size exclusion chromatography

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SIZE EXCLUSION CHROMATOGRAPHY:

Maulik kapadiya Department of pharmaceutics M.Pharm sem-1 Atmiya Institute of Pharmacy Rajkot SIZE EXCLUSION CHROMATOGRAPHY Size Exclusion Chromatography 1

Contents:

Contents Introduction Principle Objective Material Instrumentation Advantage Disadvantage Application Gel Electrophoresis Size Exclusion Chromatography 2

Introduction:

Introduction Size-exclusion chromatography (SEC) is a chromatographic method in which molecules in solution are separated based on their size. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. Large sample molecules cannot or can only partially penetrate the pores, whereas smaller molecules can access most or all pores. Thus, large molecules elute first, smaller molecules elute later, while molecules that can access all the pores elute last from the column. Size Exclusion Chromatography 3

Slide 4:

When an aqueous solution is used to transport the sample through the column, the technique is known as Gel-filtration chromatography . When an organic solvent is used as a mobile phase,the technique is known as Gel-permeation chromatography. Size Exclusion Chromatography 4

Principle:

Principle SEC separates based on the size or hydrodynamic volume of the analytes. This differs from other separation techniques which depend upon chemical or physical interactions to separate analytes. Separation occurs via the use of porous beads packed in a column. Size Exclusion Chromatography 5

Slide 6:

Size Exclusion Chromatography The smaller analytes can enter the pores more easily and therefore spend more time in these pores, increasing their retention time. Conversely, larger analytes spend little if any time in the pores and are eluted quickly. All columns have a range of molecular weights that can be separated. There is a limited range of molecular weights that can be separated by each column and therefore the size of the pores for the packing should be chosen according to the range of molecular weight of analytes to be separated. 6

Slide 7:

Size Exclusion Chromatography If an analyte is either too large or too small it will be either not retained or completely retained respectively. Analytes that are not retained are eluted with the free volume outside of the particles (V o ), while analytes that are completely retained are eluted with volume of solvent held in the pores (V i ). The total volume can be considered by the following equation Vt = Vg + Vi + Vo where ,V g is the volume of the polymer gel V t is the total volume 7

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Size Exclusion Chromatography 8

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Porous beads pores Totally excluded –eluted first Partially included Totally included –eluted last column Size Exclusion Chromatography 9

Objective of Spearation:

Objective of Spearation Proteins are extracted from animals and humans as a mixture in a serum of body fluids. When immunologists want to study a specific protein, like an antibody, hormone, or enzyme, they need to separate it from the mixture. Size Exclusion Chromatography 10

Materials:

Materials Stationary phase Mobile phase Size Exclusion Chromatography 11

Stationary Phase:

Stationary Phase Semi-permeable, porous beads with well-defined range of pore sizes . Beads are crosslinked polymers Degree of crosslinking is controlled carefully to yield different pore sizes Property includes a fractionation range (due to the different pore sizes) molecules within that molecular weight range can be separated. Size Exclusion Chromatography 12

Nature of Porous Material (stationary phase):

Nature of Porous Material (stationary phase) Porous material must swell up and imbibe/absorb the liquid phase The created solvent-filled ‘sponge’ allows diffusion of molecules Therefore, stationary phase may be hydrophilic to imbibe aqueous media, or lipophilic to imbibe non-polar organic solvents. Size Exclusion Chromatography 13

Types of Stationary Phase:

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 Size Exclusion Chromatography 14

Soft gels:

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 . Size Exclusion Chromatography 15

Semirigid Gels:

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. Size Exclusion Chromatography 16

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Size Exclusion Chromatography 17

Mobile Phase:

Mobile Phase Size Exclusion Chromatography Materials Solvents Synthetic elastomers ( polybutadiene , polyisoprene ) Toluene PS, PVC, Styrene-Butadiene Rubber, Epoxy resins Tetrahydrofuran Polyolefins Tri- chloro -benzene Polyurethane Di- methylformamide Proteins, polysaccharides Water / Buffers 18

Mobile Phase Preparation :

Mobile Phase Preparation Filter and degas mobile phase Filter mobile phase solvents using 0.5 micron filter to remove any particular impurities such as dusts, insoluble salts. This is to prevent any clog on columns. Gas in solvent contributes to baseline drift in RI response. Additives in mobile phase Antioxidant is added to trichlorobenzene to keep solvent stable in high temperature. Other additives eliminate adsorption or interaction of solutes with column packing materials Size Exclusion Chromatography 19

Sample Preparation:

Sample Preparation Concentration About 1mg/ml, usually less than 3mg/ml Samples with broad molecular weight distribution may require higher concentrations. Injection volume 50 – 100 µL/column Size Exclusion Chromatography 20

Slide 21:

Size Exclusion Chromatography Agitation and filtration Generally filtration is required to remove insoluble impurities and agitation is allowed. Do not agitate and filter samples that contain very high MW (>1 million). Consider centrifugation. 21

Instrumentation:

Instrumentation Injection valve Pump Column Detector Recorder Size Exclusion Chromatography 22

Types of Column:

Types of Column Size Exclusion Chromatography Column type Column dimension I.D.x length [mm] Analytical 8 x 300 8 x 600 Micro 4.6 x 250 Preparative 20 x 300 20 x 600 40 x 250 23

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The first column contains Sephadex G-75, which separates blue dextran and hemoglobin. The second column contain Sephadex G-10, which separates hemoglobin and riboflavin. Because there is a difference in the two packing materials, the hemoglobin molecule runs very differently in the two columns. Column 2 Size Exclusion Chromatography Column 1 24

Column - example:

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. Size Exclusion Chromatography 25

Detector:

Detector In GPC, the concentration by weight of polymer in the eluting solvent may be monitored continuously with a detector Types Concentration sensitive detectors : UV absorption,refractive index (RI) detectors, Infrared (IR) absorption and density detectors. 2) Molecular weight sensitive detectors : Low angle light scattering detectors (LALLS), Multi angle light scattering (MALLS). : Size Exclusion Chromatography 26

Schematic diagram of GPC system:

Schematic diagram of GPC system Size Exclusion Chromatography 27

Instrumental Diagram of GFC system:

Instrumental Diagram of GFC system Size Exclusion Chromatography A . sample holder, B .Column C .Pump D . Refractive Index Detector E . UV-vis Detector 28

Chromatogram:

Chromatogram extent of retention depends on size of the included molecules relative to the pores. Smallest molecules will enter all pores . 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. Initial peak contains totally EXCLUDED solutes . Final peak contains totally included solutes . Size Exclusion Chromatography 29

Description of Separation in SEC:

Description of Separation in SEC Size Exclusion Chromatography 30

Advantages of Gel Filtration:

Advantages of Gel Filtration Rapid, routine analysis Identify high mass components even in low concentration Can analyze polydisperse samples Branching studies can be done Absolute molecular weights can be obtained Size Exclusion Chromatography 31

Slide 32:

Size Exclusion Chromatography Disadvantages Filtrations must be performed before using the instrument to prevent dust and other particulates from ruining the columns and interfering with the detectors. Bad response for very small molecular weights Standards are needed. Sensitive for flow rate variation. Internal standard should be used whenever possible. High Investment cost 32

Applications :

Applications Purification Molecular weight Determination Concentration of Dilute Solutions Desalting Protein Binding study Size Exclusion Chromatography 33

Purification:

Purification Purification of viruses, proteins, enzymes, hormones, nucleic acids, antibodies and polysaccharides Separation of low molecular weight compound from mixtures e.g. Separation of Dextran from corn syrup oil Size Exclusion Chromatography 34

Molecular Weight Determination:

Molecular Weight Determination Size is approximately proportional to molecular weight, M. Elution time , V E , can be expressed by: V E = a + b log M a and b are constants Size Exclusion Chromatography 35

Concentration of Dilute Solutions:

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 . Size Exclusion Chromatography 36

Desalting:

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. Size Exclusion Chromatography 37

Protein Binding Study:

Protein Binding Study Reversible binding of a ligand to a macromolecule such as a protein including receptor proteins A sample of protein/ligand mixture is applied to column of gel which has previously equilibrated with solution of a ligand of same concentration in mixture Sample is eluted with buffer and concentration of ligand and protein in the effluent are determined Size Exclusion Chromatography 38

Gel electrophoresis:

Gel electrophoresis Gel electrophoresis is a technique used for the separation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein molecules using an electric field applied to a gel matrix. DNA Gel electrophoresis is usually performed for analytical purposes, often after amplification of DNA via PCR , but may be used as a preparative technique prior to use of other methods such as mass spectrometry , RFLP , PCR , cloning , DNA sequencing , or Southern blotting for further characterization. Size Exclusion Chromatography 39

Separation:

Separation Gel is used as matrix to separate target molecules. Gel is a crosslinked polymer whose composition and porosity is chosen based on the specific weight and composition of the target to be analyzed. When separating proteins or small nucleic acids ( DNA , RNA , or oligonucleotides ) the gel is usually composed of different concentrations of acrylamide and a cross-linker , producing different sized mesh networks of polyacrylamide . Size Exclusion Chromatography 40

Slide 41:

Size Exclusion Chromatography When separating larger nucleic acids the preferred matrix is purified agarose. " Electrophoresis " refers to the electromotive force (EMF) that is used to move the molecules through the gel matrix. By placing the molecules in wells in the gel and applying an electric field, the molecules will move through the matrix at different rates, determined largely by their mass when the charge to mass ratio (Z) of all species is uniform, toward the anode if negatively charged or toward the cathode if positively charged. 41

Visualization:

Visualization After the electrophoresis is complete, the molecules in the gel can be stained to make them visible. Ethidium bromide , silver, or Coomassie Brilliant Blue dye may be used for this process. If the analyte molecules fluoresce under ultraviolet light, a photograph can be taken of the gel under ultraviolet lighting conditions, often using a Gel Doc . Size Exclusion Chromatography 42

Application:

Application Proteins are usually analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( SDS-PAGE ), by native gel electrophoresis , by Quantitative Preparative Native Continuous polyacrylamide gel electrophoresis ( QPNC-PAGE ), or by 2-D electrophoresis . Gel electrophoresis of large DNA or RNA is usually done by agarose gel electrophoresis . Size Exclusion Chromatography 43

Slide 44:

Size Exclusion Chromatography Electrophoresis of RNA samples can be used to check for genomic DNA contamination and also for RNA degradation. 44

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Size Exclusion Chromatography