Chromatograph

y
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
plants?

the

pigments

present

How can we separate the pigments?

in

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
the most commonly used adsorbents in liquid
chromatography are silica (SiO2) and alumina
(Al2O3)

Silica and Alumina

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 result of the chromatographic
technique

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

STATIONARY PHASE

Solid; adsorbent

Gas-solid (adsorption)

Solid; molecular sieves

Liquid

TYPE

Gas-solid

Gas-liquid (partition)

Liquid Solid; adsorbent

Liquid-Solid (adsorption)

Solid; ion exchanger Ion exchanger (ion exchange)

Solid; gel

Gel permeation (steric exclusion)

Solid; immobilized
biosorbent
Liquid

Affinity (hydrogen bonding)

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
separate components of mixture

to

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
(move more slowly)

Halogenated hydrocarbons
Aromatic hydrocarbons
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

Blue-green

700 nm

Orange-red

Violet

600 nm

Yellow

Red-Violet

550 nm

Yellow-green

Red

530 nm

Blue-green

Orange

500 nm

Blue

Yellow

450 nm

Violet

400 nm

conjugation, wavelength, frequency, energy

The End
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believe according to their faith.