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

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Presentation on theme: "Kausar Ahmad Kulliyyah of Pharmacy, IIUM Physical Pharmacy 2 1 SIZE EXCLUSION CHROMATOGRAPHY."— Presentation transcript:

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

2 Contents Physical Pharmacy 2 2 Principles underlying chromatographic techniques Retention mechanism Diffusion Ficks law Types of size exclusion chromatography Gel permeation Gel filtration Applications Reliability of results

3 Objective of Separation Physical Pharmacy 2 3 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.

4 Some examples of separative techniques Physical Pharmacy Solvent extraction 2. Chromatography 3. Precipitation 4. Recrystallisation 5. Electrophoresis

5 Examples of chromatographic techniques 1. Ion-exchange chromatography 2. Size-exclusion chromatography 3. Paper chromatography 4. Thin layer chromatography 5. Affinity chromatography Physical Pharmacy 25

6 Paper Chromatography of Inks Physical Pharmacy 2 6 Inks from pens are chromatographed on paper using water as the mobile phase.

7 Chromatography of spinach extract Physical Pharmacy 2 7 Spinach extract is separated by thin layer chromatography into chlorophyll and B-carotene

8 TypeStationary phaseMobile phase Ion-exchange Based on charge Polymeric matrix – bonded with functional groups e.g. carboxylic acids, quarternary amines Liquid ionic solutes are retained by forming electrostatic chemical bonds Size-exclusion Based on size Polymeric substance with numerous pores Liquid or gaseous small solutes diffuse into pores, big solutes remain in mobile phase Affinity Based 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 Physical Pharmacy 28

9 Stationary Phase Physical Pharmacy 2 9 Semi-permeable due to porous structure of beads Semi-permeable polymer Degree of crosslinking is controlled to give different pore sizes Porous beads Define the FRACTIONATION RANGE molecules within that molecular weight range can be separated. Different pore size

10 Nature of Porous Material (stationary phase) Physical Pharmacy 2 10 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.

11 Types of Stationary Phase Physical Pharmacy 2 11 Soft gelse.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

12 Soft gels Physical Pharmacy 2 12 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.

13 Semirigid Gels Physical Pharmacy 2 13 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.

14 Mobile Phase Physical Pharmacy 2 14 The mobile phase contains a mixture of solutes. Small solutes will diffuse in and out of the pores (obeying Ficks law) Their path through the column is longer The elution time will be longer

15 Extent of retention Physical Pharmacy 2 15 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

16 Physical Pharmacy 2 16 Porous beads pores Totally excluded – eluted first Partially included Totally included – eluted last column

17 Common terms Physical Pharmacy 2 17 V 0, void volume, is the volume of mobile phase between the beads of the stationary phase inside the column V i, included volume, is the volume of mobile phase inside the porous beads V e = V o + Kv i K= 0 to 1

18 Procedure Physical Pharmacy 2 18 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

19 Equipment Equipment 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. ) Physical Pharmacy 2 19

20 Applications Physical Pharmacy 2 20 FractionationDesalting Concentration Molecular weight determination

21 Desalting Physical Pharmacy 2 21 Necessary for purification of biochemicals.Due to techniques involving buffers and precipitating reagents.Gel with low exclusion limit (MW ) 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.

22 Concentration of Dilute Solutions Physical Pharmacy 2 22 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.

23 Molecular Weight Determination Physical Pharmacy 2 23 Size is approximately proportional to molecular weight, M. Volume at which a solute is eluted, V R, can be expressed by: V R = a + b log M a and b are constants dependent on the mobile and stationary phases.

24 V R VS MW & K Physical Pharmacy 2 24

25 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 Physical Pharmacy 2 25

26 Separation based on size - precaution Physical Pharmacy 2 26 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.

27 Effect of Shape on Size Physical Pharmacy ProteinMyosinCytochrome C ShapeLong rodglobular MW530 Stokes radius

28 Advantages of Gel Filtration Physical Pharmacy 2 28 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.

29 Columns and Detectors Physical Pharmacy 2 29 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

30 Types of Column Physical Pharmacy 2 30 exclusion range for some common gel filtration chromatography media. Sephadex G kD Sephadex G kD Sephadex G kD Sephadex is a trademark of Pharmacia.

31 Column - example Physical Pharmacy 2 31 Trisacryl GF 05: Particle size 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.

32 How to check reliability? Physical Pharmacy 2 32 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.

33 Calibration for MW Determination Physical Pharmacy 2 33 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.

34 Physical Pharmacy 2 34 Calibration Kits TypeMolecular Weight Gel Filtration LMW Calibration Kit Ribonuclease A Chymotrypsinogen A Ovalbumin Bovine Serum Albumin Blue Dextran 2000 » Gel Filtration HMW Calibration Kit Aldolase Catalase Ferritin Thyroglobulin Blue Dextran 2000 »

35 Exercise Physical Pharmacy 2 35 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).

36 References Physical Pharmacy 2 36 AR Gennaro, Remington: The Science and Practice of Pharmacy 20 th 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, Atkins Physical Chemistry 7 th Ed., Oxford, New York (2002) Chapter 22 And others…..

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