GEL PERMEATION CHROMATOGRAPHY

BY : VISHNU PRAJAPATI

SUB : CHEMISTRY...

CONTENTS

• AIM

• INTRODUCTION

• WORKING PRINCIPLE OF INSTRUMENT

• MATERIALS AND METHOD FOR SAMPLE ANALYSIS

• APPLICATIONS

AIM :TO STUDY ABOUT GEL PERMEATION CHROMATOGRAPHY.

INTRODUCTION

• CHROMATOGRAPHY IS A PHYSICAL METHOD OF SEPARATION IN WHICH

THE COMPONENTS TO BE SEPARATED ARE DISTRIBUTED BETWEEN TWO

PHASES, ONE OF WHICH IS STATIONARY (STATIONARY PHASE) WHILE

THE OTHER (THE MOBILE PHASE) MOVES IN A DEFINITE DIRECTION.

• TYPES OF CHROMATOGRAPHIC TECHNIQUES:

WORKING PRINCIPLE OF INSTRUMENT:

THE SEPARATION IS BASED STRICTLY ON THE SIZE OF THE SAMPLE IN

SOLUTION.

THERE SHOULD BE NO INTERACTION WITH THE COLUMN PACKING.

THE MODE OF SEPARATION IS NOT BASED ON MOLECULAR WEIGHT, BUT

ON THE SIZE OF THE MATERIAL BEING ANALYZED (USUALLY A POLYMER)

IN SOLUTION.

IN OTHER WORDS, TO DO GPC CORRECTLY, THE SAMPLE MUST BE

DISSOLVED IN A SUITABLE SOLVENT.

MATERIALS AND METHOD FOR SAMPLE ANALYSIS

Gel

• Gels are used as stationary phase for GPC.

• The pore size of a gel must be carefully controlled in order to be able to apply the gel to a

given separation.

• Other desirable properties of the gel forming agent are the absence of ionizing groups and,

in a given solvent, low affinity for the substances to be separated.

• Commercial gels like PLgel,Sephadex,Bio-Gel (crosslinked polyacrylamide) agarose

gel and Styragel are often used based on different separation requirements.

Eluent

• The eluent (mobile phase) should be a good solvent for the polymer.

• It should permit high detector response from the polymer and should wet the packing

surface.

• The most common eluents in for polymers that dissolve at room temperature GPC

are tetrahydrofuran (THF)

SCHEMATIC OF A BASIC GEL PERMEATION CHROMATOGRAPH

This diagram illustrates how the sample is injected into the mobile phase and the path the sample takes

to the detector.

A. sample holder,

B. Column

C. Pump

D. Refractive Index Detector

E. UV-vis Detector

A typical Waters GPC instrument including

COMPONENTS1. Pump

Pumps the polymer in solution through the system.

Different polymers produce solutions of different viscosities. To compare data from one analysis

to the next, the pump must deliver the same flow rates independent of viscosity differences

2. Injector

Introduces the polymer solution into the mobile phase.

The injector must be capable of small volume injections (for molecular weight determinations)

and large volume injections (if fraction collecting is desirable). The injector should not disturb

the continuous mobile phase flow. It should also be capable of automatic multiple sample

injection when the sample volume is large.

3. Column Set

Efficiently separates sample components from one another.

High efficiency columns give maximum separating capability and rapid analyses. Every

column must provide reproducible information over extended periods for both analytical and

fraction collecting purposes.

4. Detector

Monitors the separation and responds to components as they elute from the

column.

Detectors must be nondestructive to eluting components if they are to be collected

for further analysis.

The detectors must be sensitive and have a wide linear range in order to respond to

both trace amounts and large quantities of material if necessary.

5. Automatic data processing equipment

Automatically calculates, records, and report numerical values for Mz, Mw,

Mv, Mn, and MWD.

Data systems can also provide complete control of GPC systems so that large

numbers of samples can be run unattended and raw data can be automatically

processed. Today's GPC software offerings need to be able to provide special

calculations for multi-detection processing, band broadening correction, special

calibration routines and polymer branching determination, just to name a few.

GPC SEPARATION MECHANISM

POLYMER IS PREPARED AS A DILUTE SOLUTION IN THE ELUENT

AND INJECTED INTO THE SYSTEM

THE GPC COLUMN IS PACKED WITH POROUS BEADS OF

CONTROLLED POROSITY AND PARTICLE SIZE

LARGE MOLECULES ARE NOT ABLE TO PERMEATE ALL OF THE

PORES AND HAVE A SHORTER RESIDENCE TIME IN THE COLUMN

SMALL MOLECULES PERMEATE DEEP INTO THE POROUS

MATRIX AND HAVE A LONG RESIDENCE TIME IN THE COLUMN

POLYMER MOLECULES ARE SEPARATED ACCORDING TO

MOLECULAR SIZE, ELUTING LARGEST FIRST, SMALLEST LAST

APPLICATIONS

• IT IS USED TO DETERMINE THE RELATIVE MOLECULAR WEIGHT OF POLYMER

SAMPLES.

• IT IS USED TO DETERMINE THE DISTRIBUTION OF MOLECULAR WEIGHTS OF

POLYMER SAMPLES.

• SEPARATION OF SUGARS POLYPEPTIDES, PROTEINS, LIQUIDS, BUTYL RUBBERS,

POLYSTYRENES, SILICON POLYMERS.

• SEPHADEX G-25 : FOR SEPARATION OF SALTS & AMINO ACIDS FROM PROTEINS .

• SEPHADEX G-75:PURIFICATION OF PROTEINS POLYSACCHARIDES & NUCLEIC

ACIDS.