Chromatography

Introduction
• Invented by M.Tswett a botanist in 1906
• He used for separation of coloured
compounds
• Now chromatographic separation any given
mixture can be done.
• Chromatography is greek word Chroma
means colour and graphy means writing
• It is based on the difference in the rate at
which components of the mixture move
through the porous medium ( called
stationary phase) under the influence of
solvent ( mobile phase)
Definition

Chromatography is a separation technique based on the different

interactions of compounds with two phases, a mobile phase and a
stationary phase, as the compounds travel through a supporting
medium.

Components:
mobile phase: a solvent/gas that flows through the supporting
medium

stationary phase: a layer or coating on the supporting medium that

interacts with the analytes

supporting medium: a solid surface on which the stationary phase is

bound or coated
The analytes interacting most
strongly with the stationary
phase will take longer to pass
through the system than those
with weaker interactions.

These interactions are usually

chemical in nature, but in some
cases physical interactions can
also be used.
 Classification
• Based on principle Classified as
• Adsorption chromatography
• Partition chromatography

Chromatography can be classified based on the type of

mobile phase, stationary phase and support material
1.) The primary division of chromatographic techniques is based on the type of mobile phase
used in the system:

Type of Chromatography Type of Mobile Phase

1. Gas chromatography (GC) gas
2. Liquid chromatograph (LC) liquid

2.) Further divisions can be made based on the type of stationary phase used in the system:

Gas Chromatography
Name of GC Method Type of Stationary Phase
Gas-solid chromatography solid, underivatized support
Gas-liquid chromatography liquid-coated support
Bonded-phase gas chromatography chemically-derivatized support
 Types of Chromatography

1. Liquid Chromatography
Name of LC Method
Adsorption chromatography
Partition chromatography
Ion-exchange chromatography
Size exclusion chromatography
Affinity chromatography
Type of Stationary Phase
solid, underivatized support
liquid-coated or derivatized support
support containing fixed charges
porous support
support with immobilized ligand
3.) Chromatographic techniques may also be classified based on the type of support material
used in the system:

1. Packed bed (column) chromatograph

2. Open tubular (capillary) chromatography
3. Open bed (planar) chromatography
 Theory of Chromatography
1.) Typical response obtained by chromatography (i.e., a chromatogram):

chromatogram - concentration versus elution time

Wh

Wb

Inject

Where:
tR = retention time
tM = void time
Wb = baseline width of the peak in time units
Wh = half-height width of the peak in time units
Note: The separation of solutes in chromatography depends on two factors:

(a) a difference in the retention of solutes (i.e., a difference in their time or volume of
elution
(b) a sufficiently narrow width of the solute peaks (i.e, good efficiency for the separation
system)

Peak width & peak position

determine separation of peaks

A similar plot can be made in terms of elution volume instead of elution time. If volumes
are used, the volume of the mobile phase that it takes to elute a peak off of the column is
referred to as the retention volume (VR) and the amount of mobile phase that it takes to
elute a non-retained component is referred to as the void volume (VM).
2.) Solute Retention:

A solute’s retention time or retention volume in chromatography is directly related to the

strength of the solute’s interaction with the mobile and stationary phases.

Retention on a given column pertain to the particulars of that system:

- size of the column
- flow rate of the mobile phase

Capacity factor (k’): more universal measure of retention, determined from tR or VR.

k’ = (tR –tM)/tM

or

k’ = (VR –VM)/VM

capacity factor is useful for comparing results obtained on different systems since it is
independent on column length and flow-rate.
The value of the capacity factor is useful in understanding the retention mechanisms for a
solute, since the fundamental definition of k’ is:
moles Astationary phase
k’ =
moles Amobile phase

k’ is directly related to the strength of the interaction between a solute with the stationary
and mobile phases.

Moles Astationary phase and moles Amobile phase represents the amount of solute present in each
phase at equilibrium.

Equilibrium is achieved or approached at the center of a chromatographic peak.

When k' is < 1.0, separation is poor

When k' is > 30, separation is slow
When k' is = 2-10, separation is optimum
• Thin-layer chromatography and
column chromatography and are
different types of liquid
chromatography.
• The mobile (moving) phase is a
liquid. The stationary phase is
usually silica or alumina. This phase
is very polar.
• The principle of operation is the
same!
Thin Layer Chromatography
• simple equipments
• Shorter developing time
• Wide choice of stationary phase
• Early recovery of separated components
• Superior separation
• Easy visualization of separated components
• Sensitivity
• Chemically inert stationary phase
 Materials
• Coating materials
Silica gel, Alumina, Kieselger,
Cellulose powder
• Preparation of thin layer in plates
1. Dipping
2. Pouring
3. Spraying
4. Spreading
• Activation of Adsorbent
• Sample application
• Developing Tanks
• Solvent Systems
• Development methods
• Detection of components
1. Visual Assessments
2. Determination of measuring the spot
areas
3. Direct spectrophotometry
 Thin Layer Chromatography
The surface of the plate consists of a very thin layer of silica on a plastic or
aluminum backing. The silica is very polar. This is the stationary phase. Spot
the material at the origin (bottom) of the TLC plate.

Place the plate into a glass jar with a small amount of a solvent in the glass jar.
This solvent acts as the moving phase.

Remove the plate from the bottle when the solvent is close to the top of the plate.

Visualize the spots.

Non-polar compounds will be less strongly attracted to the plate and will spend
more time in the moving phase. This compound will move faster and will appear
closer to the top of the plate.

Polar compounds will be more strongly attracted to the plate and will spend less
time in the moving phase and appear lower on the plate.
 TLC
DEVELOPING
CHAMBER

SPOT

SOLVENT

Solvent
Front

1.1 cm

5.5 cm

Origin

Distance from starting origin to center of zone

Rf =
Distance from starting origin to solvent front

5.5
= = 0.5
11
Stationary Phase: Silica (SiO2)
OH
OH
OH
OH
OH Si
Si O
Si O
Si O O
Si O O
O O
O O
O Si Si
O O
Si
Si O O O
Si O O
O
O O
O
Si
Si O
O
O O
O
Stationary Phase: Alumina

O OH OH OH OH

Al Al Al Al Al
O O O O O O

Acidic: -Al-OH
Neutral: -Al-OH + -Al-O-
Basic: -Al-O-
 Thin-Layer Chromatography:
A Two-Component Mixture

solvent front

component B Less polar!

solvent front

component B

component A More polar!

component A

origin mixture origin origin

solvent front

Increasing Development Time

 Visualization Method
• The previous slide shows colored spots.
Most of the time, the spots won’t show
unless they are visualized!
• Vizualization is a method that is used to
render the TLC spots visible.
• A visualization method can be:
– Ultraviolet light
– Iodine vapors to stain spots
– Colored reagents to stain spots
– Reagents that selectively stain spots while
leaving others unaffected.
 Thin-Layer Chromatography:
Determination of Rf Values
solvent front

Rf of component A =
dA component B

dS
dS
dB
Rf of component B =
dB component A
dS
dA
The Rf value is a decimal
origin
fraction, generally only
reported to two decimal
places
Thin-Layer Chromatography:
Qualitative Analysis

A B unknown
 Uses of TLC
• To determine how many components
there are in a mixture (is it really
pure?)
• To determine the best solvent
conditions for separation on a column
• To identify the substances being
studied
• To monitor the composition of fractions
collected from column chromatography
• To monitor the progress of a reaction
 Column Chromatography
The stationary phase (column packing) in the column is
very polar!

Polar compounds are going to be attracted to the polar

column packing by hydrogen bonding or dipole-dipole
attractions. Polar compounds are going to move slowly!

Non-polar compounds are going to come off the column

first, while the polar compounds are going to come off
the column last.

Usually, one starts will a less polar solvent to remove

the less polar compounds, and then you slowly
increase the polarity of the solvent to remove the more
polar compounds.
Principles of Separation on a
column
Principles of Separation
Principles of Separation
Principles of Separation
Principles of Separation
Principles of Separation
Principles of Separation
 REMEMBER…
• The stationary phase is POLAR
• The more polar component interacts
more strongly with the stationary
phase
• The more polar component moves
more slowly.
• The non-polar component moves
more rapidly.
 Reverse phase
chromatography
Silica is alkylated with long chain hydrocarbon groups, using 18
carbons long. This is usually referred to as C-18 silica.
 Reverse Phase column
chromatography
• The stationary phase (column packing)
is now NON-POLAR
• Non-polar compounds will move more
slowly because they are attracted to the
column packing.
• The more polar component moves more
quickly down the column.
• Polar solvents, such as water and
methanol are used in reverse phase
chromatography
• Used mainly in columns, such as HPLC
 Normal phase Column
Chromatography
The column packing in the column is very polar!

Polar compounds are going to be attracted to the polar

column packing by hydrogen bonding or dipole-dipole
attractions. Polar compounds are going to move slowly!

Non-polar compounds are going to come off the column

first, while the polar compounds are going to come off
column last.

Usually, one starts will a less polar solvent to remove

the less polar compounds, and then you slowly
increase the polarity of the solvent to remove the more
polar compounds.
 Paper Chromatography
• Partition Chromatography
• Fibers of paper is stationary phase
 Types
• Descending chromatography
• Ascending Chromatography
• Ascending Descending
chromatography
• Radial Chromatography
• Two dimensional Chromatography
Thanks