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Chromatography

Maria Salvacion A. Esmalla, M. Sc.


Biochemistry Department
Faculty of Pharmacy
University of Santo Tomas

Objectives
To separate the components of Capsicum frutescens / Moringa
oleifera
To perform column chromatography and thin layer chromatography
To identify the colored pigments present in Capsicum frutescens /
Moringa oleifera
To determine the polarity of the colored pigments
To compute the Rf value of the colored pigments

Plant Sources
Capsicum frutescens

Moringa oleifera

What are the pigments present in plants?

How can we separate the pigments?

Chromatography
separation of mixtures due to differences in the
equilibrium distribution of sample components
between stationary phase and mobile phase.
separation is based on the rates at which the
components of a mixture are carried through a
stationary phase by a mobile phase

Stationary vs Mobile Phase


Stationary Phase
It is fixed in place; either in a column or a planar
surface

Mobile Phase
Moves over or though the stationary phase
carrying the pigments in it

Stationary Phase: Adsorbent


porous or finely divided solids (or liquids) in
which a thin layer of molecules of some
substances can be adhered

organic
compounds
interact
with
the
adsorbents through the following types of
interactions: ion-dipole, dipole-dipole, hydrogen
bonding, dipole induced dipole, and van der
Waals forces
Silica and Alumina

the most commonly used adsorbents in liquid


chromatography are silica (SiO2) and alumina
(Al2O3)

Mobile Phase: Eluent


is a solvent or solvent system used to
carry the components of a mixture through
a stationary phase
polarity of the eluent/solvent affects the
relative rates at which compounds move
through the column

ELUTION

Chromatogram
pattern formed by substances that have been
separated by chromatography
output or
technique

result

of

the

chromatographic

Classification: Polarity of the Stationary


and Mobile Phase
Normal Phase chromatography
- stationary phase: polar
- mobile phase: least polar or non-polar
higher Rf, least polar

Reverse Phase chromatography

- stationary phase: least polar or non-polar


- mobile phase: polar
higher Rf, most polar

Types of Chromatography
Based on direction of movement of the solvent
1. ascending (through capillary action)
2. descending (due to action of gravity)

Based on concentration of the solvent system

1. Isocratic
2. Gradient

Classification: Mechanisms of Separation


Adsorption chromatography
- particles are adsorbed onto the surface of a polar
packing (solid support)
- the stationary phase is the surface of a finely divided
polar solid
- e.g. column chromatography, TLC
Partition chromatography

- a solute distributes between two immiscible phases


(gas-liquid or liquid-liquid)
- particles are separated on the components of the
solvent system

- e.g. paper chromatography

Classification of Chromatographic Methods


Column Chromatography
MOBILE PHASE
Gas

Liquid

STATIONARY PHASE

TYPE

Solid; adsorbent

Gas-solid (adsorption)

Solid; molecular sieves

Gas-solid

Liquid

Gas-liquid (partition)

Solid; adsorbent

Liquid-Solid (adsorption)

Solid; ion exchanger

Ion exchanger (ion exchange)

Solid; gel

Gel permeation (steric exclusion)

Solid; immobilized

Affinity (hydrogen bonding)


biosorbent

Liquid

Liquid-liquid (partition)

Classification of Chromatographic Methods


Planar Chromatography
MOBILE PHASE

Liquid

STATIONARY PHASE

TYPE

Paper

Paper (partition)

Solid; thin layer

Thin layer (adsorption)

Sample Preparation

Column Chromatography
one of the most useful methods for
the separation and purification of
solids and liquids when purifying
small quantities of material

adsorbent

the stationary phase is a powdered


adsorbent packed into a column
uses polarity differences to separate
components of mixture

cotton
plug

Column Chromatography
Elution with the Solvent System
solvent

the sample is subjected to two


opposing forces:
sample
mixture

adsorbent

(1) the

solubility of the sample in the


elution solvent system

(2) the

adsorption forces binding the


sample to the solid phase

Column Chromatography
Development of Chromatogram

Mr. A. R. M. Salcedo

Theoretical Elution Order


Alkanes
Alkenes

Ethers

Increasing polarity

Halogenated hydrocarbons
Aromatic hydrocarbons

(move more slowly)

Aldehydes and ketones


Esters

Alcohols
Amines
Carboxylic acids

Mr. A. R. M. Salcedo

Column Chromatography
Components of Capsicum frutescens
Stationary phase: silica gel

Mobile phase: (Eluents)


1. DCM:hexane (1:1)
2. DCM
3. DCM: methanol (1:1)

Column Chromatography
Components of Capsicum frutescens

Column Chromatography
Components of Capsicum frutescens
CAPSANTHIN (red)

CAPSORBIN (red-orange)

Column Chromatography
Components of Capsicum Frutescens
-CAROTENE (yellow)

Column Chromatography
Components of Capsicum frutescens

Fatty acid ester of capsanthin and capsorbin


contributes primarily to the deep red shade of
Capsicum frutescens

-CAROTENE responsible for the yellow pigment


of Capsicum frutescens

Column Chromatography
Components of Moringa oleifera
Stationary phase: silica gel

Mobile phase:
1. Hexane:acetone (7:3)
2. Acetone
3. Acetone: methanol (1:1)

Column Chromatography
Components of Moringa oleifera
lutein

zeaxanthine

Column Chromatography
Components of Moringa oleifera
Chlorophyll a (dark green)

Chlorophyll b (lime green)


O

Column Chromatography
Components of Moringa oleifera
Phaeophytin a & b
(dark gray and bluish gray)

Thin Layer Chromatography


- stationary phase used is in the form of thin layer, either a
sheet of paper or a slurry of the adsorbent and water
solvent front

adsorbent

spots of sample
origin

Thin Layer Chromatography


Some common uses of TLC
1. To determine the number of components in a
mixture.
2. To determine the identity of two substances.
3. To monitor the progress of a reaction.
4.To determine the effectiveness of a
purification.
5. To determine the appropriate conditions for a
column chromatographic separation.
6. To monitor column chromatography.

Thin Layer Chromatography


Steps:
1. Sample Loading/ Application

2. Equilibration
3. Development of Chromatogram

4. Visualization
5. Evaluation

Thin Layer Chromatography


Sample application
the sample is applied as a
concentrated spot with small
dimension
significant loss in resolution is a
consequence of applying too large
amount of the sample

Thin Layer Chromatography


Development of Chromatogram
- solvent rises by capillary action
up through the adsorbent

- differential partitioning occurs


between the components of the
mixture dissolved in the solvent and
the stationary adsorbent phase

Thin Layer Chromatography


Visualization Techniques

1. Sulfuric acid/heat: destructive,


leaves charred blots behind
2. Ceric stain: destructive, leaves a
dark blue blot behind for polar
compounds
3. Iodine: semi-destructive, iodine
absorbs onto the spots, not
permanent
4. UV light: non-destructive, short
wavelength (background green,
spots dark), long wavelength
(plate dark, compounds glow)

Thin Layer Chromatography


Documentation and Interpretation of Data

Rf =

Distance from origin to center of substance spot


Distance from origin to solvent front

Thin Layer Chromatography


Retention factor, Rf value
- is characteristic for any given compound on the same
stationary phase using the same mobile phase for
development of the plates
- it depends on the following parameters:
solvent system
adsorbent
amount of material spotted
temperature

Ultraviolet Spectroscopy
- used to detect the presence of a conjugated pi bond
system in a molecule
The Electromagnetic
Spectrum

Ultraviolet Spectroscopy
Observed Color
of Compound

Color of Light Absorbed

Approximate Wavelength
of Light Absorbed

Green

Red

700 nm

Blue-green

Orange-red

600 nm

Violet

Yellow

550 nm

Red-Violet

Yellow-green

530 nm

Red

Blue-green

500 nm

Orange

Blue

450 nm

Yellow

Violet

400 nm

conjugation, wavelength, frequency, energy

The End
Miracles are reserved to those
who believe according to their
faith.