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Transcript of chromatography
CHROMATOGRAPHYBY:DARAKHSHAN SALEEMM.S MICROBIOLOGY
According to Britannica
Chromatography
Technique for separating the components,
or solutes, of a mixture.
On the basis of
Moving fluid stream, called the Mobile Phase.
Contagious stage or Stationary Phase
Either a Solid or LiquidEither a Gas or Liquid
Laboratory technique for the Separation of
mixtures
Chroma -"color" and graphein - "to write”.
Colure bands - separation of individual compounds
Measured or analyzed.
CHROMATOGRAPHY
PURPOSE OF CHROMATOGRAPHY
PreparativePreparative: Used to purify sufficient quantities of a
substance
AnalyticalAnalytical :Determine Chemical composition of a sample
TSWETT EXPERIMENT
TERMS
• Chromatograph - equipment that enables a
sophisticated separation.(Gas chromatography or
Liquid chromatography)
• Eluent - Fluid entering column/ solvent that carries
the analyte.
• Eluate - Mobile phase leaving the column.
• Stationary phase - Immobilized phase
Immobilized on the support particles or on the inner
wall of the column tubing.
Examples : Silica layer - Thin Layer
Chromatography
• MOBILE PHASE
• The mobile phase moves through the chromatography
column (the stationary phase) where the sample interacts
with the stationary phase and is separated.
• Retention time : Time takes for a particular analyte to
pass through the system (from the column inlet to the
detector) under set conditions.
• Sample (Anylate): Sample analyzed in chromatography
• Solvent : Any substance capable of solubilizing another
substance.
Visual output of the chromatograph.
Separation - Different peaks or patterns on the
chromatogram correspond to different components
of the separated mixture.
CHROMATOGRAM
Important Steps In Chromatography
Pack Column - Column is packed with
material (resin) that can absorb molecules based
on some property (charge, size, binding affinity,
polarity)
Equilibrate Column - Column is washed with
several column volumes of buffer
Load sample - apply sample mix to column
Wash column - Molecules washed through
the column with buffer
Collect fractions - Fractions are taken, at
some point your molecule will elute
The techniques can be broadly divided into
Planar
Columnar.
CHROMATOGRAPHY TECHNIQUES
In Planar type the stationary phase is a plane surface i.e
(two dimension surface where only length and breadth
are taken as area) on which chromatograms are formed.
This method is adopted in techniques like
1. Using Paper.
2. Thin layer chromatography.
3.High performance thin layer
chromatography(HPTLC).
here is use of a column on whose walls lies a stationary
phase and the mobile phase is flushed through the
column.
The techniques which employ this method are
1. Column Chromatography.
2. Gas Chromatography.
3. HPLC
4. Size Exclusion.
5. Ion Exchange.
COLUMNAR TYPE
Planar techniques have the advantages
like faster separation,
visualization of formed chromatograms or spots
and are less expensive.
But they are not useful for preparative purposes.
PROS AND CONS
In Columnar techniques, the advantages like
better or effective separations of even complex
mixtures, possibility to yield large amount of
compounds by separation of mixtures i.e
preparative mode..
PROS AND CONS
But they have disadvantage of being
expensive, time consuming and cumbersome
(heavy).
is a chromatographic technique used to
separate the components of a mixture
using a thin stationary phase supported by
an inert backing.
THIN LAYER CHROMATOGRAPHY (TLC)
Routinely used by researcher in the field of phyto-
Chemicals, Biochemistry,Pharma and Food Industry
To identify the components in a compound mixture like
alkaloids, phospholipids, amino acids etc..
It is a semi quantitative method of analysis
APPLICATION:
TLC SYSTEM
TLC Plate
FILTER PAPER
TLC Chamber
Mobile phase
Simple, Rapid and Cheap
Allow for quantification
Better separations
Choice between different adsorbents.
Better resolution
High Pressure Thin Layer Chromatography.
This is a sophisticated advancement in Thin
Layer Chromatography (TLC).
HPTLC High Performance Thin layer Chromatography
The injection of a small volume of liquid sample into a
tube packed with tiny particles (3 to 5 micron (µm) in
diameter called the stationary phase)
where individual components of the sample are moved
down the packed tube
(column) with a liquid (mobile phase) forced through the
column by high pressure delivered by a pump.
HPLC IS A SEPARATION TECHNIQUE
These components are separated from one another by
the column packing that
involves various chemical and/or physical interactions
between their molecules and the packing particles.
• These separated components are detected at the exit
of this tube (column) by a
flow-through device (detector) that measures their
amount.
Detecting very small amounts
High resolution
Rapid analysis
Speed, efficiency, sensitivity and ease of operation
High degree of versatility
Easily separate a wide variety of chemical mixtures
400 atmospheres.
PUMP PRESSURE 1000 atmosphere
Protein separation
Insulin purification
Plasma fractionation
Enzyme purification
APPLICATION OF HPLC
Column chromatography is the actual or the basic
type of chromatography procedure which was
developed during early stages of chromatography
When a mixture of mobile phase and sample to be
separated are introduced from top of the column, the
individual components of mixture move with different
rates. Those with lower affinity and adsorption to
stationary phase move faster and eluted out first while
those with greater adsorption affinity move or travel
slower and get eluted out last.
The solute molecules adsorb to the column in a
reversible manner
Solid materials (Adsorbents) – Ability to hold
the molecules at their surface
Attractive forces (Vanderwalls & Hydrogen )
Functional groups (Hydroxyl/ Aromatic)
Silica
The stationary phase material is suitably moistened with mobile
phase and packed sufficiently in the column with a cotton or
asbestos pad at the bottom. The extract material or sample to be
separated is placed on the top of packed stationary phase with a
second cotton or asbestos pad in between.
The mobile phase is poured into the column over the sample. A
collecting beaker is placed at the bottom of column near the end
to collect the elute.
PROCEDURE:
• Gas-Liquid chromatography, (GLC)
• Mobile phase – Gas (Helium) Carrier Gas Pressure
= 4 kg/cm2
• Stationary phase - Column, which is typically
"packed" or "capillary".
• The stationary phase is adhered to the inside of a
small-diameter glass tube (a capillary column) or a
solid matrix inside a larger metal tube (a packed
column).
GAS CHROMATOGRAPHY
• High sensitivity,
• High Resolution,
• High speed
• High Accurasy,
• Highly Quantitative
ADVANTAGES
Size Exclusion Chromatography (SEC) is the separation technique
based on the molecular size of the components. Separation is
achieved by the differential exclusion from the pores of the packing
material, of the sample molecules as they pass through a bed of
porous particles. The principle feature of SEC is its gentle non-
adsorptive interaction with the sample, enabling high retention of
biomolecular activity.
Separate by size
Column packed with porous beads
As wash with buffer:
Small molecules enter the beads
Large molecules move between the beads
Elution:
Large proteins elute first
small proteins last
Size Exclusion (Gel Filtration)
SEC can be used to guide cell-line selection.
It can also discriminate between aggregate forms that may be more
or less difficult to remove during downstream purification steps
SEC can also be used extensively to guide the development of the
purification process for biopharmaceuticals
not recommended for separating proteins with only a small
difference in molecular weight.
Separate by charge
Column packed with a charged resin
Use a charged buffer
• Like charged proteins flow through with buffer
• Oppositely charged proteins bind to column
Elute protein
• Increase salt or pH to elute protein of interest
ION EXCHANGE CHROMATOGRAPHY
Separate by specificity
Column packed with: Molecules (ligands) that interact
strongly with protein of interest
As wash: Molecules of interest bind to column, other
proteins flow through
Elution: Bound proteins eluted by adding high
concentration of ligand
AFFINITY CHROMATOGRAPHY
Affinity chromatography is widely used in the pharmaceutical
industry to purify and extract molecules of interest from complex
mixtures.
These molecules tend to be enzymes, proteins or amino acids, but
other biological species can be selectively retained.
Once isolated, these biological species can be selectively
amplified to produce larger quantities, although at large
concentrations.