid chromatography
Thin layer chromatography
(TLC)
Paper chromatography Column chromatography HPLC

5. 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.
Thin layer chromatography (TLC) is an important technique for
identification and separation of mixtures of organic compounds. It is
useful in -
• Identification of components of a mixture (using appropriate standards)
• following the course of a reaction,
• analyzing fractions collected during purification,
• analyzing the purity of a compound.
Michael Tswett is credited as being
the father of liquid chromatography.
Tswett developed his ideas in the
early 1900’s.
THIN LAYER CHROMATOGRAPHY

6. As the mobile phase rises up the TLC plate by
capillary action, the components dissolve in the
solvent and move up the TLC plate.
Individual components move up at different rates,
depending on intermolecular forces between the
component and the silica gel stationary phase and the
component and the mobile phase.
THIN LAYER CHROMATOGRAPHY
The stationary phase is SiO2 and is very “polar”.
It is capable of strong dipole-dipole and H-bond donating and accepting interactions
with the “analytes” (the components being analyzed).
More polar analytes interact more strongly with the stationary phase in move very
slowly up the TLC plate.
By comparison, the mobile phase is relatively nonpolar and is capable of interacting
with analytes by stronger London forces, as well as by dipole-dipole and H-bonding.
More nonpolar analytes interact less strongly with the polar silica gel and more
strongly with the less polar mobile phase and move higher up the TLC plate.

7. • Silica gel
• Silica gel-F (Fluorescing indicator added)
• Magnesium Silicate (Florisil)
• Polyamides
• Starch
• Alumina
Adsorbents for TLC
THIN LAYER CHROMATOGRAPHY
Absorption of Solutes
The adsorption strength of compounds increases with increasing polarity of
functional groups, as shown below:
-CH=CH2, -X, -OR, -CHO, -CO2R, -NR2, -NH2, -OH, -CONR2, -CO2H.
(weakly adsorbed) (strongly adsorbed)
(nonpolar) (more polar)
Mobile phase for TLC
Depending on the polarity of the analyte
solvet has to be used.
Polar solvents- MeOH, AcOH etc
Non polar solvent- hexane, CHCl3 etc.
Mixture of solvent can be used. Eg-
hexane/EtOAc, MeOH/CHCl3 etc.

8. Silica Gel
• Silica gel is the most common adsorbent used
in TLC
• Silica gel is a polymer based on Silica to
oxygen linkages with many -hydroxyl groups
extending from this matrix
Si-O-Si-O-Si-O-Si-O-Si-O-Si-(OH)x
• They are very porous and have large
surface area
• The mode of separation is generally by adsorption or partition
• The more polar components will be adsorbed preferentially by the polar layer
• Hydrogen Bonding is the main force controlling adsorption between the silica
gel surface and the analyte functional groups
THIN LAYER CHROMATOGRAPHY
Principles -

9. THIN LAYER CHROMATOGRAPHY
Procedure:
2. Mark the plate
3. Apply the sample
4. Prepare the mobile phase
5. Develop the plate
1. Prepare a plate or select a plate
6. Detection of analyte

10. Visualization and Interpretation
• Most of the compound will not be visible to the naked eye.
• Spots can be visualized by two basic techniques:
– Ultraviolet light at 254 nm (shortwave UV). Long wave UV (340 nm) is used less
commonly.
– Staining to make spots visible
THIN LAYER CHROMATOGRAPHY
Place the TLC strip under the UV light. Room light
should be eliminated as much as possible.
If a silica gel F plate is used, the sample spots will
appear as black spots on a fluorescent green
background
Using UV:
• If the compounds have color
ie, visualized in naked eye, no
need of external thing.

11. THIN LAYER CHROMATOGRAPHY
Staining-
Zones may be made visible by staining or spraying the TLC plate
(strip) with a visualization reagent. Several different reagents can be
used. Not all will visualize your sample zone.
Stain solutionNot detectable by Naked eye After staining
You must have the correct visualization reagent to be able to view your spots
A universal visualization reagent is a 10%
sulfuric acid solution. When sprayed on your
plate, the plate is heated and your spots are
charred which can be seen by eye. Glass plate
only. Several details may need to be worked out
with this reagent.

12. THIN LAYER CHROMATOGRAPHY
Rf =
Distance travels by a particular spot
Distance travels by Solvent front
For Compound A,
Rf =
a
s
For Compound B,
Rf =
b
s
Rf reflects the comparative polarity of the spotted compounds

13. THIN LAYER CHROMATOGRAPHY
Calculation of Rf’s
The Rf is defined as the distance the center of the spot moved divided by the distance the
solvent front moved (both measured from the origin)
A B CU
x xx x
Solvent Front
Origen
Distance solvent
migrated = 5.0 cm
Distance A
migrated = 3.0 cm
Distance B
migrated = 2.0 cm
Distance C
migrated = 0.8 cm
0.8 cm
3.0 cm
Rf (A) =
Rf (B) =
Rf (C) =
Rf (U1) =
Rf (U2) =
2.0 cm
5.0 cm
= 0.40
= 0.60
= 0.16
= 0.60
= 0.16
3.0 cm
5.0 cm
0.8 cm
5.0 cm
3.0 cm
5.0 cm
0.8 cm
5.0 cm
D
x
Rf (D) = = 0.804.0 cm
5.0 cm
4.0 cm

14. THIN LAYER CHROMATOGRAPHY
Calculation of Rf’s
The Rf is defined as the distance the center of the spot moved divided by the distance the
solvent front moved (both measured from the origin)
A B CU
x xx x
Solvent Front
Origen
Distance solvent
migrated = 5.0 cm
Distance A
migrated = 3.0 cm
Distance B
migrated = 2.0 cm
Distance C
migrated = 0.8 cm
0.8 cm
3.0 cm
Rf (A) =
Rf (B) =
Rf (C) =
Rf (U1) =
Rf (U2) =
2.0 cm
5.0 cm
= 0.40
= 0.60
= 0.16
= 0.60
= 0.16
3.0 cm
5.0 cm
0.8 cm
5.0 cm
3.0 cm
5.0 cm
0.8 cm
5.0 cm
D
x
Rf (D) = = 0.804.0 cm
5.0 cm
4.0 cm

15. • Low cost
• Short analysis time
• Ease of sample preparation
• All spots can be visualized
• Sample cleanup is seldom necessary
• Adaptable to most pharmaceuticals
• Uses small quantities of solvents
• Requires minimal training
• Reliable and quick
• Minimal amount of equipment is needed
THIN LAYER CHROMATOGRAPHY
Advantages of TLC

16. COLUMN CHROMATOGRAPHY
(CC)

17. Usually a preparative technique rather than analytical
Usually silica used (range pH:2-8), particle diameter 40-60 m
Use alumina if basic solution is required.
Adsorption solvent strength parameter:
εo = measure of adsorption energy / unit area of solvent.
Admits factors other than polarity. Can choose weak and
strong solvent and mix to get required strength. Non linear
effect! Solvents must be miscible.
Van der Waals,
ionic & dipolar
interactions 
adsorption of
polar species
Vary polarity of MP to increase
or decrease time polar solutes
spend on SP  separation.
Polar surface = SP
MP ~ non-polar
Chromatographic
strength of solvent:
how fast it moves
analytes. For silica:
more polar  more
strength.
Column Chromatography
Si
O
O
O
O
Si
O
OH
Si OH
O

18. Dry pack
Voids form as silica packs more
tightly after introduction of
solvent
Avoid!
Keep column “wet” when stored
Slurry pack
Put silica in conical flask, add just enough
solvent to make a very thick slurry.
Pour into column as evenly as possible.
Fractions
Separation into
components
Mixture of
components
Mobile phase
added continuously
Acid washed sand
Glass wool
Silica
Sand
Column Chromatography

19. Carbowax (polyethylene glycol)
C18 (hydrocarbon coated silica) – reverse phase
Paper
Cellulose
Starch
Calcium sulfate
Silica
Florosil (magnesium silicate)
Magnesium oxide
Alumina (aluminium oxide)
Activated carbon
INCREASINGPOLARITY stationary phase polarity
Column Chromatography

20. Helium
Nitrogen
Petroleum ether (pentanes)
Ligroin (hexanes)
Cyclohexane
Carbon tetrachloride
Toluene
Chloroform
Dichloromethane
t-butyl methyl ether
Diethyl ether
Ethyl acetate
Acetone
2-propanol
Pyridine
Ethanol
Methanol
Water
Acetic acid
Mobile phase
polarities
Column Chromatography
INCREASINGPOLARITY

21. Alkane hydrocarbons
Alkenes (olefins)
Aromatic hydrocarbons
Ethers
Esters
Ketones
Aldehydes
Amines
Alcohols
Phenols
Carboxylic acids
INCREASINGFUNCTIONAL
GROUPPOLARITY
Elution sequence by functional group on silica or
alumina (polar) TLC & Column Chromatography
FAST
SLOW
Column Chromatography

22. TLC - Optimizing for column chromatography
Optimum: 0.2 < Rf < 0.5

23. COLUMN CHROMATOGRAPHY
* Column chromatography (CC) is an extremely
valuable technique for purification of synthetic or
natural products.
* Compounds are separated by CC through the same
mechanism as TLC; through differential
intermolecular forces between the components of the
mixture with the mobile phase, and between the
components with the stationary phase.
* A variety of adsorbents can be used as the stationary
phase; silica gel (which is very polar) is most
commonly used in organic chemistry.

24. Polar components (b) adsorb more strongly to the polar silica and elute after
the less polar components (a), which move more quickly with the non-polar
(relative to silica) solvent.
COLUMN CHROMATOGRAPHY

25. COLUMN CHROMATOGRAPHY

26. HPLC
High performance liquid Chromatography
High Pressure Liquid Chromatography

27. • HPLC is a form of liquid chromatography used to separate
compounds that are dissolved in solution. HPLC instruments consist
of a reservoir of mobile phase, a pump, an injector, a separation
column, and a detector.
• Compounds are separated by injecting a sample mixture onto the
column. The different component in the mixture pass through the
column at differentiates due to differences in their partition behavior
between the mobile phase and the stationary phase. The mobile
phase must be degassed to eliminate the formation of air bubbles.
 This technique is used for chemistry and biochemistry research analyzing complex
mixtures, purifying chemical compounds, developing processes for synthesizing
chemical compounds, isolating natural products, or predicting physical properties. It is
also used in quality control to ensure the purity of raw materials, to control and improve
process yields, to quantify assays of final products, or to evaluate product stability and
monitor degradation.
Uses of HPLC-
 In addition, it is used for analyzing air and water pollutants, for monitoring materials that
may jeopardize occupational safety or health, and for monitoring pesticide levels in the
environment. Federal and state regulatory agencies use HPLC to survey food and drug
products, for identifying confiscated narcotics or to check for adherence to label claims.

28. Components

29. • Higher resolution and speed of analysis
• HPLC columns can be reused without repacking or regeneration
• Greater reproducibility due to close control of the parameters
affecting the efficiency of separation
• Easy automation of instrument operation and data analysis
• Adaptability to large-scale, preparative procedures
• Advantages of HPLC are result of 2 major advances:
– stationary supports with very small particle sizes and large
surface areas
– appliance of high pressure to solvent flow
• Separation of enantiomers are possible with ease.
Advantages to HPLC