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Gel filtration Principles and Methods
1 Gel filtration Principles and Methods
Contents lntroducetion n abbreviations Chapter 1 Gel filtration in practice 9 Introduction. Separation by gel filtration Resolution in gel filtration 13 da elecrto Buffer composition and Denaturing (chaotropic)agents and detergents. Column and media preparation . 33 Sample application . .23 Elution and flow rates. .24 Method development for high resolution fractionation. 26 Care of gel filtration media .27 Equipment selection 27 Scaling up BioProcess Media for large-scale production Troubleshooting. .29 Chapter 2 Gel filtration media Components of gel filtration media. Superdex:first choice for high resolution,short run times and high recovery. 6 Separation options. aration examples 43 Media characteristics Chemical stability 44 torage. Sephacryl:fast,high recovery separations at laboratory and industrial scale . eparation options Media characteristics 51 Chemical stability. 51 storage. Superose:broad fractionation range,but not suitable for industrial scale separations.52
2 Contents Introduction . 5 Symbols . 6 Common abbreviations . 7 Chapter 1 Gel filtration in practice . 9 Introduction . 9 Separation by gel filtration . 9 Resolution in gel filtration . 13 Media selection . 16 Sample preparation . 20 Buffer composition and preparation . 21 Denaturing (chaotropic) agents and detergents .22 Column and media preparation . 23 Sample application . 23 Elution and flow rates . 24 Method development for high resolution fractionation . 26 Care of gel filtration media . 27 Equipment selection . 27 Scaling up . 27 BioProcess Media for large-scale production . 29 Troubleshooting . 29 Chapter 2 Gel filtration media . 35 Components of gel filtration media . 35 Superdex: first choice for high resolution, short run times and high recovery. 36 Separation options . 38 Separation examples . 39 Performing a separation .42 Cleaning .43 Media characteristics .44 Chemical stability . 44 Storage . 44 Sephacryl: fast, high recovery separations at laboratory and industrial scale . 45 Separation options . 48 Separation examples . 48 Performing a separation .49 Cleaning .50 Media characteristics .51 Chemical stability . 51 Storage . 51 Superose: broad fractionation range, but not suitable for industrial scale separations . 52
Separation options mple ing a sep Media characteristics 56 Chemical stability. 56 Storage .56 mple 57 61 Scale up and processing large sample volumes .6 nn Packing cal stability 69 Chapter 3 Gel filtration in theory .71 Defining the process. Selectivity curves and media selection. .74 Resolution .75 Chapter 4 Molecular weight determination and molecular weight distribution analysis. .79 Chapter adex LH-20 81 Packing a column 83 Performing a separation. 84 Transferring Sephadex LH-20 between organicent 85 Chapter 6 Gel filtration in a Purification Strategy(CPP). 81 Applying CiPP. 8 Selection and combination of purification techniques .88 Gel filtration as a polishing step. .91
3 Separation options . 53 Separation examples . 54 Performing a separation .54 Cleaning .55 Media characteristics .56 Chemical stability . 56 Storage . 56 Sephadex: rapid group separation of high and low molecular weight substances, such as desalting, buffer exchange and sample clean up . 57 Separation options . 59 Separation examples . 61 Performing a separation .61 Scale up and processing large sample volumes .66 Media characteristics .68 Column Packing . 68 Cleaning .68 Chemical stability . 68 Storage . 69 Chapter 3 Gel filtration in theory . 71 Defining the process . 71 Selectivity curves and media selection . 74 Resolution . 75 Chapter 4 Molecular weight determination and molecular weight distribution analysis . 79 Chapter 5 Sephadex LH-20 . 81 Separation options . 82 Separation examples . 82 Packing a column . 83 Performing a separation .84 Cleaning .84 Medium characteristics .84 Chemical stability . 85 Storage . 85 Transferring Sephadex LH-20 between organic solvents .85 Chapter 6 Gel filtration in a Purification Strategy (CIPP) . 87 Applying CIPP . 87 Selection and combination of purification techniques . 88 Gel filtration as a polishing step .91
93 Columns fo pa ing gel filtration media. 9 Checking column efficiency. .95 octinpereprepe and 95 Column packing for group separations using Sephadex. .9 102 Appendix 3 Sample preparation. .103 Sample stability 103 Sample clarification 104 Specific sample preparation steps. 105 Removal of lipoproteins. ,108 Removal of phenol red. .108 Removal of low molecular weight contaminants ,108 of puricalon 109 Appendix 5 Converting from linear flow(cm/hour)to volumetric flow rates (ml/min)and vice versa . 110 data:proteins,column pressures .111 Appendix 7 Table of amino acids .112 Appendix 8 Analytical assays during purification . .114 higlogical samples. 116 Additional reading and reference material .117 Ordering information .118
4 Appendix 1 Column packing and preparation . 93 Columns for packing gel filtration media . 93 Checking column efficiency . 95 Column packing for high resolution fractionation using Superdex prep grade and Sephacryl High Resolution . 95 Column packing for group separations using Sephadex . 98 Appendix 2 Sephadex and Darcy's law . 102 Appendix 3 Sample preparation. 103 Sample stability .103 Sample clarification .104 Specific sample preparation steps . 105 Removal of lipoproteins. 108 Removal of phenol red . 108 Removal of low molecular weight contaminants . 108 Appendix 4 Selection of purification equipment . 109 Appendix 5 Converting from linear flow (cm/hour) to volumetric flow rates (ml/min) and vice versa . 110 Appendix 6 Conversion data: proteins, column pressures . 111 Appendix 7 Table of amino acids . 112 Appendix 8 Analytical assays during purification . 114 Appendix 9 Storage of biological samples . 116 Additional reading and reference material . 117 Ordering information . 118
Introduction Biomolecules are purified using chromatography techniques that separate them according to differeces in their speific properties,s sho wn in Figure 1. Property Technique Size Gel firation (GF).also called size exclusion Charge lon exchange chromatography (EX) Hydrophobicity Biorecognition(ligand specificity) Affinity chromatography (AC) Gel filtration :. For more than forty years since the introduction of SephadexM gel filtration has played a key role in the purification of enzymes,polysaccharides,nuclei acids,proteins and othe biological macromolecules.Gel filtration is the simplest and mildest of all the chromato graphy techniques and separates molecules on the basis of differences in size.The technique can be applied in two distinct ways: 1.Group separations:the components of a sample are separated into two major groups according to size range.A group separation can be used to remove high or low molecular weight contaminants(such as phenol red from culture fluids)or to desalt and exchange buffers. 2.High resolution fractionation of biomolecules:the components of a sample are separated according to differences in their molecular size.High r solution fractionation can be used to isolate ne or more moleular wight or torfo
5 Introduction Biomolecules are purified using chromatography techniques that separate them according to differences in their specific properties, as shown in Figure 1. Property Technique Size Gel filtration (GF), also called size exclusion Charge Ion exchange chromatography (IEX) Hydrophobicity Hydrophobic interaction chromatography (HIC) Reversed phase chromatography (RPC) Biorecognition (ligand specificity) Affinity chromatography (AC) Gel filtration Hydrophobic interaction Ion exchange Affinity Reversed phase Fig. 1. Separation principles in chromatography purification. For more than forty years since the introduction of Sephadex™, gel filtration has played a key role in the purification of enzymes, polysaccharides, nucleic acids, proteins and other biological macromolecules. Gel filtration is the simplest and mildest of all the chromatography techniques and separates molecules on the basis of differences in size. The technique can be applied in two distinct ways: 1. Group separations: the components of a sample are separated into two major groups according to size range. A group separation can be used to remove high or low molecular weight contaminants (such as phenol red from culture fluids) or to desalt and exchange buffers. 2. High resolution fractionation of biomolecules: the components of a sample are separated according to differences in their molecular size. High resolution fractionation can be used to isolate one or more components, to separate monomers from aggregates, to determine molecular weight or to perform a molecular weight distribution analysis. Gel filtration can also be used to facilitate the refolding of denatured proteins by careful control of changing buffer conditions
Gel filtration is a robust technique that is well suited to handling biomolecules that are sensitive to changes in pH,concentration of metal ions or co-factors and harsh environmental conditions.Separations can be performed in the presence of essential ions or cofactors deterg guanidine hydrochloride,at high or low ionic strength,at 37Cor in the his handbook describes the use of gel filtration for the purification and sepa media biomolec u applications are included step towards a successful separation is to select the correct medium and this handbook focuses on the most up-to-date gel filtration media and prepacked columns. The biocompatibility,stability and utility of gel filtration media from Amersham Biosciences have made these products the standard choice in practically every laboratory using the technique.A wide variety of prepacked columns and ready to use media is available. The illustration on the inside cover shows the range of handbooks from Amersham Biosciences that have been produced to ensure that chromatography and other separation techniques are used easily and effectively at any scale,in any laboratory and for any application. Symbols this symbol indicates general advice which can improve procedures or provide recommendations for action under specific situations. 业 this symbol denotes advice which should be regarded as mandatory and gives a warning when special care should be taken. this symbol highlights troubleshooting advice to help analyse and resolve difficulties that may occur. chemicals,buffers and equipment. experimental protocol
6 Gel filtration is a robust technique that is well suited to handling biomolecules that are sensitive to changes in pH, concentration of metal ions or co-factors and harsh environmental conditions. Separations can be performed in the presence of essential ions or cofactors, detergents, urea, guanidine hydrochloride, at high or low ionic strength, at 37 °C or in the cold room according to the requirements of the experiment. This handbook describes the use of gel filtration for the purification and separation of biomolecules, with a focus on practical information for obtaining the best results. The media available, selection criteria and examples with detailed instructions for the most common applications are included, as well as the theoretical principles behind the technique. The first step towards a successful separation is to select the correct medium and this handbook focuses on the most up-to-date gel filtration media and prepacked columns. The biocompatibility, stability and utility of gel filtration media from Amersham Biosciences have made these products the standard choice in practically every laboratory using the technique. A wide variety of prepacked columns and ready to use media is available. The illustration on the inside cover shows the range of handbooks from Amersham Biosciences that have been produced to ensure that chromatography and other separation techniques are used easily and effectively at any scale, in any laboratory and for any application. Symbols this symbol indicates general advice which can improve procedures or provide recommendations for action under specific situations. this symbol denotes advice which should be regarded as mandatory and gives a warning when special care should be taken. this symbol highlights troubleshooting advice to help analyse and resolve difficulties that may occur. chemicals, buffers and equipment. experimental protocol
Common abbreviations In chromatography GF:gel filtration (sometimes referred to as SEC:size exclusion chromatography) IEX:ion exchange chromatography (also seen as IEC) AC:affinity chromatography RPC:reverse phase chromatography HIC:hydrophobic interaction chromatography CIPP:Capture,Intermediate Purification and Polishing MPa:megapascals psi:pounds per square inch SDS:sodium dodecyl sulphate CIP:cleaning in place A2sonm,A214nm:UV absorbance at specified wavelength M:relative molecular weight N:column efficiency expressed as theoretical plates per meter ovoismead fom the chromaogram and relates to the molecula V:void volume is the elution volume of molecules that are excluded from the gel filtration medium because they are larger than the largest pores in the matrix and pass straight through the packed bed V:total column volume is equivalent to the volume of the packed bed (also referred to as CV) R:resolution,the degree of separation between peaks Ka and logM,:partition coefficient and log molecular weight,terms used when defining the selectivity of a gel filtration medium In product names HMW:high molecular weight LMW:low molecular weight HR:high resolution Pg:prep grade PC:precision column SR:solvent resistant
7 Common abbreviations In chromatography GF: gel filtration (sometimes referred to as SEC: size exclusion chromatography) IEX: ion exchange chromatography (also seen as IEC) AC: affinity chromatography RPC: reverse phase chromatography HIC: hydrophobic interaction chromatography CIPP: Capture, Intermediate Purification and Polishing MPa: megapascals psi: pounds per square inch SDS: sodium dodecyl sulphate CIP: cleaning in place A280nm, A214nm: UV absorbance at specified wavelength Mr: relative molecular weight N: column efficiency expressed as theoretical plates per meter Ve: elution volume is measured from the chromatogram and relates to the molecular size of the molecule. Vo: void volume is the elution volume of molecules that are excluded from the gel filtration medium because they are larger than the largest pores in the matrix and pass straight through the packed bed Vt : total column volume is equivalent to the volume of the packed bed (also referred to as CV) Rs: resolution, the degree of separation between peaks Kav and logMr: partition coefficient and log molecular weight, terms used when defining the selectivity of a gel filtration medium In product names HMW: high molecular weight LMW: low molecular weight HR: high resolution pg: prep grade PC: precision column SR: solvent resistant
Chapter 1 Gel filtration in practice Introduction Gel filtration separates molecules according to differences in size as they pass through a gel filtration medium packed in a column.Unlike ion exchange or affinity chromatography, molecules do not bind to the chromatography medium so buffer composition does not directly affect resolution (the degree of separation between peaks).Consequently,a significant advantage of gel filtration is that conditions can be varied to suit the type of sample or the requirements for further purification,analysis or storage without altering the separation on is well suited for biomolecules that may be sensitive to chan ions or co-fact nd harsh env tors onment l Separation 10 requirements of the experiment.Purified proteins can be collected in any chosen buffer. This chapter provides general guidelines applicable to any gel filtration separation.A key step towards successful separation is to select the correct medium,so selection guides for the most up-to-date gel filtration media and prepacked columns are included.Other application examples and product-specific information are found in Chapter 2. Separation by gel filtration To performa separation,ge fitrationmispaked into a colm to forma ped bed.The t have bee for their che ical sta bility,and in tparuide The ligid imil the pormm as the stationary phase and this liquid is in equilibrium with the liquid outside the particles referred to as the mobile phase.It should be noted that samples are eluted isocratically,i.e. there is no need to use different buffers during the separation.However,a wash step using the running buffer is usually included at the end of a separation to facilitate the removal of any molecules that may have been retained on the column and to prepare the column for a new run.Figure 2 shows the most common terms used to describe the separation and Figure 3illustrates the separation process of gel filtration. -
9 Chapter 1 Gel filtration in practice Introduction Gel filtration separates molecules according to differences in size as they pass through a gel filtration medium packed in a column. Unlike ion exchange or affinity chromatography, molecules do not bind to the chromatography medium so buffer composition does not directly affect resolution (the degree of separation between peaks). Consequently, a significant advantage of gel filtration is that conditions can be varied to suit the type of sample or the requirements for further purification, analysis or storage without altering the separation. Gel filtration is well suited for biomolecules that may be sensitive to changes in pH, concentration of metal ions or co-factors and harsh environmental conditions. Separations can be performed in the presence of essential ions or cofactors, detergents, urea, guanidine hydrochloride, at high or low ionic strength, at 37 °C or in the cold room according to the requirements of the experiment. Purified proteins can be collected in any chosen buffer. This chapter provides general guidelines applicable to any gel filtration separation. A key step towards successful separation is to select the correct medium, so selection guides for the most up-to-date gel filtration media and prepacked columns are included. Other application examples and product-specific information are found in Chapter 2. Separation by gel filtration To perform a separation, gel filtration medium is packed into a column to form a packed bed. The medium is a porous matrix in the form of spherical particles that have been chosen for their chemical and physical stability, and inertness (lack of reactivity and adsorptive properties). The packed bed is equilibrated with buffer which fills the pores of the matrix and the space in between the particles. The liquid inside the pores is sometimes referred to as the stationary phase and this liquid is in equilibrium with the liquid outside the particles, referred to as the mobile phase. It should be noted that samples are eluted isocratically, i.e. there is no need to use different buffers during the separation. However, a wash step using the running buffer is usually included at the end of a separation to facilitate the removal of any molecules that may have been retained on the column and to prepare the column for a new run. Figure 2 shows the most common terms used to describe the separation and Figure 3 illustrates the separation process of gel filtration. Fig. 2. Common terms in gel filtration. Void volume Vo Total column V –t Vo volume Vt Vo V – V t o Vt Low molecular weight Interacting with medium AbsorbanceSample injection High molecular weight Intermediate molecular weight Ve