CHROMATOGRAPHY

CHROMATOGRAPHY
khromatos (color) + graphia (recording or
description)
separation technique for gases, liquids and
dissolved substance
based on the different degrees of attractive
interaction of molecules to either the mobile
phase or the stationary phase

Stationary Phase is a layer or coating on the

supporting medium (column or planar
surface) which interacts with the analytes
Mobile Phase is the part of the chromatographic
system which carries the solute across the
stationary phase.

HOW DOES IT WORK?

Polarity
Hydrophobicity
Ionic interactions
Hydrogen bonding
Particle size
Structural complimentarity

CLASSIFICATION BASED ON NATURE

OF MOBILE AND STATIONARY PHASE
A. Normal
Stationary phase: POLAR
Mobile phase: NON-POLAR

B. Reverse
Stationary phase: NON-POLAR
Mobile phase: POLAR

TYPES OF CHROMATOGRAPHY
Based on direction of movement of solvent
1. Ascending (capillary action of solvent)
2. Descending (gravitational pull to the solvent)
Based on concentration of solvent
1. Isocratic
2. Gradient

CLASSIFICATION BASED ON
MECHANISM OF SEPARATION
Partition
Particles are separated on the components of the
solvent system
e.g. paper chromatography

Adsorption
Particles are adsorbed in the solid support
e.g. silica gel chromatography, thin layer
chromatography

PAPER CHROMATOGRAPHY

THIN LAYER CHROMATOGRAPHY (TLC)

RETENTION FACTOR (Rf)

Distance travelled be solute divided by
distance travelled by solvent
quantitative measure of affinity to the mobile
phase

COLUMN CHROMATOGRAPHY

HIGH PERFORMANCE/PRESSURE
LIQUID CHROMATOGRAPHY

GAS CHROMATOGRAPHY

CHROMATOGRAM

ION EXCHANGE CHROMATOGRAPHY

GEL FILTRATON/SIZE EXCLUSION

CHROMATOGRAPHY

AFFINITY CHROMATOGRAPHY

AFFINITY CHROMATOGRAPHY

Siling Labuyo
(Capsicum frutescens)

EXTRACTION OF
PIGMENTS

Malunggay
(Moringa olifeira)

Column Chromatography
DCM-Hexane
DCM
DCM-MetOH

Hexane-Acetone
Acetone
Acetone-MeOH

Eluate 1

Eluate 2

Eluate 3

INCREASING POLARITY

Eluate 4

STEPS IN THIN LAYER

CHROMATOGRAPHY
1.
2.
3.
4.

Preparation of the sample and TLC plate

Blotting/Spotting of samples
Developing
Visualization

THIN LAYER CHROMATOGRAPHY (TLC)

For column chromatography, give the order of

elution of the pigments of siling
labuyo/malunggay.

For thin layer chromatography, arrange the

pigments of siling labuyo/malunggay in terms of
increasing Rf values.

THE COLUMN
Silica gel (silicon dioxide)
Polar
Stationary Phase

PIGMENTS IN SILING LABUYO

Carotene (yellow)

Xanthophyll
Capsanthin (red)

Cabsorubin (red-orange)

PIGMENTS IN MALUNGGAY
Xanthophyll (yellow)
Lutein
Zeaxanthin

Chlorophyll (green)
Chlorophyll a (dark green)
Chlorophyll b (lime green)

Phaeophytin
Phaeophytin a (dark gray)
Phaeophytin b (bluish gray)