Kausar Ahmad Kulliyyah of Pharmacy, IIUM Physical Pharmacy 2 1 SIZE EXCLUSION CHROMATOGRAPHY

Kausar AhmadKulliyyah of Pharmacy, IIUM

http://staff.iiu.edu.my/akausar

Physical Pharmacy 21

SIZE EXCLUSION CHROMATOGRAPHY

http://staff.iiu.edu.my/akausar

Contents


Principles underlying chromatographic techniquesRetention mechanism

○ DiffusionFick’s law

Types of size exclusion chromatographyGel permeationGel filtration

Applications Reliability of results

Objective of Separation


Proteins are extracted from animals and humans as a mixture in a serum of body fluids.

To study a specific protein, like an antibody, hormone, or enzyme, need to separate from the mix.

Some examples of separative techniques


1. Solvent extraction

2. Chromatography

3. Precipitation

4. Recrystallisation

5. Electrophoresis

Examples of chromatographic techniques

1. Ion-exchange chromatography

2. Size-exclusion chromatography

3. Paper chromatography

4. Thin layer chromatography

5. Affinity chromatography


Paper Chromatography of Inks


Inks from pens are chromatographed on paper using water as the mobile phase.

Chromatography of spinach extract


Spinach extract is separated by thin layer chromatography into chlorophyll and B-carotene

Type Stationary phase Mobile phase

Ion-exchangeBased on charge

Polymeric matrix – bonded with functional groups e.g. carboxylic acids, quarternary amines

Liquidionic solutes are retained by forming electrostatic chemical bonds

Size-exclusionBased on size

Polymeric substance with numerous pores

Liquid or gaseoussmall solutes diffuse into pores, big solutes remain in mobile phase

AffinityBased on biorecognition (ligand specificity)

A specific ligand e.g. antibody is bound to stationary phase

A mixture of solute containing an antigen, will bind strongly to the ligand


Stationary Phase


• Semi-permeable due to porous structure of beadsSemi-permeable

polymer

• Degree of crosslinking is controlled to give different pore sizesPorous beads

• Define the FRACTIONATION RANGE• molecules within that molecular

weight range can be separated.

Different pore size

Nature of Porous Material(stationary phase)


Porous material must swell up & imbibe/absorb the liquid phase

This created solvent-filled ‘sponge’ that allows diffusion of molecules

Therefore, stationary phase may be hydrophilic to imbibe aqueous media, or lipophilic to imbibe non-polar organic solvents.

Types of Stationary Phase


Soft gels

e.g. Polyacrylamide gels, dextran (natural glucose polymer)

• Separation of proteins

Semirigid or rigid gels

• e.g. 1) Polystyrene gels• Separation of non-polar polymers in non-polar solvents

• e.g. 2) Porous glass gels• Separation of polar systems

Soft gels


Before column is packed, gel is imbibed by enough liquid to completely swell.

• These gels are used with aqueous mobile phase.

Once column is packed, the composition of the mobile phase cannot be altered to prevent shrinkage or bursting of the packed column.

• Because of low structural strength, they cannot be used under high pressure.

classified as gel filtration.

Semirigid Gels


Made from crosslinked polystyrene, glass beads or alkylated dextran.

Used for separation of organic-soluble polymers.

Non-aqueous mobile phases e.g. chloroform, acetone, pyridine or tetrahydrofuran.

Classified as gel permeation.

Mobile Phase


The mobile phase contains a mixture of solutes.

Small solutes will diffuse in and out of the pores (obeying Fick’s law)

• Their path through the column is longer• The elution time will be longer

Extent of retention


extent of retention depends on size of the included molecules relative to the pores.

Smallest molecules

• enter all pores -> totally included -> FINAL peak

Intermediate molecules, due to velocity of mobile phase, will not be able to diffuse into the pores that they may fit, thus will be retained less effectively.

• Enter some pores -> partially included -> INTERMEDIATE peaks

Big molecules

• Could not enter any -> totally EXCLUDED -> INITIAL peak


Porous beads

pores

Totally excluded –eluted

first

Partially included

Totally included –eluted

last

column

Common terms


V0, void volume, is the volume of mobile phase between the beads of the stationary phase inside the column

Vi , included volume, is the volume of mobile phase inside the porous beads

Ve = Vo + Kvi

K= 0 to 1

Procedure


Equilibrate column

with mobile phase

Pass mobile phase

through column

Load sample

onto column & allow to

enter resin

Pass mobile phase

through column to separate &

elute sample

Collect fractions

eluted from column

Larger solutes elute

earlier and smaller

ones elute later

EquipmentEquipment for running size exclusion chromatography.The buffer is pumped through the column by a computer controlled device

Illustrative description of separation in SEC. (From Introduction to Modern Liquid Chromatography, 2nd edition by L. Snyder and J. J. Kirkland, © 1979 by John Wiley & Sons, Inc. )


Applications


Fractionation Desalting

ConcentrationMolecular

weight determination

Desalting


Necessary for purification of biochemicals.

Due to techniques involving buffers and precipitating reagents.

Gel with low exclusion limit (MW 1000-2000) is used.

Short column and high flow rate can be used because of the vast difference in size of solutes and contaminants.

Macromolecules will be eluted with little dilution and salts retained on the column.

Concentration of Dilute Solutions


Exclusion limit of gels less than MW of solutes.

Solution is mixed with a small quantity of dry gel that will absorb 10 to 20 times its weight in water.

Some salts and small molecules are taken up also.

Final macromolecules in a solution of almost unchanged pH and ionic strength but significantly decreased volume.

Molecular Weight Determination


Size is approximately proportional to molecular weight, M.

Volume at which a solute is eluted, VR,

can be expressed by:

VR = a + b log M

a and b are constants dependent on the mobile and stationary

phases.

VR VS MW & K


Partition coefficient, K

• glutamate dehydrogenase (totally excluded), K=0

• cytochrome c (totally included)K = 1

• other proteins, which are within the fractionation range for the column.

0 > K > 1


Separation based on size - precaution


Proteins are separated according to their molecular weight

• because this is the major contribution to molecular size.

However, the shape will affect its apparent size in solution.

Hence, gel filtration is NOT recommended for separating proteins with only a small difference in molecular weight.

Effect of Shape on Size


194.6

69.2

Protein Myosin Cytochrome CShape Long rod globularMW 530 530Stokes radius 194.6 69.2

Advantages of Gel Filtration


Can handle biomolecules that are sensitive to changes in pH, concentration of metal ions or harsh environmental conditions.

Separations can be performed in the presence of essential ions, detergents, urea,, at high or low ionic strength, at 37 °C or in the cold room according to the requirements of the experiment.

Columns and Detectors


Detection of the solute zones as they emerge from the column can be achieved by spectrophotometric monitoring of the eluate by measurement of refractive index of eluate

Collection of aliquots for later analysis

Mobile phase is allowed to flow by gravity

Very high flow rate not suitable for soft gels

Types of Column


exclusion range for some common gel filtration chromatography media.

Sephadex G-50 1-30 kD



Sephadex is a trademark of Pharmacia.

Column - example


Trisacryl GF 05: Particle size 40-80 µm exclusion limit 3,000 Da fractionation range 200-2,500 Da

Physical form: Aqueous suspension in 1 M NaCl and 20% ethanol

Application: Highly hydrophilic beaded poly(N-tris[hydroxymethyl]methyl acrylamide) suitable for medium pressure separations of small molecules and peptides. Highly resistant to acid environments, sensitive to strong alkaline agents.

How to check reliability?


Calibrate

Use standards

Choice of standards depends on application

Available in low and high molecular weight ranges supplied lyophilized in individual vials.

Kits include Blue Dextran 2000 to determine the column void volume and to check column packing.

Calibration for MW Determination


Calibrate using large molecule such as blue dextran to establish void volume of the system.

Use deuterium oxide or sucrose to determine retention time for a totally included solute.

Use a series of standard proteins or polymers to calibrate regions between these two limits.


Calibration Kits Type Molecular Weight

Gel Filtration LMW Calibration Kit

Ribonuclease A 13 700

Chymotrypsinogen A 25 000

Ovalbumin 43 000

Bovine Serum Albumin 67 000

Blue Dextran 2000 » 2 000 000

Gel Filtration HMW Calibration Kit

Aldolase 158 000

Catalase 232 000

Ferritin 440 000 Thyroglobulin 669 000

Blue Dextran 2000 » 2 000 000

Exercise


Consider the separation of a mixture of

glutamate dehydrogenase (MW 290,000),

lactate dehydrogenase (MW 140,000),

serum albumin (MW 67,000),

ovalbumin (MW 43,000),

cytochrome c (MW 12,400)

on a gel filtration column: fractionation range 15,000 - 150,000).

References


AR Gennaro, Remington: The Science and

Practice of Pharmacy 20th Ed., Lippincott

Williams & Wilkins (2000) Part 4

DG Peters, JM Hayes, GM Hieftje, Chemical

Separations and Measurements, Saunders,

Philadelphia(1974) Chapter 17

Peter Atkins & Julio de Paula, Atkin’s Physical

Chemistry 7th Ed., Oxford, New York (2002)

Chapter 22

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Kausar Ahmad Kulliyyah of Pharmacy, IIUM Physical Pharmacy 2 1 SIZE EXCLUSION CHROMATOGRAPHY