8/13/2019 2nd week 111113

1/43

8/13/2019 2nd week 111113

2/43

8/13/2019 2nd week 111113

3/43

tr

RD

to=tm tr B tr A

A

B

Chromatogram:

the record of a

separation

produced by a

recorder or

integrator based

on the signalobtained from

the detector.

Large K, more time spent in the stationary phase,more time spent in the coloumn (Tr, time of retention)

Chromatography

Classification ???

8/13/2019 2nd week 111113

4/43

Chromatographyis

a separation technique

based on the different

interactions of

compounds with two

phases,

a mobile phaseanda stationary phase,

as the compounds

travel through a

supporting medium.

mobile phase:

Liquid /gas that flows through

the supporting medium

stationary phase:

2. Liquid a layer coating on the

supporting medium (solid support)that interacts with the analytes

supporting medium (solid support) is

An inert solid on which the

stationary phase is bound orcoated

1. Solid adsorbent

8/13/2019 2nd week 111113

5/43

8/13/2019 2nd week 111113

6/43

Phases involved

Mob. Phase Stat. Phase Type of Chrmt

Gas Solid GSC

Gas Liquid GLC

Liquid Solid LSC

Liquid Liquid LLC

8/13/2019 2nd week 111113

7/43

Geometry of the system

1. Column chromatography the stationary phase is

contained in a tube called the column.

2. Planar chromatography

(thin-layer chromatography)

a thin film of a stationary phase of solid particles bound

together for mechanical strength with a binder, such as

calcium sulfate, is coated on a glass plate or plastic ormetal sheet.

8/13/2019 2nd week 111113

8/43

Principle of separation

1. Adsorption chromatography

2. Partition chromatography

3. Ion-exchange chromatography

4. Exclusion chromatography

5. Affinity chromatography

8/13/2019 2nd week 111113

9/43

Mode of operation (Development)

Elution

Frontal

Displacement

8/13/2019 2nd week 111113

10/43

PhaseL S Chrmtg

G S Chrmtg

L L Chrmtg

G L Chrmtg

Coloum Planar Ads Prts IE Ekls Aff Eltn Frntl Dspl

GC

HPLC

ICSCC

TLC

STLC Analytical Chromatography

Preparative Chromatography

Normal Phase Chromatography Reverse Phase Chromatography

8/13/2019 2nd week 111113

11/43

8/13/2019 2nd week 111113

12/43

8/13/2019 2nd week 111113

13/43

Ion-exchange chromatography Chromatography in which separation is basedmainly on differences in the ion-exchange

affinities of the sample components.

In this type of chromatography, the

use of a resin (the stationary solidphase) is used to covalently attach

anions or cations onto it.

Solute ions of the opposite charge in

the mobile liquid phase are attracted

to the resin by electrostatic forces.

8/13/2019 2nd week 111113

14/43

8/13/2019 2nd week 111113

15/43

8/13/2019 2nd week 111113

16/43

Exclusion Chromatography /

gel-permeation chromatography (GPC)/

size-exculsion chromatography (SEC) /

gel-filtration

A separation technique in which separation

mainly according to the hydrodynamic volume

of the molecules or particles takes place in a

porous non-adsorbing material with pores of

approximately the same size as the effectivedimensions in solution of the molecules to be

separated.

This type of chromatography lacks an

attractive interaction between the

stationary phase and solute.

The liquid or gaseous phase passes

through a porous gel which separates

the molecules according to its size.

The pores are normally small and

exclude the larger solute molecules,

but allows smaller molecules to enter

the gel, causing them to flow through

a larger volume.

This causes the larger molecules to

pass through the column at a faster

rate than the smaller ones.

8/13/2019 2nd week 111113

17/43

8/13/2019 2nd week 111113

18/43

Exclusion Chromatography

In exclusion chromatography/gel-filtration chromatography,proteins migrate as a function of their molecular weights.

The solid matrix (beads) contains pores of various sizes. The

probability of entering the pores of the matrix is inversely

proportional to the size of the protein. In fact proteins that are

larger than a given size (depending on the resin that is used) are

totally excluded from entering the beads. Therefore, larger proteinshave a more direct route to the bottom of the column, by simply

going around all of the beads rather than entering the beads.

Notice in the figure at left that the large molecules elute first.

Size-exclusion chromatography can be used to determine the

molecular weight of a particular protein if appropriate standards are

available. As well see in a couple of slides, the elution volume is

inversely proportional to the log of the molecular weight.

8/13/2019 2nd week 111113

19/43

Exclusion chromatography

A separation technique in which

separation mainly according to the

hydrodynamic volume of the

molecules or particles takes place in a

porous non-adsorbing material with

pores of approximately the same size

as the effective dimensions in

solution of the molecules to be

separated.

8/13/2019 2nd week 111113

20/43

Size-exculsion chromatography(SEC), also called gel-filtration or gel-

permeation chromatography (GPC), uses porous particles to separate

molecules of different sizes.

It is generally used to separate biological molecules, and to determine

molecular weights and molecular weight distributions of polymers. Molecules that are smaller than the pore size can enter the particles and

therefore have a longer path and longer transist time than larger molecules

that cannot enter the particles.

Molecules larger than the pore size can not enter the pores and elute

together as the first peak in the chromatogram.

This condition is called total exclusion.

Molecules that can enter the pores will have an average residence time in

the particles that depends on the molecules size and shape.

Different molecules therefore have different total transit times through the

column.

This portion of a chromatogram is called the selective permeation region. Molecules that are smaller than the pore size can enter all pores, and have

the longest residence time on the column and elute together as the last peak

in the chromatogram.

This last peak in the chromatogram determines the total permeation limit.

http://www.chemistry.adelaide.edu.au/external/soc-rel/content/chromato.htmhttp://www.chemistry.adelaide.edu.au/external/soc-rel/content/chromato.htm

8/13/2019 2nd week 111113

21/43

Affinity chromatographyThis uses a specific ligand (such as an

antigen) bonded to the stationary phase

to separate a specific substance such as

the corresponding antibody.

This is the most selective type of

chromatography employed.

It utilizes the specific interaction between

one kind of solute molecule and a second

molecule that is immobilized on a

stationary phase. For example, the immobilized molecule

may be an antibody to some specific

protein.

When solute containing a mixture of

proteins are passed by this molecule, only

the specific protein is reacted to this

antibody, binding it to the stationary

phase.

This protein is later extracted by changing

the ionic strength or pH.

8/13/2019 2nd week 111113

22/43

Affinity Chromatrography

In affinity chromatography,

proteins are separated according totheir ability to bind to a specific

ligand that is connected to the

beads of the resin.

After the proteins that do not bindthe ligand are washed through the

column, the bound protein of

interest is eluted by a solution

containing free ligand.

El ti

8/13/2019 2nd week 111113

23/43

Elution

Fasa

Diam

Sampel 1X FASAGERAK

SAMPLE

8/13/2019 2nd week 111113

24/43

Based on the type of sample development:

Frontal development Displacement

development

Elution development

(under equilibrium)

(with displacer in the

Mobile phase)

(solutes continuously

Introduced with mobile

phase).

8/13/2019 2nd week 111113

25/43

8/13/2019 2nd week 111113

26/43

separation of mixtures due to differences

in the distribution coefficient / partition coefficient

(the equilibrium constant)

of sample components between 2 different phases

(mobile phase and stationary phase)

Distribution Coefficient (the equilibrium constant )

Concentration molar of component X in stationary phase

Concentration molar of component X in mobile phaseKx=

8/13/2019 2nd week 111113

27/43

X

X

X

Distribution of analytes between phases

[Cx]s

Kx = -----------

[Cx]m

K (The equilibrium constant) is termed the distribution coefficient;

defined as the molar concentration of analyte (x) in the stationary

phase (Cx)s divided by the molar concentration of the analyte in the

mobile phase (Cx)m

MP

SP

Migration of Analyte (X)

8/13/2019 2nd week 111113

28/43

8/13/2019 2nd week 111113

29/43

8/13/2019 2nd week 111113

30/43

8/13/2019 2nd week 111113

31/43

Classification of column chromatography

General classification Specific method Stationary phase Type of equilibrium

Gas chromatography

(GC)

Gas-liquid

Gas-solid

Liquid adsorbed or

bonded to a solid

surface

Solid

Partition between gas

and liquid

Adsorption

Liquid chromatography

(LC)

Liquid-liquid

Liquid-solid

Ion exchange

Size exclusion

Affinity

Liquid adsorbed or

bonded to a solid

surface

Solid

Ion exchange resin

Porous polymers

Bonded specific

ligand

Partition

Adsorption

Ion exchange

Partition/sieving

Partition

Supercritical fluid

chromatography

(SFC)

Organic species

bonded to a solid

surface

Partition

8/13/2019 2nd week 111113

32/43

8/13/2019 2nd week 111113

33/43

8/13/2019 2nd week 111113

34/43

SOLVENTS

Polar Solvents

Water > Methanol > Acetonitrile > Ethanol >

Oxydipropionitrile

Non-polar Solvents

N-Decane > N-Hexane > N-Pentane >

Cyclohexane

8/13/2019 2nd week 111113

35/43

MECHANISM OF ION-EXCHANGE CHROMATOGRAPHY OF

AMINO ACIDS

SO3-

SO3-

a+

COO-

H3+

a+

COOH

H3+

pH2

pH4.5

Ion-exchange Resin

8/13/2019 2nd week 111113

36/43

ION-EXCHANGE CHROMATOGRAPHY

SO3-

Na+

Separation in Ion-exchange Chromatography is based on the competition of different

ionic compounds of the sample for the active sites on the ion-exchange resin

(column-packing).

8/13/2019 2nd week 111113

37/43

LIQUID SOLID CHROMATOGRAPHY

Si - O - H

Normal phase LS

Reverse phase LS

Silica Gel

The separation mechanism in LSC is based on the

competition of the components of the mixture

sample for the active sites on an absorbent such as

8/13/2019 2nd week 111113

38/43

LIQUID SOLID CHROMATOGRAPHY

Si - OH

HEXANE

OH

C-CH3

CH3

CH3- C

CH3

CH3

OH

OH

CH3

CH3

8/13/2019 2nd week 111113

39/43

8/13/2019 2nd week 111113

40/43

Is a technique used to separate andidentify the components of a mixture.

Works by allowing the molecules present in themixture to distribute themselves between astationary and a mobile medium.

We can use chromatography to separate thecomponents of inksand dyes, such as those found in

pens, markers, clothing, and even candy shells.

Chromatography can also be used to separate the

colored pigments in plants or used to determine the

chemicalcompositionof many substances.

8/13/2019 2nd week 111113

41/43

The history

Chromatography was invented by the Russian botanist Mikhail Tswett in 1903. He

employed the technique to separate various plant pigments, including chlorophylls

andxanthophylls, by passing the extract through a glass column packed with finely

divided calcium carbonate.

Mikhail Tswett

8/13/2019 2nd week 111113

42/43

8/13/2019 2nd week 111113

43/43