EXPERIMENT 4

CHROMATOGRAPHIC
ANALYSIS

O R G A N I C C H E M I S T R Y
CHROMATOGRAPHY
a versatile technique used for the examination and chemical
separation of complex mixtures

separation is based upon the rates a which the components of a

mixture are carried through a stationary phase by a mobile phase

basically involves the separation of mixtures due to differences in the

distribution coefficient (equilibrium distribution) of sample components
between 2 different phases.
USES OF CHROMATOGRAPHY

Qualitative Analysis

for recognizing the presence or absence of components

in mixture

Quantitative Analysis

determining the amount of each component in the

mixture
THE STATIONARY and MOBILE PHASE

STATIONARY PHASE

is a phase that is fixed in place either in a column or on a

planar surface

MOBILE PHASE

is a phase that moves over or through the stationary phase,

carrying the analyte with 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
ELUENTS/SOLVENTS
Petroleum ether
Eluting Solvents for Cyclohexane
Liquid Chromatography
Carbon tetrachloride
Toluene
Benzene
Methylene chloride
Increasing eluting power
Chloroform
(More powerful eluents) Ethyl ether (anhydrous)
Ethyl acetate (anhydrous)
Acetone (anhydrous)
Propanol
Ethanol (anhydrous)
Methanol (anhydrous)
Water
Acetic acid
THE CHROMATOGRAM
pattern formed by substances that have been separated by
chromatography
output or result of the chromatographic technique
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
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

the Rf value for each spot is calculated as:

Distance from origin to center of substance spot

Rf =
Distance from origin to solvent front
THIN LAYER CHROMATOGRAPHY
Rate of Flow, 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
 THIN LAYER CHROMATOGRAPHY
O B O C O
A
H H
OH N N

O
Aspirin, acidic OH O
Acetaminophen/paracetamol ,
weakly acidic phenacetin, non-
O
O polar
D OH O
OH F CH3
H3C N
H N
N
E O N N
A B C D E F
CH3

Caffeine, basic
Ibuprofen, acidic Mefenamic, acidic &
basic, amphoteric
 THIN LAYER CHROMATOGRAPHY
INTERACTIONS OF COMPOUNDS AND ADSORBENTS

With silica gel, the dominant interactive forces

between the adsorbent and the materials to be
separated are of the dipole-dipole type.

Highly polar molecules interact fairly strong

with the polar SiO bonds of these adsorbents
and will tend to stick or adsorb onto the fine
particles of the adsorbent while weakly polar
molecules are held less tightly.
COLUMN CHROMATOGRAPHY
one of the most useful methods for the
separation and purification of solids and
liquids when purifying small quantities of
adsobent
material

the stationary phase is a powdered

adsorbent packed into a column

it uses polarity differences to separate

components of mixture cotton
COLUMN CHROMATOGRAPHY
Elution with the Solvent System
solvent
the sample is subjected to two
opposing forces:

sample mixture
(1) the solubility of the sample in the
elution solvent system
(2) the adsorption forces binding the
adsobent sample to the solid phase
COLUMN CHROMATOGRAPHY
Development of Chromatogram
COLUMN CHROMATOGRAPHY
Components of Capsicum frutescens
COLUMN CHROMATOGRAPHY
Components of Capsicum Frutescens

CAPSANTHIN

CAPSORUBIN
COLUMN CHROMATOGRAPHY
Components of Capsicum Frutescens

LUTEIN

-CAROTENE
THEORETICAL ELUTION ORDER

Alkanes
Alkenes
Ethers
Increasing polarity Halogenated hydrocarbons
Aromatic hydrocarbons
(move more slowly) Aldehydes and ketones
Esters
Alcohols
Amines
Carboxylic acids
A mixture of the following compounds, trans-stilbene [1], benzil
[2], and cinnamic acid [3] were analyzed by TLC. Match
chromatograms A, B and C with compounds 1,2 and 3.

O
C

B [1] trans-stilbene
[2] benzil
CO2H
A

[3] cinnamic acid

A sample ink dye, P, was subjected to TLC using silica gel and
ethylacetate:methanol:acetic acid (25:1:1) as solvent system. Identify its
components using the 3 standards, r, b, and g. Compute the Rf of each
standards.

Among the 3 standards, r, b, and g, which is the most

polar? ____

Which is the least adsorbed? _____

Which is the least polar? _____

Which is the most adsorbed? _____

Probable components of P: ________________

Rf Values: Rf r =
Rf b =
p r b g

Rf g =