intro to chromatography

Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.

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INTRODUCTION TO CHROMATOGRAPHY


What is Chromatography?


Chromatography:Chromatography operates on the same principle as extraction, but one phase is held in place while the other moves past it.

Phase 2

Phase 1

Solvent extraction:Extraction is the transfer of a solute from one phase to another

Extraction of Uranyl Nitrate from water into ether Partition

coefficient:

S(in phase 1) S(in phase 2)

K

[S]

2

[S]

1



Chromatography:Chromatography operates on the same principle as extraction, but one phase is held in place while the other moves past it.

Air flow


Air flow




Initial bandA and B solutes

Fresh solvent(eluent)

Column packing(stationary phase)

Suspended in solvent(mobile phase)

Solvent flowing out(eluate)

A

B

B

emerges

A emerges


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It is the collective term for a family of laboratory techniques for the separation of mixtures by distribution of its components between a mobile and stationary phase over time

All chromatographic separations depend on the reversible sorption and desorption of the

components of the mixture in the stationary and mobile phase

Mobile phase:solvent moving through the plate or column(liquid or gas)

Stationary phase:substance which is fixed in place for the chromatographic procedure

The partitioning of solutes between the mobile and stationary phases gives rise to separation


Chromatographic principle

Stationary phase

Mobile phase

Samplemixture

Equilibrium established at each point (ideally)

The molecules of the mixture interact with the molecules of the

Mobile and Stationary Phase

Retardation of rate of

movement of molecules

Each molecule interacts differently with MP and SP

Different distribution coefficients and different

net rates of migration


Purpose of Chromatography

The two are not mutually exclusive

Analytical ChromatographyAnalytical Chromatography – separate and identify or measure the relative proportions of components in a mixture

Preparative ChromatographyPreparative Chromatography - purify and collect one or more components of a sample for further use

300 L

Purifies 1 Kg of material

Scaling Up:

Large column radius

Small column radius=

Large mass

Small mass

2


Types of Chromatography

Chromatography is divided into categories on the basis of the

mechanism of interaction of the solute with the stationary phase


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Adsorption Chromatography

Competition between a solid adsorbent and the

mobile phase

Gas Column Gas-Solid Chromatography GC/GSC

Liquid

Column

Liquid column Chromatography

High Performance Liquid Chromatography

LCHPLC

Planar layer

Thin Layer ChromatographyPaper Chromatography

TLCPC

Partition Chromatography

Competition between a liquid stationary phase and the mobile phase

Gas ColumnGas-Liquid Chromatography

Supercritical Fluid Chromatography

GC/GLCSFC

Liquid

Column

Liquid-Liquid Chromatography

High Performance Liquid Chromatography

LC/HPLC

Ion Exchange Chromatography

Competition between an ionic exchange resin stationary phase and liquid mobile phase

Liquid

Column

Ion-Exchange Chromatography

High Performance Ion Chromatography

IECIC/HPIC

Permeation Chromatography

Competition between a polymer matrix

stationary phase and liquid mobile phase

Liquid

ColumnGel Permeation

ChromatographyGPC

Stationary PhaseMobilePhase

StationaryPhase

SupportType of

Chromatography

Classification based on the chromatographic principle

Acronym


Column: Stationary Phase held in a tube and Mobile Phase forced through

Planar: Stationary Phase supported on a flat plate or in pores of paper. Mobile Phase moves through Stationary Phase by capillary action, gravity or electrosatic forces

Classification of Chromatography

Based on Stationary Phase Support


Chromatography

Mobile Phase = LiquidLiquid Chromatography

Mobile Phase = GasGas Chromatography

Stationary Phase = Solid

LSC

StationaryPhase = Liquid

LLC

StationaryPhase = Solid

GSC

StationaryPhase = Liquid

GLC

Adsorption or ion exchange

Partition of analytes

between two phases

Adsorption

Partition of analytes

between two phases

Classification according to the phases


Molecular characteristic

Physical propertySeparation technique

Polarity

volatility GLC

solubility LLC

adsorptivity LSC

Ionic chargeIon Exchange

Chrom.Electrophoresis

Size (mass)diffusion

Gel Permeation Chrom.Dialysis

sedimentation Ultracentrifugation

Shape ligand binding Affinity Chrom.

Classification according to physical features of the analytes


Methods based on Ionic Nature of the sample

Ion nature of the

sample is affected

by:

•Number & nature of ionisable groups•pH environment

•Presence of other ions

And results in:

Association of ions with opposite

charge

Movement in an electric field

Ion-exchange chromatography

ElectrophoresisCapillary

electrophoresis

Competition for ionic binding sites

on a resin

Direction & velocity of movement

depends on the sign & intensity of the

ionic charge


AdsorptionChromatography

Methods based on polarity (i.e. affinity of like-molecules for each other)

Solid/Liquid Liquid/Liquid Liquid/Vapour

Adsorption Solubility Solubility

Solid adsorbents

Two immiscibleliquids

A solutionand its vapour

LiquidChromatography

Gas-LiquidChromatography

Partition between two phases:

A major factor in separation is:

And the methods involve:

Methods are generallyknown as:


Movement due to this may be:

Restricted bypore size

Methods based on size

Diffusion Sedimentation

Dialysis

Ultracentrifugation

Fixed pore size

Gel filtrationMolecular exclusion (largest molecules come outfirst as not held by pores)

Size affects the rate of:

Balanced byeach other

Reduced by high density solvents

Maximal in lowdensity solvents

Gel permeationchromatography

Variable pore size

Isodensity Velocity

Diffusion


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intro to chromatography

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