Presented By,

SORPTION :

Hoh Shee Ching
Mohd Zulfakri

Adsorption

Nina Karina
Nur Ashikin

Chromatography Syafiqah Athirah

 ADSORPTIONS ???
Refers to the phenomenon of attracting and retaining the molecules
of a substance on the surface of a liquid or solid.
ABSORPTION : when the molecule of substance are uniformly
distrusted throughout the body of a solid or liquid.
ADSORBENT the substance on which surface the absorbate is
adsorbed.
ADSORBATE the substance which is adsorbed on the surface
 ADSORBENTS ???
Most adsorbents can be made from natural (zeolites) or
synthetic material (carbons fiber).
Have amorphous or microcrystalline structure. Have
properties of both a solid and a liquid. (eg: Mayonnaise)
Commonly : cellulose based adsorbents, silica gel based
adsorbents, synthetic resins and agarose based
adsorbents.
 ADVANTAGES DISADVANTAGE
- High degree of purity - It is a surface phenomenon
can be obtained. - Separation is carried out in
- The process is batch mode only
reproducible - May result in loss of biological
activity of the solute
 SEPARATION MECHANISM
Ion Exchange
Based on electrostatic interactions between molecule and
adsorbent.
Cation exchange adsorbent bind with positively charged
molecules and vice versa.
The binding of molecule on ion exchange adsorbents is
depends on pH, ionic strength and buffer type.
Example: Separation and Purification of blood components
such as albumin, recombinant growth factors enzymes.
 AFFINITY BINDING
Based on specific binding interaction between an immobilized
ligand and its binding partner (eg: shape of the ligand is
complimentary to the shape of the entire target molecule)
Binding - held by hydrogen bonding, hydrophobic interaction
and van der Waals.
Specific affinity adsorption: ligand binds to only one type of
molecule. (eg: antigen-antibody binding).
General affinity adsorption: ligand binds a range of
molecules having certain similarities.
 AFFINITY BINDING
Example of affinity binding:
Amino acids are used as ligands for purifying proteins and
nucleic acids.
Lectins and Concanavalin-A are used for binding carbohydrates
and glycol-proteins.
REVERSE PHASE ADSORPTION
used for adsorption of non-polar molecules
adsorbent: hydrophobic, low polarity substances such as
aliphatic hydrocarbon chains chemically bound to solid support
material such as silica
when feed solution is contacted with the reverse phase
adsorbent, the non-polar molecules will bind to hydrocarbon
layer while the other polar molecules will wash through and
eluted first.
the non-polar molecules can subsequently be eluted using non-
polar solvents such as acetonitrile and isopropanol
 APPLICATION USING REVERSE
PHASE ADSORPTION
Purification of recombinant insulin
 HYDROPHOBIC
INTERACTION ADSORPTION
used for protein separation
adsorbent: alkyl and aromatic hydrocarbon groups on the agarose.
anti-chaotropic salts are added (to remove the water molecules
making up the structured layers around the hydrophobic patches of
protein molecules and adsorbent).
create hydrophobic environment for the binding of hydrophobic amino
acid of protein molecules while other impurities will be eluted first.
desorption of bound protein can be achieved simply by passing
solution with low salt concentration
APPLICATION USING HYDROPHOBIC
INTERACTION ADSORPTION
Separation of recombinant epidermal growth factor (rEGF)
DIFFUSIONAL LIMITATIONS IN
ADSORPTION PROCESSES
External mass transport (which is controlled by diffusion
from the bulk solution to the surface of the adsorbent)
Internal mass transport (which is controlled by diffusion
within a porous adsorbent)
 BATCH ADSORPTION
the feed solution and the adsorbent particles are brought into contact until
equilibrium binding is achieved.
The raffinate which also contains the impurities is then separated from the
adsorbent by using an appropriate solid-liquid sparation technique ex.
filtration or centrifugation.
The solid material obtained (i.e. the adsorbent with the bound solute) is
frequently washed for complete removal of impurities.
The adsorbed solute can then be eluted from the adsorbent by adding a
liquid which favours desorption of the solute which can then be separated
from the adsorbent by using appropriate solid-liquid separation techniques.
PACKED BED ADSORPTION
Step 1: Adsorption Step
Pumping of a predetermined amount of feed
solution through the packed bed.
Step 2: Washing Step
Passing the wash liquid through the packed bed to
remove impurities.
Step 3: Elution Step
Pumping desorption solution to wash out the target
molecules.
EFFLUENT COMPOSITION PROFILE
IN PACKED BED ADSORPTION
 WHAT IS
CHROMATOGRAPHY?
A process in which a chemical mixture carried by a liquid or
gas is separated into components as a result of differential
distribution of the solutes as they flow around or over a
stationary liquid or solid phase
A technique to separate the components of a mixture by
passing the mixture through a stationary phase.
 PRINCIPLE
Usually consist of mobile phase and stationary phase
Mobile phase mixtures of substance to be separated
dissolved in a liquid or a gas
Stationary phase a porous solid matrix through which the
sample contained in the mobile phase percolates
The interaction between the mobile phase and the stationary
phase results in the separation of the compound from the
mixture
 APPLICATION
Used for the separation of amino acid , proteins &
carbohydrates
Also used for analysis of drugs, hormones & vitamins
Helpful in qualitative & quantitative analysis of complex
mixtures
Also used in determination of molecular weights of proteins
TYPES OF CHROMATOGRAPHY
Gas Chromatography (GC)
Ion Exchange Chromatography
Paper Chromatography.
Liquid Chromatography (Lc)
Thin Layer Chromatography
Gel Chromatography
Column Chromatography
Affinity Chromatography
Gas Chromatograph (GC)
An analytical instrument that measures the content
of various components in a sample.
The analysis performed by a gas chromatograph is
called gas chromatography.
 Principle of Gas Chromatography
The sample solution injected into the instrument enters a gas stream
which transports the sample into a separation tube known as the
"column." (Helium or nitrogen is used as the so-called carrier gas.)
The various components are separated inside the column.
The detector measures the quantity of the components that exit the
column.
To measure a sample with an unknown concentration, a standard
sample with known concentration is injected into the instrument.
The standard sample peak retention time (appearance time) and area
are compared to the test sample to calculate the concentration.
 High-performance Liquid
Chromatography (HPLC)
A form of liquid chromatography used to separate compound that
are dissolved in solution.
HPLC instrument consist of a reservoir of mobile phase, a pump,
an injector , a separation column and a detector.
The different component in the mixture will pass through the
column at differentiates due to differences in their partition
behaviour between the mobile phase and the stationary phase.
The mobile phase must be degassed o eliminate the formation of
air bubbles.
 Principle of HPLC
a separation technique that involves :
The injection of small volumes of liquid samples
Into a tube packed with tiny particles of 3 to 5 micrometre
(m) in diameter called the stationary phase
Where the individual components of the sample are moved
down the packed tube (column) with a liquid forced through
the column by high pressure delivered by a pump.
 COMPARISON BETWEEN
GC AND HPLC
Gas Chromatography (GC)
Gas as mobile phase
Less operation & maintenance
cost
Separation carried out at
elevated temperature
GC columns longer & narrower
High-performance Liquid
Chromatography (HPLC)
Liquid as mobile phase
High operation & maintenance
cost
Separation carried out at
ambient temperature
HPLC columns are shorter &
wider due to high pressure.
THANK YOU