chromatography intro

7/28/2019 Chromatography Intro

1/29

CHROMATOGRAPHYTHEORY & PRACTICE

INTRODUCTION

PROF. DERICK CARBOO

DEPARTMENT OF CHEMISTRY

UNIVERSITY OF GHANA, LEGON

O244682712


2/29

OUT LINE1. What is chromatography?

2. History of chromatography3. Nomenclature for chromatography

4. Classification of chromatography Classification based on mechanism of interaction of

solute with stationary phase Classification based on development technique

Classification based on physical state of mobile phase

5. The chromatogram Retention time

Capacity factor

Selectivity factor

1/28/2010 2


3/29

6. Theories of chromatography1) Plate theory (Theoretical plate theory)

Plate height (HETP) Resolution

Theory of band broadening

2) The rate theory

The van-Deemter equation Van Deemter plots

Resolution

7. Liquid chromatography

Stationary phase materialsEluotropic series and Eluents

Gradient and isochratic elution techniques

1/28/2010 3


4/29

8. High performance liquid chromatography (HPLC)Characteristics of a HPLC systemEssential components of HPLC

Pump & Injection systemsEluentColumn

Stationary phase : TypesNormal phaseBonded phase

Reversed phaseDetectors

SpectrophotometricRefractometricConductometric

9. Choice of Mode of HPLC10. Ion Chromatography; Reversed Phase Chromatography, Bonded

Phase Chromatography etc,

1/28/2010 4


5/29

11. Gas chromatography

1/28/2010 5


6/29

Definitions:Chromatography

is the collective term for a set of techniques forthe separation of mixtures.

It involves passing a mixture dissolved in a"mobile phase" through a stationary phase,

which separates the components in the mixturebased on differential partitioning between themobile and stationary phases.

Subtle differences in the partition coefficient ofcompounds results in differential retention onthe stationary phase and thus changing theseparation.

1/28/2010 6


7/29

In chromatography two or more solutes

distribute themselves between twoimmiscible phases, such as:

liquid/liquid system

liquid/solid systemgas/liquid system

gas/solid systems

During chromatography the varioussolutes show different affinities for the

individual phases.1/28/2010 7


8/29

History of chromatography The Russian botanist Mikhail Tswett has been credited with the

invention of chromatography. He used the technique to separateplant pigments (e.g. chlorophylls and xanthophylls) by passing

solution of them through glass columns packed with finely divided

CaCO3 .

Chromatography became developed substantially as a result of the

work of A.J.P. Martin and R.L.M. Synge during the 1940s and 1950s.They established the principles and basic techniques of partition

chromatography, and their work encouraged the rapid

development of several types of chromatography method: paper

chromatography, gas chromatography, and what would become

known as high performance liquid chromatography.

They were awarded the Nobel prize in Chemistry in 1950 for their

work the development of modern chromatography.

1/28/2010 8


9/29

NomenclatureRef: IUPAC Recommendation: Pure & Appl, Chem., Vol. 65, No. 4,

pp. 819 - 872, 1993.

1. Stationary Phase

The stationary phase is one of the two phases forminga chromatographic system. It may be a solid, a gel ora liquid.

If a liquid, it may be distributed on a solid support.This solid support may or may not contribute to theseparation process.

The liquid may also be chemically bonded to thesolid (Bonded Phase) or immobilized onto it(Immobilized Phase).

1/28/2010 9


10/29

2. Mobile Phase

A fluid which percolates through or along the

stationary bed, in a definite direction. It may be a liquid (LC) or a gas (GC) or a supercritical

fluid (S-F C).

In GC the expression Carrier Gas may be used for

the mobile phase. In elution chromatography the expression Eluent is

also used for the mobile phase.

3. Eluate (Effluent)The mobile phase leaving the column.

1/28/2010 10


11/29

4. Elute (verb):

To chromatograph by elution chromatography.

5. Chromatograph (verb):

To separate by chromatography

6. Chromatograph (noun):

The assembly of apparatus for carrying outchromatographic separation.

7. Chromatogram :

A graphical presentation of detector response (on

the ordinate) versus retention time or effluent

volume

In planar chromatography "chromatogram" may

refer to the paper or layer with the separated zones.1/28/2010 11


12/29

7. Retention time :

The time between sample injection and an analyte

peak reaching a detector at the end of the column.

8. Solute

A term referring to the sample components in

partition chromatography.

1/28/2010 12


13/29

CLASSIFICATION OF CHROMATOGRAPHY

1. according to Mechanisms of interaction of solute with

stationary phase:

Adsorption chromatography

Partition Chromatography

Ion-exchange chromatography

Size exclusion chromatography

Affinity chromatography

1/28/2010 13


14/29

Adsorption chromatography : Separation is based

mainly on differences between the adsorption

affinities of the sample components for the

surface of the solid stationary phase.

Here solute is adsorbed on the surface of the solid

stationary phase. The mobile phase is eitherliquid or gas. Equilibrium is established between

the mobile phase and stationary phase .

The solute with the higher adsorption affinity

remains longer on the stationary phase.

1/28/2010 14


15/29

Partition Chromatography :

In Liquid Chromatography: Separation is based

on differences between the solubilities of thecomponents in the mobile phase and liquidstationary phase

or

In Gas Chromatography (specifically, GLC):Separation is based mainly on differencesbetween the solubilities of the samplecomponents in the liquidstationary phase.

So the solute with the higher solubility in thestationary phase remain longer on the column.

1/28/2010 15


16/29

Ion-Exchange Chromatography

Here the stationary phase is a resin with such

functional groups as -COO- , -SO3-, that can exchangecations (cation exchanger) or,

the resin may contain -NR3+ and can exchange

anions (anion exchanger).

The functional groups are covalently bonded to theresin and the counter ions are attached byelectrostatic force.

Separation is based mainly on differences in the

ion exchange affinities of the sample components Modern ion-exchange chromatography on small particle,

high efficiency columns, and usually utilizingconductometric or spectroscopic detectors is oftenreferred to as Ion Chromatography (IC).1/28/2010 16


17/29

Size exclusion chromatography(SEC)

separates molecules according to their size (or

more accurately according to their

hydrodynamic diameter or hydrodynamic

volume) on porous gel (stationary phase).

Smaller molecules are able to enter the pores

of the stationary phase and, therefore, take

longer to elute, whereas larger molecules areexcluded from the pores and elute faster.

1/28/2010 17


18/29

SEC can be carried out using two differenttechniques:

a) Gel filtration chromatography when themobile phase is an aqueous solution.

b) Gel permeation chromatography when anorganic solvent is used as a mobile phase.

SEC is usually applied to separation of large

molecules or macromolecular complexes suchas proteins and industrial polymers

1/28/2010 18


19/29

Affinity chromatography

Employs a selective non-covalent interaction

between one kind of solute molecule and asecond that is covalently attached (immobilized)

to the stationary phase.

The method is most selective kind of chromatography

Example: the immobilized molecule (or ligand) may be

an antibody to particular protein. When a mixture of

many proteins is passed through the column, only the

one protein that reacts with the antibody is bound to thecolumn. The desired protein is dislodged from the

column after all the others have been washed away.

1/28/2010 19


20/29

Affinity chromatography is used in the separation of

biochemical mixtures, biomolecules, purification of

proteins etc. Ligand availability and the cost of the specialized

media are usually prohibitive at large-scale.

1/28/2010 20


21/29

2. Classification based on the method of Development of the

chromatogram : COLUMN or PLANAR Chromatography

1. Column Chromatography: is a separation technique in whichthe stationary phase is held within a tube called column.

In packed column the particles of the solid stationary phase or

the support coated with a liquid stationary phase may fill the

whole inside volume of the tube.

In open tubular column the stationary phase is

concentrated on or along the inside tube wall leaving an open,

unrestricted path for the mobile phase in the middle part of

the tube .

The mobile phase can be liquid (LC) or gas (GC) or super

critical fluid (SFC)

1/28/2010 21


22/29

2. Planar chromatography

is a separation technique in which the stationary phase is

present as or on a (two-dimensional) plane. The plane can bea paper, serving as such or impregnated by a substance as the

stationary phase (paper chromatography), or

a layer of solid particles spread on a support such as a glass

plate (thin layer chromatography).

The mobile phase moves through the stationary phase by

capillary action or by gravity.

Different compounds in the sample mixture travel different

distances according to how strongly they interact with the

stationary phase as compared to the mobile phase. The

specific Retention factor (Rf) of each chemical can be used to

aid in the identification of an unknown substance.

1/28/2010 22


23/29

1/28/2010 23

Diagram 1shows begin

of tlc experiment


24/29

1/28/2010 24

Diagram 2 shows the plate afterthe solvent has moved about

half way up it.


25/29

3. Classification based on type of mobile phase

In Liquid chromatography the mobile phase is always

liquidBut the stationary phase can be liquid or solid, and

the stationary phase can be held in a column as incolumn liquid operation or on a plate as in TLC or

paper chromatography.In liquid-bonded phase chromatography: the liquid

stationary phase is chemically bonded to a solidsupport. Usually the silanol group (Si-OH) of the

silica gel is reacted with dimethyl alkyl chlorosilane, (Cl-Si(CH3)2R. R can be varied butusually is octadecyl (C18H37)

1/28/2010 25


26/29

Gas chromatography (GC): Here themobile phase is a gas (carrier gas).

The stationary phase is a solid matrixinside a larger metal tube (apackedcolumn). Or

solid matrix adhered to the inside of asmall-diameter glass tube (a capillarycolumn oropen tubular column)

Liquid stationary phase adsorbed or

covalently bound onto a solid matrix andpacked or adhered to the inside of thecolumn (GLC)

1/28/2010 26


27/29

Supercritical Fluid Chromatography (SFC) Here the mobile phase is a supercritical fluid. This is a fluid

with properties between a gas and a liquid. Typical example iscarbon dioxide in its supercritical state.

SFC uses all the common instruments employed by GC and

LC in addition to those required to generate and maintain the

supercritical fluid. SFC is best suited for separating compounds that are not

volatile , or non-polar or that are thermally unstable. It is

intermediate between GC and LC.

Other advantages is that removal of the mobile phase is very

easy and environmental friendly.

1/28/2010 27


28/29

Supercritical Fluid

The critical point (CP) marks the end of the vapor liquid

coexistence curve. A fluid is termed supercritical when thetemperature and pressure are higher than the correspondingcritical values.

Above the critical temperature, there is no phase transition inthat the fluid cannot undergo a transition to a liquid phase,

regardless of the applied pressure. A supercritical fluid (SCF) is characterized by physical and

thermal properties that are between those of the pureliquid and gas.

The fluid density is a strong function of the temperature and

pressure. The diffusivity of SFC is much higher than for a liquid and SCF

readily penetrates porous and fibrous solids. Consequently, SCF can offer good catalytic activity.

1/28/2010

28


29/29

1/28/2010 29

Supercritical Fluids

chromatography intro

Documents