GEL PERMEATİON CHROMATOGRAPHY

1. Gel Permeation
Chromatography
(GPC)
Prepared by:
Sami FARAJALLAH
Karadeniz Teknik Üniversitesi
Supervisor:
Prof. Dr. Murat KÜÇÜK

2. Chromatography is an important biophysical technique that enables the
separation, identification, and purification of the components of a mixture for
qualitative and quantitative analysis.
Chromatography Types
1. Gas Chromatography.
2. High Performance Liquid Chromatography.
3. Gel Permeation Chromatography.
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3. GPC
1. Size-exclusion chromatography (SEC)
2. Molecular sieve chromatography
3. Gel-filtration chromatography
4. Gel chromatography
5. molecular sieve chromatography
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4. Discovery of GPC
• İnvented in 1955 by Grant Henry Lathe and Colin R Ruthven, working at
Queen Charlotte's Hospital, London.
• Lathe and Ruthven used starch gels as the matrix.
• Jerker Porath and Per Flodin later introduced dextran gels ,agarose and
polyacrylamide.
• J. C. Moore of the Dow Chemical Company - preparation of (GPC) columns
based on cross-linked polystyrene
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5. Components of Gel Permeation Chromatography
1.Stationary Phase
2.The Mobile Phase
3.The Columns
4.The Pump
5.Detectors
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6. Applications of GPC
• For the analysis of large molecules such as proteins or polymers
• To analyze the molecular weight distribution of organic-soluble polymers.
• Other technique should not be confused with gel electrophoresis, where an electric
field is used to "pull" or "push" molecules through the gel depending on their
electrical charges.
• The amount of time a solute remains within a pore is dependent on the size of the pore.
Larger solutes will have access to a smaller volume and vice versa.
• A smaller solute will remain within the pore for a longer period of time compared to a
larger solute.
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7. Applications of GPC
• To examine the stability and characteristics of natural organic matter in
water.
• Widely utilized to study natural organic material.
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8. GPC Theory
• Molecules in solution are separated by their size or molecular weight.
• Applied to large molecules or macromolecular complexes such as proteins and
industrial polymers.
• Organic solvent is used as a mobile phase to transport the sample through the column.
• Column is packed with fine, porous beads, composed of dextran, agarose,
or polyacrylamide polymers.
• The pore sizes of these beads are used to estimate the dimensions of macromolecules.
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9. GPC Theory
• Works by trapping smaller molecules in the pores of the adsorbent
("stationary phase") in the column which typically consists of a hollow tube
tightly packed with micron-scale polymer beads containing pores of different
size.
• The larger the particles, the faster the elution. The larger molecules simply
pass by the pores because those molecules are too large to enter the pores .
• Widely used polymer characterization method because of its ability to
provide good molar mass distribution (Mw) results for polymers.
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10. GPC

11. GPC
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19. Chromatogram
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20. • The resulting chromatogram is therefore a weight distribution of
the polymer as a function of retention volume.
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22. Stationary phase
• It is composed of semi-permeable, porous polymer gel beads with a well-defined range of
pore sizes with the following properties:
1. Chemically inert
2. Mechanically stable
3. With ideal and homogeneous porous structure (wide pore size give low resolution).
4. A uniform particle and pore size
• EXAMPLE : Dextran (Sephadex) gel: An α 1-6-polymer of glucose natural gel.
• Agarose gel: A 1,3 linked β-D-galactose and 1,4 linked 3,6-anhydro-α, L-galactose natural gel.
• Acrylamide gel: A polymerized acrylamide, a synthetic gel.
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23. Mobile phase (Eluent )
• Composed of a liquid used to dissolve the bio-molecules to make the mobile phase
permit high detection response and wet the packing surface.
• Good solvent for the polymer, should permit high detector response from the
polymer.
• Common eluents for polymers that dissolve at room temperature.
• Tetrahydrofuran (THF), o-dichlorobenzene and trichlorobenzene at 130–150 °C for
crystalline polyalkynes and m-cresol.
• o-chlorophenol at 90 °C for crystalline condensation polymers such
as polyamides and polyesters.
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24. Pump
• There are two types of pumps available for uniform delivery of relatively
small liquid volumes for GPC:
1. piston pumps
2. peristaltic pumps
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25. PİSTON PUMP
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26. Peristaltic Pump
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27. Detector
• Detector types can be divided into two main categories.
1. Concentration sensitive detectors which includes UV
absorption, differential refractometer (DRI) or refractive index
(RI) detectors, infrared (IR) absorption and density detectors.
2. Molecular weight sensitive detectors, which include low angle
light scattering detectors (LALLS) and multi angle light
scattering (MALLS).
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28. Detector
• The most sensitive detector is the differential UV photometer
and the most common detector is the differential refractometer
(DRI).
• When characterizing copolymer, it is necessary to have two
detectors in series.
• For accurate determinations of copolymer composition at least
two of those detectors should be concentration detectors.
• The determination of most copolymer compositions is done
using UV and RI detectors, although other combinations can be
used.
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29. A size exclusion column
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30. Column
• Columns mostly made from hydrophilic gels
of dextran, agarose or polyacrylamide
• The column used for GPC is filled with a microporous packing
material called gel.
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31. Advantages of GPC
• Short analysis time.
• Well defined separation.
• Narrow bands and good sensitivity.
• There is no sample loss.
• The small amount of mobile phase required.
• The flow rate can be set.
• good separation of large molecules from the small molecules with a
minimal volume of eluate
• determining the molecular weight of polymer macromolecules.
• The separation is based on the analyte molecular sizes since the gel behaves
like a molecular sieve.
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32. Disadvantages of Gel Permeation chromatography
• The number of peaks that can be resolved within the short time frame of a
GPC run is small.
• For a satisfactory resolution of peaks, GPC as a technique needs at least a
10% difference in molecular weight.
• The molecular masses of most of the polymer chains are too close together
for the GPC separation to produce anything other than large peaks.
• For polymers it needs filtration prior to use to prevent dust and other
particulates from destroying the columns and interfering with the detectors.
• limited number of bands can be accommodated because the time scale of
the chromatogram is short, as 10% difference in molecular mass to have a
good resolution.
• This relative data can be used to calculate molecular weights within 5%
precision if comparable criteria are used.
• To calibrate the GPC, polystyrene standards with disparities of less than 1.2
are commonly used.
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33. GPGC(GAZA POWER GENERATİNG COMPANY)
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