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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
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
Mobile Phase
Moves over or though the stationary phase
carrying the pigments in it
ELUTION
Chromatogram
pattern formed by substances that have been
separated by chromatography
output or result of the chromatographic
technique
Types of Chromatography
Based on direction of movement of the solvent
1. ascending (through capillary action)
2. descending (due to action of gravity)
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)
TYPE
Gas-solid
Gas-liquid (partition)
Liquid-Solid (adsorption)
Solid; immobilized
biosorbent
Liquid
Liquid-liquid (partition)
Classification of Chromatographic
Methods
Planar Chromatography
MOBILE PHASE
Liquid
Paper
Paper (partition)
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
to
cotton
plug
Column Chromatography
Elution with the Solvent System
solvent
adsorbent
(1) the
(2) the
Column Chromatography
Development of Chromatogram
Mr. A. R. M. Salcedo
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
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)
Column Chromatography
Components of Moringa oleifera
Phaeophytin a & b
(dark gray and bluish gray)
adsorbent
spots of sample
origin
Rf =
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
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
The End
Miracles are reserved to those who
believe according to their faith.