SIZE EXCLUSION CHROMATOGRAPHY Physical Pharmacy 2 4/1/2017 SIZE EXCLUSION CHROMATOGRAPHY Kausar Ahmad Kulliyyah of Pharmacy, IIUM http://staff.iiu.edu.my/akausar Physical Pharmacy 2 KBA

Contents Principles underlying chromatographic techniques Physical Pharmacy 2 4/1/2017 Contents Principles underlying chromatographic techniques Retention mechanism Diffusion Fick’s law Types of size exclusion chromatography Gel permeation Gel filtration Applications Reliability of results Physical Pharmacy 2 KBA

Objective of Separation Physical Pharmacy 2 4/1/2017 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. Physical Pharmacy 2 KBA

Some examples of separative techniques Physical Pharmacy 2 4/1/2017 Some examples of separative techniques Solvent extraction Chromatography Precipitation Recrystallisation Electrophoresis Physical Pharmacy 2 KBA

Examples of chromatographic techniques Physical Pharmacy 2 4/1/2017 Examples of chromatographic techniques Ion-exchange chromatography Size-exclusion chromatography Paper chromatography Thin layer chromatography Affinity chromatography Physical Pharmacy 2 KBA

Paper Chromatography of Inks Physical Pharmacy 2 4/1/2017 Paper Chromatography of Inks Inks from pens are chromatographed on paper using water as the mobile phase. Physical Pharmacy 2 KBA

Chromatography of spinach extract Physical Pharmacy 2 4/1/2017 Chromatography of spinach extract Spinach extract is separated by thin layer chromatography into chlorophyll and B-carotene Physical Pharmacy 2 KBA

ionic solutes are retained by forming electrostatic chemical bonds Physical Pharmacy 2 4/1/2017 Type Stationary phase Mobile 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 2 KBA

Semi-permeable polymer Physical Pharmacy 2 4/1/2017 Stationary Phase 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 Physical Pharmacy 2 KBA

Nature of Porous Material (stationary phase) Physical Pharmacy 2 4/1/2017 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. Physical Pharmacy 2 KBA

Types of Stationary Phase Physical Pharmacy 2 4/1/2017 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 Physical Pharmacy 2 KBA

Physical Pharmacy 2 4/1/2017 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. Physical Pharmacy 2 KBA

Physical Pharmacy 2 4/1/2017 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. Physical Pharmacy 2 KBA

Mobile Phase The mobile phase contains a mixture of solutes. Physical Pharmacy 2 4/1/2017 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 Physical Pharmacy 2 KBA

Physical Pharmacy 2 4/1/2017 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 Physical Pharmacy 2 KBA

Totally included –eluted last Physical Pharmacy 2 4/1/2017 Porous beads column Totally included –eluted last Partially included pores Totally excluded –eluted first The above animation describes the duration of molecules of different sizes in the column. Small molecules will diffuse into the porous beads due to concentration gradient. As mobile phase is continuously supplied, concentration gradient is reestablished. Physical Pharmacy 2 KBA

Common terms Ve = Vo + Kvi K= 0 to 1 Physical Pharmacy 2 4/1/2017 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 Ve = Vo + Kvi If K=0, i.e. the molecule cannot get into any of the pores, and thus totally excluded, Ve = Vo Hence, eluted volume = void volume. If K=1, i.e. the molecule gets into ALL the pores, and thus totally included, Ve = Vo + Vi GO TO slide 23 & 24 Physical Pharmacy 2 KBA

Procedure Equilibrate column with mobile phase Physical Pharmacy 2 4/1/2017 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 SMALL proteins can fit inside all the pores in the beads: included. have access to mobile phase inside the beads & mobile phase between beads and elute last LARGE proteins that cannot fit into any of the pores: excluded have access only to the mobile phase between the beads: elute first Proteins of intermediate size are partially included - can fit inside some of the pores in the beads. These proteins will then elute between the large ("excluded") and small ("totally included") proteins. Physical Pharmacy 2 KBA

Equipment Physical Pharmacy 2 Physical Pharmacy 2 4/1/2017 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 KBA

Molecular weight determination Physical Pharmacy 2 4/1/2017 Applications Fractionation Desalting Concentration Molecular weight determination Physical Pharmacy 2 KBA

Desalting Necessary for purification of biochemicals. Physical Pharmacy 2 4/1/2017 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. Physical Pharmacy 2 KBA

Concentration of Dilute Solutions Physical Pharmacy 2 4/1/2017 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. Exclusion limit of gel is less than MW of solutes. The stationary phase has a fractionation range. Solutes that are bigger than the biggest pore are EXCLUDED. Thus, to do concentration of dilute solutions, the solutes must not enter any of the pores, i.e. they must be totally excluded. This can only be achieved if the size of pores are smaller than the smallest solute. OR, the size of the biggest pore is smaller than the MW of solutes. Physical Pharmacy 2 KBA

Molecular Weight Determination Physical Pharmacy 2 4/1/2017 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. From slide 17, Volume at which solute is eluted, Ve = Vo + Kvi Using blue dextran to calibrate to get Vo, Ve = Vr = Vo And using sucrose to calibrate to get Vi, Ve = Vo + Vi, Hence, Vi = Ve – Vo MW of blue dextran and sucrose is known. Thus a & b, can be resolved Physical Pharmacy 2 KBA

VR VS MW & K Physical Pharmacy 2 Physical Pharmacy 2 4/1/2017 Vr = a + b (log M) Vr is determined from experiment. Thus the molecular weight, M, can be calculated, or obtained from the plot. Physical Pharmacy 2 KBA

Partition coefficient, K Physical Pharmacy 2 4/1/2017 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 KBA

Separation based on size - precaution Physical Pharmacy 2 4/1/2017 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. Physical Pharmacy 2 KBA

Effect of Shape on Size Protein Myosin Cytochrome C Shape Long rod Physical Pharmacy 2 4/1/2017 Effect of Shape on Size Protein Myosin Cytochrome C Shape Long rod globular MW 530 Stokes radius 194.6 69.2 194.6 69.2 Physical Pharmacy 2 KBA

Advantages of Gel Filtration Physical Pharmacy 2 4/1/2017 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. Physical Pharmacy 2 KBA

Physical Pharmacy 2 4/1/2017 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 Physical Pharmacy 2 KBA

Physical Pharmacy 2 4/1/2017 Types of Column exclusion range for some common gel filtration chromatography media. Sephadex G-50 1-30 kD Sephadex G-100 4-150 kD Sephadex G-200 5-600 kD Sephadex is a trademark of Pharmacia. Physical Pharmacy 2 KBA

Physical Pharmacy 2 4/1/2017 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. What is the difference between exclusion limit and fractionation range? How do you modify the exclusion limit? Physical Pharmacy 2 KBA

How to check reliability? Physical Pharmacy 2 4/1/2017 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. Physical Pharmacy 2 KBA

Calibration for MW Determination Physical Pharmacy 2 4/1/2017 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. Physical Pharmacy 2 Plots of calibration curves for standard proteins. The data were obtained on the column and under the conditions described in Figure 5.5.1. The protein standards were thyroglobulin, apoferritin, yeast alcohol dehydrogenase, serum albumin, ovalbumin, myoglobin, and insulin. Stokes radii for these proteins are listed in Table 5.5.3(except for alcohol dehydrogenase, with RS = 4.55 nm). (A) A plot of RS versus Kd in which all the data were used. (B) A plot of log RS versus Kd. In the latter plot the top and bottom values of Kd (thyroglobulin and insulin) were omitted. Vo was determined to be 6.67 ml from the elution peak of blue dextran.Vt was determined to be 12.28 ml from the elution peak of sodium azide. KBA

Calibration Kits Type Molecular Weight Physical Pharmacy 2 4/1/2017 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  For media that have very large fractionation range, blue dextran cannot be used to determine a void volume. Alternative methods include the use of raw dextrans (MW 50 MDa) polystyrene microspheres Physical Pharmacy 2 KBA

Exercise Consider the separation of a mixture of Physical Pharmacy 2 4/1/2017 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). When the protein mixture is applied to the column, glutamate dehydrogenase would elute first because it is above the upper fractionation limit. Therefore it is totally excluded from the inside of the porous stationary phase and would elute with the void volume (V0). Proteins larger than the exclusion range of the resin (i.e. not within the fractionation range) are unable to enter the pores and pass quickly through the column in the spaces between the resin. This is known as the void volume of the column. Cytochrome c is below the lower fractionation limit and would be completely included, eluting last. The other proteins would be partially included and elute in order of decreasing molecular weight. Physical Pharmacy 2 KBA

Physical Pharmacy 2 4/1/2017 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 http://www.chromatography.amershambiosciences.com/ And others….. Physical Pharmacy 2 KBA