CHAPTER 7

BASIC CHROMATOGRAPHY
 Learning Outcomes
At the end of the chapter, student should be able to explain:
INTRODUCTION OF
CHROMATOGRAPHY
CHROMATOGRAPHY is Technique employed for separation
of the components of mixture by continuous distribution of the
components between 2 phases.
CLASSICAL SEPARATION METHODS
• Masking
• Precipitation
• Distillation
• Solvent extraction
• Ion exchange
• Chromatography
• Electrophoresis
CHROMATOGRAPHY

FUNCTION
 The stationery phase can be paper, silica,
coated silica and derivatized silica.

The mobile phase can be a gas or a liquid

( organic solvent or water)
 Illustration of Chromatography Seperation,phases
so 2 diff mobile

Ada 4 diff components - YG RINGAN PI DULU, BERAT LMBT GERAK

Red, blue, yellow, black - IF MP IS POLAR SO YG KELUAR DULU IS MORE POLAR
 INTERACTION BETWEEN SOLUTES,
STATIONERY PHASE, MOBILE PHASE

Differences in the interaction between solutes and stationery and

mobile phase enable separation
 USES OF CHROMATOGRAPHY (TECHNIQUE)

Chromatography is used by scientists to:

• Analyze
– examine a mixture, its components, and their relations to one
another.
• Identify
– determine the identity of a mixture or components based on
known components.
• Purify
– separate components in order to isolate one of interest for
further study.
• Quantify
– determine the amount of the a mixture and/or the components
present in the sample.
PRINCIPLE OF CHROMATOGRAPHY

• techniques use to separate the components of

a mixture based on the rates at which they are
carried through a stationary phase by gaseous
or liquid phase known as mobile phase.
HOW TO MEASURE RATE
 Terms:

Signal

X-axis - retention time

Y-axis – signal corresponding to the
response created by the analytes existing
in the system
R.T.
Types of chromatography on the basis of
interaction of the analyte with stationary
phase
 1) ADSORPTION

Nak tau adsorbtivity refer polarity components towards SP

 ADSORPTION
CHROMATOGRAPH
Y

grey
Stationary
phase: solid
Mobile
phase: liquid
Pink = surface of stationery.
Analyte attracted to the surface

Black dot

solute

Black dot attracted on surface, so we

called ADSORBTION.
o Example:
1. Thin-layer chromatography (TLC)
- stationary phase: plane (a solid
supported on an inert plate) TLC paper solid
- mobile phase: liquid (solvent) MP yg letak dlm bekas

2. Column chromatography
- Stationary phase: silica gel (solid)
- Mobile phase: solvent (liquid)

3. Gas solid chromatography

- Stationary phase: solid
- Mobile phase: gas
• Partition = The distribution (by solving) the component
between 2 immiscible phase. Separation of the components
will based on relative solubility of the components in the
mobile phase and stationery phase. (Paper chromatography)

• Adsorption = A chromatography where the analytes separated

based on their different degree of adsorption on a solid
stationery phase. (column chromatography and tlc)
 2) PARTITION Lebih kpd solubility not polarity

Macam oil & water campur lah camna pun akan terpisah
 PARTITION CHROMATOGRAPHY
Mobile phase:
liquid

Dia x bercampur depends on polarity.

Biru lg byk dkat pink so dia lebih attract kpd
SP

Stationary solute
phase: liquid
supported on
solid

Attracted by the light blue

Examples:
i. High performance liquid chromatography (HPLC)
ii.Paper chromatography (exp7)
iii.Gas chromatography (GC)

HPLC GC Paper chromatography

Means, yg tertarik kpd MP is nonpolar sbb MP non polar

Eluted = remove an adsorbed substance

by washing with solvent which is MP
TYPES OF CHROMATOGRAPHY
BASED ON TECHNIQUES

PLANAR CHROMATOGRAPHY
COLUMN CHROMATOGRAPHY (CC)
TYPES OF CHROMATOGRAPHY
COLUMN AND PLANAR CHROMATOGRAPHY
2 TYPES OF CHROMATOGRAPHY

Column or elution
chromatography
Liquid solvent ;
benzene (liquid)
narrow tube

PC – an organic solvent
( in liquid )
Planar chromatography PC = water adsorbed
i. Paper on paper (liquid)
chromatography PC
TLC – a solid TLC – a solvent
ii. Thin Later
Chromatography TLC adsorbent ; alumina , ( in liquid )
Silica gel (solid)
 Mobile phase sama cuma bezanya pada stationary phase

Separation column
 Chromatography

Column Plana
chromatography

Thin layer Paper

chromatography chromatography
TLC PC

Ada thick aluminium

foil @ plastic coated on
TLC
(means ada solid
attach to TLC plate)
 1. PLANAR CHROMATOGRAPHY

i. Paper chromatography (PC)

A sheet or a narrow strip of paper serves as the
stationary phase.

ii. Thin-layer chromatography (TLC)

A thin film of a stationary phase of solid particles
bound together for mechanical strength with a
binder, such as calcium sulfate, coated on a glass
plate or plastic or metal sheet
PC- water absorbed on paper, involve liquid
TLC- solid absorbent (more to adsorbtion
mechanism)
 i) Paper
Chromatography
Purpose:
Use the technique of paper chromatography to separate a
homogeneous mixture into its individual components
Uses: PC – component to be separated is PARTITION
- Separation not adsorption.
- Identification Between stationery phase water adsorbent on
paper and mobile phase (solvent)
Advantages:
Cheap, fast , sensitive, wide uses.
Disadvantages:
- Reproducibility of the results is low.
- Cannot withstand corrosive chemicals.
 i) Paper
Chromatography

So easily absorb water

If SP polar
MP less polar
 i) Paper
Chromatography
Principles and techniques of PC

Spotted
sample

Origin line
 The more soluble a component,
The further it moves

solvent

Place small a spot of the

mixture to be analysed. Dip
the paper in the solvent.
3) Measuring Retention factor Rf value

Rf = distance solute moves (b)

distance solvent front moves (a)

Solvent front

(b) (a)

Origin line

 The distance the solute moves, b is measured at the center of

the solute spot or at its maximum density
 Yg lagi tinggi/jau
-is eluted faster becau
- strong attraction towards mobile p
- The retention time is sho

(Stationary phase is polar compo

chromatograph

Yg rendah
-less polar and strongly ad
a -More affinity so interact more strongl
Contains -So compound retarded and le
both
compound
b a&b

Rf a = 0.85
Rf b = 0.42
A = Polar compound. The compound is soluble.
B = nonpolar. The compound is less soluble
 Same Rf value = Same
component present in the sample
 Paper chromatography

Measuring
Retention
factor (Rf) value

Solvent:
spots move
mobile
upward by
phase
Filter capillary
paper: action
stationary
phase
• Retention factor (Rf) value
The distance travelled by the compound divided by the
distance travelled by the solvent.

Rf value = distance moved by substance

@ MP @
distance moved by eluent SOLVENT

If poor = component differ with MP

= Solvent x sesuai dgn component
• Example of calculation of Rf value:

Distance Solvent
by spot distance

So, it is a good result

 TRY
• Calculate the Rf values for blue, purple and yellow spots.

Solvent front

blue

purple
10 cm
8 cm
6 cm
yellow

2 cm

Origin line
ii) Thin Layer Chromatography
(TLC)

Technique same dgn PC but

difference of the SP bcoz ada solid
adsorbent TLC
Solid Adsorbents of
TLC
Adsorbents of
TLC
Principles of separation using a
TLC
 Principles of separation using a
TLC
• If the mobile phase is a non-polar solvent and
stationary phase is a polar.

- The less polar compound is more soluble in the mobile

phase. So, it will be travelled faster up the plate.
- The more polar compound will strongly adsorbed to the
stationary phase and will travel slower up the plate.
 Proses in Thin Layer
Chromatography (TLC)
(a) Preparing and activating TLC plates

(b) Spotting the TLC plates with sample

(c) Developing the TLC plates

(d) Drying the plates

(e) Visualizing the substance spots

(f) Measuring the Rf value

(a) Preparing and activating TLC plates
Preparing TLC plates
• The steps involved are as follows:
a)The glass plates are cleaned with soaps and any trace of
grease/oil on the plates is removed by using acetone.
to remove

b)An aqueous slurry of powder (SiO2 or Al2O3) (in water) is

prepared by mixing with a binder such as plaster of paris,
gypsum, or poly(viny alcohol) to help it adhere to the
backing material.
Slurry = a semi-liquid mixture

c)The slurry is spread on the plate to form a thin film (0.1–

0.3 mm thick) analytical purposes and around 0.5 –
2.0 mm for preparative TLC by using a spreader to assure
uniform thickness.
Activating TLC plates
 spreader
Spreader machine = to to make it
silica or alumina is in uniform ……
(b) Spotting the TLC plates with sample

 Spotting a TLC plate first involves preparing a glass

spotting capillary (available commercially).

 These capillaries are made using melting point capillary

which has been elongated and thinned down to a small
diameter capillary tube by heating.

 To spot a TLC plate, obtain a silica gel TLC plate (4 cm x

10 cm), draw a straight line parallel to the short
dimension of the plate with a pencil.
Spotting the TLC plates with sample
(c) Developing the TLC plates

Yg drawn guna
pencil td
TLC Developing Chambers Developing the TLC plates
Solvent must
below the line
(e) Visualizing the substance spots

eg; dia moving but we cant see so these are the methods

( 1-2 sec )
Sulfuric Acid
 UV lamp

TLC plate under TLC plate under TLC plate inside the Spraying TLC plate
UV light (365 UV light (254 iodine chamber with ninhydrin
nm) nm)
Variety of TLC
Advantages of TLC
over PC

PC separation x smoothly

seperate

Tahan lasak
 2. COLUMN CHROMATOGRAPHY

SP can be liquid @ solid but dlm Narrow Tube

Mcm burette
but no marks
 2. COLUMN CHROMATOGRAPHY
Eluent = mobile phase. Term shj berbeza.
Eluent is to carried the component of mixture through stationary phase

Adsorbtion = ada
interaction dgn SP

Adsorption ni dia attract ON surface

Adsorb : liquid – solid
Partition : liquid - liquid
 2. COLUMN CHROMATOGRAPHY

Macam MP

Depends on
Stationery Phase.
SP = powder

SP = slurry,
kepekatan tinggi
Analyze untuk tau component exist
in the sample?
eg; tryglicerine/ prot/ fat ke,
 Principles of chromatographic separations
Sample Mobile phase
1) Sample injected in column.
Dlm coumn already ada SP.
2) masukkan MP as eluent
SAME macam TLC
& PC but this one
put in column Component A
Stationary
Component B
phase
Component C

That’s y CompA move slowly

Separation of compounds based on polarity

If MP = polar, so SP = non-polar.
If sample polar so move faster. If non-polar slower sbb attracted to SP so retarded
 Separation of compounds based on polarity
2. Perform a column chromatography using a polar
stationary phase and a non-polar mobile phase.

•Compounds will be separated due to different solubility in

mobile phase and stationary phase.
•The less polar compound is more soluble in the mobile phase.
So, it will be eluted faster.
•The more polar compound is more attracted to the stationary
phase. It will retained stronger to the stationary phase and will
be eluted slower.
•Less polar compound will eluted first followed by the polar
compound.
 ADVANTAGES DISADVANTAGES
Low cost and disposability of
( 1- 2 days )
the stationary phase.

Can be scaled to the project

at hand.

Determine the number of

components of a mixture.

Separate and purify

substantial quantities of
those components for
subsequent analysis.
 Comparison between CC, PC and TLC
Column Paper chromatography Thin layer chromatography
chromatography

Stationary phase Stationary phase is Water trapped in the A solid adsorbent such as
placed in a narrow paper or paper strip alumina (Al2O3) or silica gel
tube or column. which made up of (SiO2).
cellulose fibers (made-
up of hydroxyl groups).

Mobile phase Liquid Liquid Liquid

m. Type of Adsorption Partition Adsorption
SP chromatography
Type of Column Planar chromatography Planar chromatography
ue chromatography chromatography

Type of force Gravity Capillary Capillary

that moves the
mobile phase
APPLICATIONS OF PLANAR
AND COLUMN
CHROMATOGRAPHY
 Applications of column chromatography
 Pharmaceutical Company
- To determine amount of chemical in new product

 Hospital
- To detect types of blood or alcohol levels

 Law Enforcement
- To compare a sample found at a crime scene with samples from
suspects

 Environmental Agency
- To determine the level of pollutants in the water supply

 Manufacturing Plant
- to purify a chemical needed to make a product
 Applications of TLC
• To Analyze ceramides and fatty acids.

• To Detect types of pesticides or insecticides in food and

water.

• To separate the dye composition in fibers.

• To Identify the medicinal plants and their constituents.

• In food industry, TLC method is used for separation and

identification of colours, preservatives and sweetening agent.
Applications of Planar
Chromatography