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.