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Gel filtration

Principles and Methods

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8 th edition 18-1022-18

Gel filtration Principles and Methods

ISBN 91-97-0490-2-6

Contents Introduction ............................................................................................. 4 Principles ................................................................................................. 6 Gel filtration ...................................................................................... 6 Gel media .......................................................................................... 6 Separation by size ............................................................................... 6 Experimental parameters ..................................................................... 7 Sample concepts ........................................................................... 7 Column parameters ...................................................................... 7 Eluent parameters ......................................................................... 8 Running conditions ...................................................................... 8 Characterization of solute behaviour ................................................ 8 Theoretical considerations ........................................................... 12 Deviations from ideal behaviour in gel filtration ............................. 13 Application examples ..................................................................... 14 Fractionation by size ................................................................... 14 Separation of monomers from dimers and higher aggregates ............ 16 MW estimation, native and other forms ........................................ 17 Determination of molecular weight distribution of polymers ............ 18 Determination of equilibrium constants ........................................ 19 Desalting ................................................................................... 19 Industrial applications ................................................................... 21 Properties of gel filtration media ............................................................ Sephacryl HR ................................................................................. Chemical and physical properties ................................................. Chromatographic properties ........................................................ Availability ................................................................................ Further information .................................................................... Superdex ......................................................................................... Chemical and physical properties ................................................. Chromatographic properties ........................................................ Availability ................................................................................ Further information .................................................................... Superose .......................................................................................... Chemical and physical properties ................................................. Chromatographic properties ........................................................ Availability ................................................................................ Further information ....................................................................

22 22 22 24 27 27 27 27 29 30 30 31 31 32 34 34

Sephadex ......................................................................................... Chemical and physical properties ................................................. Chromatographic properties ........................................................ Availability ................................................................................ Further information .................................................................... Sepharose ....................................................................................... Chemical and physical properties ................................................. Chromatographic properties ........................................................ Availability ................................................................................ Further information .................................................................... Sepharose CL ................................................................................. Chemical and physical properties ................................................. Chromatographic properties ........................................................ Availability ................................................................................ Further information ....................................................................

35 35 36 37 38 38 38 40 41 41 41 41 42 43 43

Experimental design ............................................................................... Performance ................................................................................... Resolution ................................................................................. Separation time ........................................................................ High performance gel filtration .................................................... Capacity .................................................................................... Process considerations ................................................................. Choice of gel .................................................................................. The purpose of the experiment ..................................................... Solute characteristics ................................................................... Sample characteristics ................................................................. Choice of running conditions ......................................................... Choice of column ....................................................................... Flow rate ................................................................................... Sample characteristics ................................................................. Eluent ....................................................................................... Optimization .............................................................................

44 44 44 44 45 45 47 47 48 51 51 52 52 54 55 58 58

Performing a gel filtration experiment ................................................... 61 Preparing the gel ............................................................................. 61 Pre-swollen media (Sephacryl HR, Superose prep grade, Sepharose CL and Sepharose) ...................................................... Media which require swelling (Sephadex G-types) .......................... Preparation of Sephacryl HR, Superose prep grade or Sepharose CL for use in organic solvents ....................................... Gel filtration in organic solvetns ...................................................

61 61

62 63 Packing a column ........................................................................... 63 2

Packing Sephadex G types Sepharose and Sepharose CL .................. Packing Sephacryl HR and Superose prep grade ............................. Adaptors ................................................................................... Checking the packed bed .............................................................

63 68 70 71

Using pre-packed columns .............................................................. 72 Sample application ......................................................................... 72 Sample application withour an adaptor ......................................... 72 Sample application with an adaptor .............................................. 73 Elution ........................................................................................... 77 Flow rates .................................................................................. 78 Cleaning gels and packed columns ................................................. 80 General cleaning procedures ........................................................ 80 Procedures to remove specific contaminants .................................. 83 Storage of gels and columns ........................................................... Prevention of microbial growth .................................................... Antimicrobial agents ................................................................... Storage of unused media .............................................................. Storage of used media ................................................................. Storage of packed columns ..........................................................

84 84 85 86 86 87

Fault finding chart ................................................................................. 89 References .............................................................................................. 98 Ordering information ............................................................................ 102

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Introduction For more than thirty years since the introduction of Sephadex (l,2), gel filtration has occupied a key position in the purification of thousands of enzymes, polysaccharides, nucleic acids, proteins and other biological macromolecules. Its continuing value depends partly on the special nature of the macromolecules studied by the biochemist and partly on the reliability and simplicity of gel filtration as a separation technique. Biological macromolecules form a class of substances with special functions which are controlled in vivo by small changes in the environment. Changes in the pH, concentrations of metal ions, cofactors etc may have a profound effect on the molecules being studied and it is clearly necessary to have available mild separation techniques which operate independently of these factors. Gel filtration is one of these techniques. A gel filtration separation can be performed in the presence of essential ions or cofactors, detergents, urea, at high or low ionic strength, at 37 °C or in the cold room according to the requirements of the experiment. The stability of gel filtration media from Amersham Pharmacia Biotech and their inertness towards biopolymers under a wide range of conditions have made them the standard in practically every biochemistry laboratory. No less valuable than its widespread usefulness, is the reliability and simplicity of gel filtration as an experimental procedure. Little equipment is required, the procedure is straightforward and good separations and yields are usually obtained even in the first experiment. The reliability of gel filtration stems from the reliability of Sephadex and other gel filtration media from Amersham Pharmacia Biotech, backed by over 30 years of experience in developing biochemical separation techniques and by thousands of publications describing their use. Among the new developments described in this handbook are Sephacryl HR for standard chromatography and Superdex and Superose for high speed gel filtration. These new media are the result of continuing efforts of Pharmacia to improve the separation techniques which you need for your work. This handbook is designed as a laboratory aid in the selection and practical use of gel filtration media from Amersham Pharmacia Biotech. For specific separation problems where the substances in question may have special properties, or for theoretical aspects where ideas are undergoing continual evolution, it is essential to refer to the original literature.

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Our aim remains to provide better tools for the life sciences and we believe that to achieve this it is important to maintain close cooperation between and manufacturer. We hope that this handbook will aid our cooperation and that you will find the information helpful. Should we at any time be able to give you further information, please us.

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Principles Gel filtration In gel filtration molecules in solution are separated according to differences in their sizes as they through a column packed with a chromatographic medium which is a gel.

Gel media A gel is a heterogeneous phase system in which a continuous liquid phase, usually aqueous, is contained within the pores of a continuous solid phase, the gel matrix. In gels made for gel filtration, the pores have a carefully controlled range of sizes, and the matrix is chosen for its chemical and physical stability, and inertness (lack of adsorptive properties). Gels may be formed from polymers by cross-linking to form a three-dimensional network; for example Sephadex which is formed by cross-linking dextran. Some polymers, like agarose, form gels spontaneously under the appropriate conditions. Composite gels may be prepared by, for example, grafting a second polymer onto a pre-formed matrix. Superdex is such a gel. Dextran chains are covalently bonded to a highly cross-linked agarose gel matrix. Composite gels are of interest since they can combine valuable properties from more than one gel-forming system.

Separation by size The pores in the gel matrix which are filled by the liquid phase are comparable in size to the molecules we may wish to separate. Relatively small molecules can diffuse into the gel from a surrounding solution, whereas relatively large molecules will be prevented by their size from diffusing into the gel to the same degree. Sufficiently large molecules are completely unable to diffuse into the gel and are thus confined to the solution outside. In a gel filtration column, gel particles in bead form are packed to form a separation bed through which a buffer solution, the eluent, is ed. 6

Sample molecules which are to be separated are added in solution as a zone to the top of the bed. The sample zone moves down the bed as eluent is added to the top. The small molecules which diffuse into the gel beads are delayed in their age down the column compared with the large molecules which cannot diffuse into the gel and move continuously down the column in the flowing eluent. The large molecules thus leave the column first followed by the smaller molecules in the order of their sizes.

Experimental parameters Sample concepts Characteristics of the sample which are important for the result, apart from the solutes which are to be separated, include its volume and viscosity. The volume of the sample will influence the size of column which will be needed, and the viscosity must not be so large as to cause hydrodynamic instability (see below). It is the viscosity which places an upper limit on the sample concentration which is permissible. Note that the pH, ionic strength and composition are not significant as long as they do not affect the sizes or stability of the molecules to be separated and are not outside the, wide, stability range of the gel filtration medium.

Column parameters The most important characteristic of a gel filtration column is the way in which the gel filtration medium has been packed. If the column is evenly packed so the sample zone is not unnecessarily broadened as it es down the column then good results can be obtained. If the column is packed unevenly then good results will never be obtained from it. The length of the column, cm, is significant since it affects both the resolution and the time taken to elute it. Resolution

α

√column length

Elution time

α

column length

and

The volume of the column, ml, is a direct measure of its loadability under otherwise comparable conditions and is chosen depending on the sample volume. 7

Eluent parameters Since the separation depends only on the sizes of the molecules being separated, the composition of the eluent is unimportant for the separation mechanism. The eluent can thus be whatever is convenient with regard to the overall requirements of the experiment. Usually it is a buffer solution with a well defined pH and ionic composition chosen to preserve the structure and biological activity of the substances of interest. An ionic strength of 0.15 or greater is generally used to avoid any unwanted ionic interactions between the solute molecules and the gel matrix.

Running conditions The experimental variable of significance which remains to be considered is the rate at which the eluent flows through the column. This affects not only the speed at which the separation is obtained but also the resolution which can be achieved. Generally speaking, the lower the flow rate the better the resolution, at least for large molecules. Flow rates are measured in simple volume , e.g. ml/min, but when comparing results between columns of different sizes it is useful to use the linear superficial flow rate, e.g. cm/ hour. Linear superficial flow rate (cm/h) = Volume flow rate (ml/min) x 60 Cross-sectional area of the column (cm2) The flow rate defined in this was is usually simply referred to as the linear flow rate. Results obtained at the same linear flow rate will be comparable as far as the effects of flow rate are concerned.

Characterization of solute behaviour Results in gel filtration are typically expressed in the form of an elution diagram showing the variation of solute concentration in the eluent with the volume of eluent ed through the column (Fig. 1). For protein and nucleic acid work and in many other applications continuous detection using a UV-monitor (e.g. Pharmacia Monitor UV-l, UV-M II or Uvicord S II) and a recorder gives an immediate permanent record, a chromatogram. From this diagram the elution volume (Ve ) of a given solute can be obtained. Different criteria are used for the determination of elution volume (Fig. 2). 8

Fig. 1. Gel filtration of an oligosaccharide mixture from the acetolysis and hydrolysis of cellulose on Sephadex G-25. The numbers above the peaks indicate the degree of polymerisation. Column, 4.5x150 cm; eluent, water; flow rate, 1.9 ml.cm-2.h-1 (Flodin, P., Aspberg, K., Biological Structure and Function 1 (1961) 345-349. Reproduced by kind permission of the Authors and the Publisher).

Fig. 2. Measurement of elution volume, Ve A. Sample size negligible compared with bed volume. B. Sample size not negligible compared with bed volume. C. Sample giving plateau elution curve.

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• When very small samples are applied (small enough to be neglected compared with the elution volume), the position of the peak maximum in the elution diagram should be taken as Ve (Fig. 2A). • If the sample volume cannot be neglected compared with the elution volume, the elution volume is measured from half the sample volume to the position of the peak maximum (Fig. 2B). • When very large sample volumes are used (giving a plateau region in the elution curve), the volume eluted from the start of sample application to the inflexion point (or half height) of the rising part of the elution peak should be taken as Ve. This criterion is often incorrectly applied to samples not giving plateau regions. (Fig. 2C). In gel filtration solutes normally give symmetrical peaks. Elution volumes are, therefore, easily determined by these methods. More sophisticated criteria are seldom useful in practice. Ve is not in itself sufficient to define the behaviour of the sample substance, since this parameter varies with the total volume of the packed bed (Vt) and with the way the column has been packed. By analogy with other types of partition chromatography the elution of a solute is best characterized by a distribution coefficient (Kd) The volume of the mobile phase is equal to the void volume, Vo, the elution volume of molecules which are confined to the mobile phase because they are larger than the largest pores in the gel. The volume of the stationary phase, Vs, in gel filtration is equal to Vi, the volume of solvent inside the gel which is available to very small molecules, i.e. the elution volume of a solute which will distribute freely between the mobile and stationary solvent phases minus the void volume. Thus Kd represents the fraction of the stationary phase which is available for diffusion of a given solute species. In practice the volume of the stationary phase defined in this way is rather difficult to determine. Methods which can be used often involve measurement of the elution volumes of radioactive ions such as 23Na. It is much more convenient to substitute the term (Vt-Vo) for Vs, when we obtain Kav = (Ve - Vo)/(Vt-Vo) Since (Vt-Vo) includes the volume of the gel forming substance, which is inaccessible to all solute molecules, Kav is not a true partition coefficient (Fig. 3). However, for a given gel there is a constant ratio of Kav:Kd which is independent of the nature of the solute or its concentration. Kav is easily determined and, like Kd, defines solute behaviour independently of the bed dimensions and packing. Other methods of normalizing data give values which vary depending upon how well the column is packed. The approximate relationships between some of these are shown in Figure 4. 10

Fig. 3. Diagrammatic representation of Vt and Vo. Note that Vt-Vo will include the volume of the solid material which forms the matrix. (Fischer, L. Laboratory Techniques in Biochemistry and Molecular Biology. Vol. 1 part II. An introduction to Gel Chromatography. North Holland Publishing Company, Amsterdam. Reproduced by kind permission of the Authors and the Publisher).

Fig. 4. Relationship between several expressions used for normalizing elution behaviour.

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Theoretical considerations The use of gel filtration for the determination of molecular weight or size, particularly of proteins, is well documented (3). In practice it is found that for a series of compounds of similar molecular shape and density a sigmoidal relationship exists between their Kav values and the logarithms of their molecular weights (MW). Calibration curves constructed in this way for a particular gel type are often termed selectivity curves (see p 25, 30, 33, 34, 37, 40), and over a considerable range there is a conveniently linear relationship between Kav and log MW. In ideal gel filtration behaviour no molecules can be eluted with a Kav greater than 1 or less than 0. If the Kav is greater than 1, some kind of adsorption is indicated. If the Kav is less than 0 after calibration then channelling in the chromatographic bed is indicated and the column must be repacked. Several models have been proposed to describe the behaviour of solutes during gel filtration (3, 4). Most have regarded the partition of solute molecules between the gel particles and surrounding fluid as an entirely steric effect. Thus Flodin (5) divided the gel into permitted and forbidden regions. The larger the molecular dimensions of the solute the greater is the proportion of the gel which is forbidden. In the permitted region the concentration of solute is assumed to be identical to that in the surrounding liquid. The steric approach has been extended in various ways. Porath (6) derived a theoretical relationship between Kd and Stokes radius assuming that the pores in Sephadex are conical. In another treatment Squire (7) considered pores and crevices as well as cones. An interesting approach by Laurent and Killander (8) assumes that the gel network is composed of rigid rods randomly arranged. Good correlation was found between Kav and molecular radius with this model. All of these models have been successfully applied to predict the elution behaviour of solutes. However, as has been pointed out by Ackers (9), none of them may be accurate in a structural sense. Results are equally in accordance with a formulation where the fractions of the stationary phase available to molecules of different radii are defined by a Gaussian probability curve and no assumption is made about the geometric shape of the pores. From a practical stand-point, for molecular weight determination, it is still most common to construct a calibration curve and perform the estimations graphically. 12

Deviations from ideal behaviour in gel filtration In ideal gel filtration the only effects which contribute to the behaviour of solute molecules are steric effects. Insensitivity of gel filtration to the composition of the eluent is a major advantage and is observed in the vast majority of cases in aqueous systems at neutral pH in the presence of electrolyte. However, deviation from a Kav:log MW calibration curve may still occur if a compound being studied does not have the same molecular shape as the standards. Under certain conditions, factors other than the size and shape of the molecules being studied can influence the separation. These effects can usually be avoided and are generally only significant when chromatographing highly acidic or basic substances at low ionic strength or aromatic materials on those gels which have a high matrix content. Often they have proved highly advantageous. Thus an important application of Sephadex types G-10, G-15 and G-25 is in the separation of aromatic peptides and other substances which may differ only slightly in molecular weight. Further details of these effects can be found under the chromatographic properties of the different gels (see pages 22–43).

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Application examples Fractionation by size The special ability of gel filtration to separate macromolecules on the basis of small differences in size between them makes it an essential fractionation tool. Here the correct choice of gel and operating conditions are critical if good results are to be obtained. The peptide separation shown in Figure 5

Fig. 5. Gel filtration of a tryptic digest of citraconylated totally reduced and 14C alkylated Fd' fragments of IgG3 on Sephadex G-50 Fine. Column, 1x96 cm; eluent, 10 % formic acid. (Michaelsen, T.T., Frangione, B., Franklin, E.C., J. Biol. Chem. 252 (1977) 883-889: Reproduced by kind permission of the Authors and the Publisher).

illustrates this type of experiment. With the proper experimental design, a protein can be separated from other species differing in molecular weight by a factor of two or slightly less (Fig. 6). Practical simplicity, excellent recovery, free choice of elution conditions and straight forward interpreta14

tion of results make fractionation by gel filtration an invaluable part of any purification scheme. Details about experimental design are given on p 44– 60. Practical details are given on p 61–87.

Fig. 6. Separation of IGF-1 (MW 7 600) from its fusion partner ZZ (MW 14 500) and uncleaved fusion protein on Superdex 75 HR 10/30. The secreted fusion protein, ZZ-IGF-1, was initially purified by affinity chromatography on IgG-Sepharose Fast Flow and subsequently cleaved with hydroxylamine (2 M, pH 9.2). Sample concentration, 5 mg/ml; sample volume, 100 µl; eluent, ammonium acetate (0.25 M, pH 6.0); flow rate, 0.5 ml/min. (Work by Pharmacia LKB Biotechnology, Uppsala, Sweden, in collaboration with KabiGen, Stockholm, Sweden.)

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Separation of monomers from dimers and higher aggregates It is frequently found that proteins with a high degree of homogeneity with respect to the protein species present contain dimers and higher aggregates. High resolution gel filtration provides an excellent and gentle means for separating the monomer from the aggregates, either as part of an analysis or for their final purification. Gel filtration is thus especially useful as a polishing step in a more complex purification scheme and finds widespread use for this purpose in the purification of recombinant and other proteins in industrial processes (Fig. 7) where protein aggregates must be reduced to below a specified level in the final product.

Fig. 7. Final purification of a mouse monoclonal antibody, IgG2a, on Superdex 200 prep grade. Hybridoma cell culture supernatant was concentrated by ultrafiltration and clarified by centrifugation and age through a 0.22 µm filter. The concentrate was desalted and purified initially by cation exchange on BioPilot Column S Sepharose High Performance 60/100. Peaks, 1. IgG dimers; 2. IgG monomers; 3. transferrin; column, BioPilot Superdex 200 prep grade 60/600; sample concentration, 11.37 mg/ml; sample volume, 50 ml; eluent, PBS (pH 7.5) containing sodium azide (0.05 %); flow rate, 14 ml/min. Work by Pharmacia LKB Biotechnology, Uppsala, Sweden.

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MW estimation, native and other forms A calibrated column of Sephacryl HR, Superdex, Superose, Sephadex or Sepharose provides a simple and well-documented way of determining the molecular weights of proteins during a natural stage in their purification (10–14). Unlike electrophoretic techniques, gel filtration provides a means of determining the molecular weight or size (Stokes radius) of native or denatured globular proteins under a wide variety of conditions of pH, ionic strength, temperature etc., freeing the researcher from the constraints imposed by the charge state of the molecules. Gel filtration in the presence of urea or guanidine hydrochloride, dissociating agents which transform polypeptides and proteins to a random coil configuration reducing structural differences, has proved particularly useful for molecular weight determinations. Figure 8 illustrates this kind of application.

Fig. 8. Gel filtration of proteins on Sepharose CL-6B under denaturing conditions. Peaks, 1. Blue Dextran; 2. bovine serum albumin; 3. rabbit IgG H chain; 4. α-chymotrypsinogen; 5. cytochrome c; 6. insulin; 7. B chain of insulin; 8. DNP-ala: Column, 1.5x90 cm: Eluent, 6 M guanidine-HCI, 0.1 m sodium phosphate, pH 8.0; flow rate, 6.8 ml.cm-2.h-1. (Ansari, A.A., Mage, R.G., J. Chromatogr. 140 (1977) 98-102. Reproduced by kind permission of the Authors and the Publisher).

Gel filtration also eliminates the need to set up a separate experiment for each determination as the calibrated column can be used for extended periods, both for molecular weight determination and for routine separations. In the experiment depicted in Figure 8, for example, Ansari and Mage (14) used the same column packed with Sepharose CL-6B for 17

molecular weight determination in 6M guanidine hydrochloride over a period of 10 months, without any change in the elution pattern. The only special requirements for molecular weight determination are a column packed with the appropriate gel and a series of protein standards to calibrate it. Gel Filtration Calibration Kits HMW and LMW from Pharmacia provide a series of well characterized globular protein standards carefully chosen to give reliable calibration points in the molecular weight range 13 700 to 669 000 (see p 87–88). An elution profile obtained with Sephadex G-200 Superfine is shown in Figure 9.

Fig. 9. Calibration proteins being used to calibrate Sephadex G-200 Superfine. Peaks, 1. catalase; 2. aldolase; 3. bovine serum albumin; 4. ovalbumin; 5. chymotrypsinogen A; 6. ribonuclease A: column, K 26/70: eluent, 0.05 M potassium phosphate, pH 6.8, containing 0.1 M NaCI and 2 % sodium azide. (Work by Pharmacia LKB Biotechnology, Uppsala, Sweden.

Determination of molecular weight distribution of polymers The molecular weight distribution is very important for characterization of natural and synthetic polymers. Distribution analysis by classical methods is difficult and tedious as it involves fractionation of the macromolecules by precipitation and determination of molecular weight and amount of substance in every fraction. 18

The ability of gel filtration to fractionate molecules according to molecular size offers an improved method for distribution analysis of many polymers (15). The elution curve can be recorded continuously or determined by investigating individual fractions. It is unnecessary to determine molecular weights of the polymers in the effluent fractions of individual experiments, as the chromatographic behaviour of the columns is very reproducible. A calibration curve once determined for the column can be applied to a large number of runs. The precision of this method of distribution analysis is very high (16). The method is used for the determination of molecular weight distributions of e.g. dextrans, polyvinylpyrrolidones, gelatin preparations and other water-soluble polymers (3).

Determination of equilibrium constants Gel filtration has proved to be a valuable technique for the study of chemical equilibria. In the case of slow reactions, where the reactants and the products can be separated on a gel filtration column, these substances can be quantitatively determined in the effluent, thereby establishing the position of the equilibrium (17–19). Gel filtration can also be used to determine the position of equilibrium of complex formation where the reactions are rapid. In this case, one of the reactants is chromatographed in an eluent containing the other reactant. From the elution curve of the reactants the complex formation can be studied. This method is of great value in the study of protein binding of low molecular weight substances such as drugs (20–26) and can be extended to the study of, for instance, competition of two molecules for the same site (22). Gel filtration has also been applied to the estimation of reaction rates (27, 28).

Desalting Since proteins and other biomacromolecules differ greatly in size from salts and other small molecules, gel filtration is particularly efficient for many everyday laboratory operations including: • buffer exchange. Enzymic reactions, assays and other analytical procedures require that the sample be adjusted to the proper ionic conditions; this is most efficiently done by gel filtration. Gel filtration is the most efficient way to adjust the ionic composition of a sample to the required species and concentration prior to ion exchange chromatography, hydrophobic interaction chromatography, affinity 19

chromatography and other LC techniques which use an aqueous mobile phase. • phenol removal from preparations of nucleic acids. • removal of unincorporated nucleotides during DNA sequencing (29, 30). • removal of free low molecular weight labels, e.g. 125I, FITC, from solutions of labelled proteins (Fig. 10).

Fig. 10. Removal of free 125l and Chloramine T from labelled albumin. Column, Fast Desalting Column HR 10/10; sample concentration, 0.5 mg/ml human serum albumin; sample volume, 350 µl; eluent, sodium phosphate (0.05 M, pH 7.4) containing Tween 20 (0.05 %); flow rate, 4 ml/min. (Work by Pharmacia LKB Biotechnology, Uppsala, Sweden).

• termination of reactions between macromolecules and low molecular weight reactants. • removal of products, cofactors, inhibitors etc. from enzymes. • phenol red removal from culture fluids prior to anion exchange chromatography. 20

Industrial applications The advantages of gel filtration for the separation of biological molecules can be equally well realized in large-scale applications as they are in the research laboratory. In particular, scale-up from pilot-plant to production capacity has proved a straightforward and trouble-free operation. Pharmacia has built up considerable experience in this area and you are invited to us directly should you require further information.

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Properties of gel filtration media Sephacryl HR Chemical and physical properties Sephacryl High Resolution (HR) is a composite gel prepared by covalently cross-linking allyl dextran with N,N'-methylene bisacrylamide to form a hydrophilic matrix of high mechanical strength (Fig. 11).

Fig. 11. Hypothetical partial structure of Sephacryl HR.

22

The porosity of the gel is controlled by the dextran component to give five types with different fractionation ranges. The wet bead diameter is between 25–75 µm, with an average bead diameter of approximately 50 µm. Sephacryl HR types are supplied pre-swollen and ready to use. Table 1 summarizes some of the properties of Sephacryl HR. Table 1. Properties of Sephacryl HR Gel type

Sephacryl S-100 HR Sephacryl S-200 HR Sephacryl S-300 HR Sephacryl S-400 HR Sephacryl S-500 HR

Bead size µm 25 – 75 25 – 75 25 – 75 25 – 75 25 – 75

Fractionation range Globular proteins 1 000 – 100 000 5 000 – 250 000 10 000 – 1 500 000 20 000 – 8 000 000 ND

Fractionation range Exclusion Dextrans limit DNA ND 1 000 – 80 000 2 000 – 400 000 10 000 – 2 000 000 40 000 – 20 000 000

ND 118 118 271 1078

Chemical stability Sephacryl HR is stable in all aqueous buffers commonly used in biochemistry within the pH range 3–11. Short term exposure, e.g. for cleaning, to pH´s in the range 2-13 has no adverse effect on subsequent chromatographic properties. The separation properties of the gel are not affected by detergents e.g. 1% SDS, chaotropic salts or dissociating agents e.g. 8 M urea and 6 M guanidine HCI. Strong solutions of NaOH (0.5M) can be used for cleaning if the gel is washed immediately afterwards with buffer or water. Sephacryl HR can also be used with organic solvents. A method for transferring Sephacryl HR from water to organic solvents is described on page 62. Table 2 lists the gel volumes in various organic solvents for an original 100 ml of gel sedimented in water. Table 2. Bed volumes of Sephacryl HR-types in common organic solvents starting from 100 ml sedimented gel. Gel type

Formamide

DMSO

Methanol

Ethanol

Acetone

Sephacryl S-100 HR Sephacryl S-200 HR Sephacryl S-300 HR Sephacryl S-400 HR Sephacryl S-500 HR

110 115 100 100 100

100 110 90 90 95

100 100 100 100 100

100 100 95 95 100

85 85 85 85 90

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Physical stability

Sephacryl HR may be autoclaved repeatedly at 121 °C, pH 7 for 30 minutes without significantly affecting its chromatographic properties. Typical flow rates for a 60 cm long bed can be seen in the pressure/flow diagram in Fig. 12.

Fig. 12. Pressure drop as a function of flow rate for Sephacryl HR. Bed height approximately 60 cm; eluent distilled water; temperature 25 °C. To calculate the volumetric flow rate, multiply the linear flow rate by the cross-sectional area of the column (2 cm2 for XK 16 or 5.3 cm2 for XK 26).

Columns of all dimensions, including wide diameter production columns with total bed heights of 60-100 cm, can be packed and equilibrated successfully at high flow rates, resulting in short overall separation times (31). Likewise, the mechanical rigidity of Sephacryl HR allows even relatively viscous eluents, such as 8 M urea, to be run at practicable flow rates.

Chromatographic properties Selectivity The five Sephacryl HR types have different fractionation ranges. Sephacryl S-100 HR is excellent for gel filtration of peptides and small proteins in the molecular weight range 1 x 103–100 x 103. When fractionating proteins in the molecular weight range 5 x 103 –250 x 103 or 10 x 103 –1.5 x 106, 24

including monoclonal antibodies and serum proteins, use Sephacryl S-200 HR or S-300 HR respectively. Sephacryl S-400 HR and S-500 HR are the choice for separation of larger proteins, polysaccharides, nucleic acids, DNA fragments and small particles e.g. plasmids. Table 1 gives the fractionation ranges for Sephacryl HR. Figure 13 shows the selectivity curves for globular proteins and dextrans respectively.

Fig. 13. Selectivity curves for Sephacryl HR in phosphate buffer (0.05 M, pH 7.0) containing NaCl (0.15 M).

The narrow particle size distribution of Sephacryl HR allows columns to be packed easily and with high efficiency giving > 9000 plates per metre if the packing instruction is followed. A typical example of results obtainable with Sephacryl HR is shown in Figure 14. 25

Fig. 14. Separation of integral membrane proteins from human erythrocytes on Sephacryl S-300 HR. Human erythrocyte membrane were solubilized in phosphate buffer (0.1 M, pH 7.4) containing SDS (0.1 M), EDTA (1 mM), DTE (1 mM). Peaks, 1. dimeric anion transporter protein; 2. monomer of the anion transporter protein; 3. dimeric glycophorin A; 4. glucose transporter protein; 5. possibly lipids and SDS; column, K 26/70; bed height, 61 cm; sample concentration, 2 mg/ml; sample volume, 2 ml; eluent, phosphate buffer (0.1 M, pH 7.4) containing SDS (0.05 M), EDTA (1 mM), DTE (1 mM); flow rate, 58 ml/hour. (Work by E. Greijer and P. Lundahl, Institute of Biochemistry, University of Uppsala, Uppsala, Sweden.)

Adsorption Tests in our laboratories have shown that Sephacryl S-100 HR gives yields of at least 96% (UV absorption at 280 nm, using 0.05 M phosphate, 0.15 M NaCl, pH 7.0) with the following model substances: Blue Dextran 2000, ferritin, catalase, aldolase, BSA, ovalbumin, β-lactoglobulin A+B, chymotrypsinogen A, myoglobin, Iysozyme, ribonuclease A and cytochrome c. For the best results eluents with an ionic strength of at least 0.15 should be used.

FPLC and industrial scale Columns packed with Sephacryl can be used with with standard chromatography instrumentation and with FPLC system. Its high resolution and high chemical stability make Sephacryl HR ideally suited for use in industry, both in pilot and in process scale. The gels provide rapid cycle times and can be sanitized in place which is of particular advantage. 26

Availability Sephacryl HR types are supplied pre-swollen as a suspension containing 20% ethanol in packs of 750 ml. Sephacryl S-100, S-200 and S-300 are also supplied pre-packed in XK-columns with the designation HiLoad columns.

Further information Further information on Sephacryl HR is given in the data sheet: “Sephacryl High Resolution gel filtration media” with details of applications. For information on scale-up and the operation of large scale chromatography systems, please Pharmacia.

Superdex Chemical and physical properties Superdex is based on highly cross-linked porous agarose beads to which dextran has been covalently bonded. Superdex is thus a composite gel (Fig. 15) in which the high physical and chemical stabilities are chiefly due to the agarose matrix and the gel filtration properties are principally determined by the dextran chains. Different types with different fractionation ranges are available. Superdex 30 prep grade, Superdex 75 prep grade and Superdex 200 prep grade have

Fig. 15. Structure of Superdex. Dextran chains are covalently linked to a highly cross-linked agarose matrix.

27

an average wet bead diameter of 34 µm with a range of 24–44 µm. Superdex peptide, Superdex 75 and Superdex 200 for high performance gel filtration have a bead size of 13 µm (mean). Table 3 summarizes some of the properties of Superdex and Superdex prep grade; HiLoad columns can be used both with standard chromatography systems and with FPLC system. Superdex packed in HR columns is for use with FPLC system. Table 3. Properties of Superdex Gel type Superdex peptide Superdex 30 prep grade Superdex 75 prep grade Superdex 75 Superdex 200 prep grade Superdex 200

Bead size µm 11 – 15 24 – 44 24 – 44 11 – 15 24 – 44 11 – 15

Fractionation range Globular proteins 100 – 7 000 – 10 000 3 000 – 70 000 3 000 – 70 000 10 000 – 600 000 10 000 – 600 000

Fractionation range Dextrans – – 500 – 30 000 500 – 30 000 1 000 – 100 000 1 000 – 100 000

Chemical stability Superdex can be used with all aqueous buffers commonly used in biochemistry within the pH range 3–12, and withstands strong bases, e.g. 0.2 M NaOH, and acids, e.g. 0.01 M HCI or 0.1 M acetic acid. Short term exposure to extremes of pH (1-14) e.g. for cleaning, has no adverse effect on subsequent chromatographic properties. The separation properties of the gel are not affected by detergents, e.g. 1% SDS, chaotropic salts or dissociating agents, e.g. 8 M urea or 6 M guanidine HCI. Strong solutions of NaOH (0.5 M) or HCl (0.5 M) can be used for cleaning if the gel is immediately washed with buffer or water. The high stability of Superdex prep grade makes it very suitable for use in industrial processes where high flow rates and effective cleaning-in-place are required. Superdex can also be used with organic solvents. A method for transferring Superdex from water to organic solvents is described on page 62.

Physical stability Columns pre-packed with Superdex may be used at temperatures in the range +4–40 °C. Exposure to temperatures outside this range will destroy the efficiency of the packed bed and the column will need to be re-packed. Typical flow rates for a bed 60 cm long packed with Superdex prep grade 28

Fig. 16. Pressure drop as a function of flow rate for HiLoad columns packed with Superdex prep grade. Bed height approximately 60 cm in distilled water at 25 °C. To calculate volumetric flow rate, multiply linear flow rate by cross-sectional area of column (2 cm2 for XK16, 5.3 cm2 for XK26).

can be seen in the pressure/flow diagrams in Figure 16. Superdex can be equilibrated at flow rates which are much higher than those recommended for the best resolution, resulting in short overall cycle times. Similarly, its mechanical rigidity allows even relatively viscous eluents, such as 8 M urea, to be run at practicable flow rates.

Chromatographic properties Efficiency HiLoad Superdex prep grade columns are supplied packed to an efficiency of >13 000 plates/metre. Superdex 75 and Superdex 200 HR 10/30 columns are supplied with an efficiency of > 30 000 plates/metre.

Selectivity The fractionation ranges of Superdex 75 and Superdex 200 correspond closely to the fractionation ranges of Sephadex G-75 and Sephadex G-200 respectively (Fig. 17). Superdex 30 prep grade is recommended for separations of peptides, oligonucleotides and small proteins in the molecular weight range up to 10000. Superdex 75 is particularly suited for 29

Fig. 17. Selectivity curves for Superdex.

the separation of a wide range of recombinant DNA products. Superdex 200 prep grade is a suitable choice when the molecular weight of the protein of interest is unknown. It is especially suitable for the separation of monoclonal antibodies from dimers and from contaminants of lower molecular weight, for example albumin and transferrin.

Adsorption Non-specific interactions between proteins and Superdex are negligible under normal chromatographic conditions using buffer solutions with ionic strengths in the range 0.15 to 1.5. At very low ionic strengths, the presence of a small number of negatively charged groups leads to retardation of basic proteins and exclusion of acidic proteins.

Availability Superdex prep grade is supplied in packs of 150 ml, 1 litre, 5 litres or prepacked in HiLoad and larger BioPilot columns. Superdex with a bead size of 13 µm is supplied pre-packed in HR 10/30 columns.

Further information Further information on Superdex with details of applications is given in the technical brochure “HiLoad columns”. For information on scale-up and the operation of large scale chromatography systems, please Pharmacia. 30

Superose Chemical and physical properties Superose (32) is composed of highly cross-linked porous agarose beads in two different particle sizes and two different fractionation ranges. The average wet bead diameter is 10 ±2 µm or 13 ±2 µm for Superose 6 and Superose 12 respectively, and 20–40 µm for the corresponding prep grades. Table 4 summarizes some of the properties of Superose. Table 4. Properties of Superose Gel type Superose 12 prep grade Superose 12 Superose 6 prep grade Superose 6

Bead size µm

Fractionation range Globular proteins

20 – 40 8 – 12 20 – 40 11 – 15

1 000 – 300 000 1 000 – 300 000 5 000 – 5 000 000 5 000 – 5 000 000

Fractionation range Dextrans ND ND ND ND

Chemical stability Superose can be used with all aqueous buffers commonly used in biochemistry within the pH range 3–12. Short term exposure to extremes of pH (1–14) e.g. for cleaning, has no adverse effect on subsequent chromatographic properties and the media withstand strong bases (33, 34) e.g. 0.2 M NaOH, and acids e.g. 0.01 M HCI and 1 M acetic acid. The separation properties of the gels are not affected by detergents e.g. 1% SDS, chaotropic salts or dissociating agents e.g. 8 M urea and 6 M guanidine HCl. Strong solutions of NaOH (0.5 M) or HCl (0.5 M) can be used for cleaning if the gel is immediately washed with buffer or water. Superose can also be used with organic solvents. Superose prep grade may be transferred from water to organic solvents by the method described on page 62.

Physical stability

Superose prep grade may be autoclaved repeatedly at 121 °C, pH 7 for 30 minutes without significantly affecting its chromatographic properties. Prepacked columns must not be exposed to temperatures outside the range +4 –40 °C. Exposure to temperatures outside this range will destroy the efficiency of the packed bed .

31

Recommended flow rates for Superose prep grade are in the range 0.3–0.5 ml/min for a column 16 mm i.d., 50 cm long. Pressure/flow diagrams for Superose 6 HR 10/30 and Superose 12 HR 10/30 columns are shown in figure 18.

Fig. 18. Typical pressure-flow relationships for Superose 6 and Superose 12 HR 10/30 columns in aqueous buffer solutions at room temperature.

The mechanical rigidity of Superose prep grade allows even relatively viscous eluents, such as 8 M urea, to be run at practicable flow rates.

Chromatographic properties Efficiency Superose 6 HR10/30 and 12 HR10/30 columns are supplied packed to an efficiency of >30 000 plates/metre.

Selectivity Gels with two different fractionation ranges are available, Superose 6 and Superose 12, with selectivity curves as shown in figures 19, 20 and 21. The selectivities of the corresponding prep grades are the same. 32

Fig. 19. Selectivity curves of Superose 6 and 12, globular proteins.

Fig. 20. Selectivity curves of Superose 6 and 12, dextrans.

33

Fig. 21. Selectivity curves of Superose 6 and 12, DNA-fragments.

Adsorption Ionic interactions between solutes and Superose are negligible at ionic strengths above 0.15 M. Some hydrophobic interactions have been recognized, i.e. some compounds (e.g. smaller hydrophobic and/or aromatic peptides, membrane proteins or lipoproteins) may be eluted later than predicted (these interactions can be of considerable value to the resolution). The degree of interaction is less for Superose prep grade than on prepacked Superose and Superose 6 shows weaker hydrophobic effects than Superose 12.

Availability Superose prep grades are available pre-swollen in packs of 125 ml. Superose 6 HR 10/30 and Superose 12 HR 10/30 columns are supplied prepacked.

Further information Further information on Superose with details of applications is given in the data file: “Superose 6, Superose 12”. For information on scale-up and the operation of large scale chromatography systems, please Pharmacia Biotech. 34

Sephadex Chemical and physical properties Sephadex is a bead-formed gel prepared by cross-linking dextran with epichlorohydrin (Fig. 22). Table 5 lists the different G-types of Sephadex and their physical properties.

Fig. 22. Partial structure of Sephadex.

Sephadex swells in aqueous solutions, and in dimethylsulphoxide and formamide. Dimethylformamide may be used with Sephadex G-10 and G-15 and mixtures of water with the lower alcohols may be used with Sephadex G-10, G-15, G-25 and G-50. It should be noted that the degree of swelling in organic solvents or their mixtures will not be the same as in water alone. Sephadex LH-20 (described in a separate booklet), Sephacryl HR and Sepharose CL are recommended for gel filtration in organic solvents. 35

Table 5. Properties of Sephadex. Gel type

Dry bead size µm

Sephadex G-10 40 – 120 Sephadex G-15 40 – 120 Sephadex G-25 Coarse 100 – 300 Sephadex G-25 Medium 50 – 150 Sephadex G-25 Fine 20 – 80 Sephadex G-25 Superfine 10 – 40 Sephadex G-50 Coarse 100 – 300 Sephadex G-50 Medium 50 – 150 Sephadex G-50 Fine 20 – 80 Sephadex G-50 Superfine 10 – 40 Sephadex G-75 40 – 120 Sephadex G-75 Superfine 10 – 40 Sephadex G-100 40 – 120 Sephadex G-100 Superfine 10 – 40 Sephadex G-150 40 – 120 Sephadex G-150 Superfine 10 – 40 Sephadex G-200 40 – 120 Sephadex G-200 Superfine 10 – 40

Fractionation range Globular proteins – 700 – 1 500 1 000 – 5 000 1 000 – 5 000 1 000 – 5 000 1 000 – 5 000 1 500 – 30 000 1 500 – 30 000 1 500 – 30 000 1 500 – 30 000 3 000 – 80 000 3 000 – 70 000 4 000 – 150 000 4 000 – 100 000 5 000 – 300 000 5 000 – 150 000 5 000 – 600 000 5 000 – 250 000

Fractionation range Dextrans

– 700 – 1 500 100 – 5 000 100 – 5 000 100 – 5 000 100 – 5 000 500 – 10 000 500 – 10 000 500 – 10 000 500 – 10 000 1 000 – 50 000 1 000 – 50 000 1 000 – 100 000 1 000 – 100 000 1 000 – 150 000 1 000 – 150 000 1 000 – 200 000 1 000 – 150 000

Swelling factor ml/g 2– 3 2.5 – 3.5 4– 6 4– 6 4– 6 4– 6 9 – 11 9 – 11 9 – 11 9 – 11 12 – 15 12 – 15 15 – 20 15 – 20 20 – 30 18 – 22 30 – 40 20 – 25

Chromatographic properties The G-types of Sephadex differ in their degree of cross-linking and hence in their degree of swelling and fractionation range (Fig. 23 and 24). Sephadex is available in different particle size grades. The different grades give chromatographic beds with different efficiencies and operating pressures. The highest efficiencies, and operating pressures, are obtained with the Superfine grade. The Superfine grade is also suitable for thin layer gel filtration. The Fine grade is recommended for preparative purposes. The Coarse and Medium grades are intended for preparative chromatographic processes where a high flow rate at a low operating pressure is essential. In addition the Coarse grade is suitable for batch procedures. Sephadex G-10, G-15, G-25 and G-50 are recommended for separations of peptides and other small biomolecules. Sephadex G-75, G-100, G-150 and G-200 are useful in work with proteins and other macromolecules where their relatively poor physical stability is not a hindrance. The DNA grade of Sephadex G-25, G-50 or G-100 should be used for work with DNA or oligonucleotides. 36

Fig. 23. Selectivity curves of Sephadex G-types Superfine, globular proteins.

Fig. 24. Selectivity curves of Sephadex G-types, globular proteins.

Availability All Sephadex G-types are supplied as dry powders in 100 g and 500 g packs. In addition: Sephadex G-25 Superfine is supplied pre-packed in Fast Desalting Column HR 10/10; Sephadex G-25 Medium is supplied pre-packed in disposable PD-10 Columns; Sephadex G-25 DNA grade is supplied pre-packed in disposable NAP Columns; and Sephadex G-50 DNA grade is supplied pre-packed in disposable NICK Columns and NICK Spin Columns. 37

Further information Further information on Sephadex with details of applications is given in the data sheet: “Sephadex gel filtration media”. For information on scale-up and the operation of large scale chromatography systems, please Pharmacia.

Sepharose Chemical and physical properties Sepharose is a bead-formed gel prepared from agarose. In its natural state agarose occurs as part of the complex mixture of charged and neutral polysaccharides referred to as agar. The agarose used to make Sepharose is obtained by a purification process which removes the charged polysaccharides to give a gel with only a very small number of residual charged groups. A gel forms spontaneously as a hot solution of agarose is cooled. The individual polysaccharide chains form double helices which subsequently aggregate to form bundles during the formation of a stable gel (Fig. 25).

Fig. 25. Gel structure of agarose. (Låås, T. Doctoral thesis. Acta Universitatis Upsaliensis 1975. Reproduced by kind permission of the Author.)

The individual polysaccharide chains may be represented as polymers of the repeating unit shown in Figure 26. 38

Fig. 26. Structure of the repeating unit of agarose. Note the presence of the unusual sugar 3,6-anhydro-L-galactose.

Sepharose is available in three types with different agarose concentrations and different fractionation ranges (Table 6). Table 6. Properties of Sepharose. Gel type

Approx. % agarose

Bead size µm

Fractionation range Globular proteins.

Fractionation range Dextrans

Sepharose 6B Sepharose 4B Sepharose 2B

6 4 2

45 – 165 45 – 165 60 – 200

10 000 – 4 000 000 60 000 – 20 000 000 70 000 – 40 000 000

10 000 – 1 000 000 30 000 – 5 000 000 100 000 – 20 000 000

Chemical stability Although the gel structure of Sepharose is stabilized by hydrogen bonding and not by covalent cross-links, it can still be used under most of the conditions encountered in gel filtration. It is stable in water and salt solutions over the pH range 4–9 and in the absence of oxidizing agents. Sepharose may be used for long periods in dissociating media, such as guanidine hydrochloride and urea, but for the best results Sepharose CL (cross-linked Sepharose, page 41) is recommended for these applications. Chaotropic salts, such as KSCN, should be avoided, but may be used with Sepharose CL.

Physical stability

Sepharose melts on heating above 40 °C and the bead structure may be irreversibly damaged on freezing. Consequently Sepharose cannot be sterilized by autoclaving, but it may be sterilized chemically, for example, by treatment with diethylpyrocarbonate. 39

The mechanical strength of Sepharose depends on the agarose concentration in the beads. Thus Sepharose 6B, (approx. 6% agarose) is considerably stronger than Sepharose 2B (approx. 2% agarose). Practical details on flow rates obtainable with Sepharose are given on page 66.

Chromatographic properties Selectivity The fractionation ranges for the different types are shown in Table 6 and Figure 27 shows the selectivity curves. The absence of suitable standards

Fig. 27. Selectivity curves for Sepharose and Sepharose CL, globular proteins.

makes it difficult to estimate the exclusion limits for proteins with confidence and these figures should be taken as a guide only.

Adsorption Sepharose contains a very small number of sulphate and carboxyl groups which may cause adsorption of basic proteins at low ionic strengths. These effects can be eliminated by using eluents with an ionic strength exceeding 0.15 and effects due to protein adsorption are seldom encountered in practice. Certain nucleic acids can be separated on Sepharose and this forms the basis of a number of schemes for their purification. For example tRNA species may be resolved on Sepharose 4B in high concentrations of ammonium sulphate (35). Similarly DNA, tRNA and high molecular weight RNA from a variety of species may be separated on Sepharose in 1.5 M NaCl (36). 40

Availability Sepharose is supplied as a ready-to-use suspension in 1 litre packs containing 20% ethanol as a preservative.

Further information Further information on Sepharose with details of applications is given in the data sheet: “Sepharose and Sepharose CL gel filtration media”. For information on scale-up and the operation of large scale chromatography systems, please Pharmacia.

Sepharose CL Chemical and physical properties Sepharose CL is prepared from Sepharose by reaction with 2,3 dibromopropanol under strongly alkaline conditions. This produces a cross-linked agarose gel with substantially the same porosity as the parent gel, but with greatly increased thermal and chemical stability. After cross-linking, the gel is desulphated by alkaline hydrolysis under reducing conditions yielding a gel with an extremely low content of ionizable groups (37). The resultant cross-links have the same structure as those present in Sephadex (5) and in the epichlorohydrin cross-linked agarose gels described by Porath, Janson and Låås (37). Sepharose CL is available in three types corresponding to the parent gels Sepharose 2B, 4B and 6B (Table 6).

Chemical stability Stability in aqueous media

Sepharose CL can be used in aqueous media in the range pH 3–13. Its stability in alkaline media is particularly high (37). Short term exposure to extremes of pH (2–14) e.g. for clening, has no adverse effect on subsequent Table 7. Stability of Sepharose CL in the presence of 3 M KSCN. Sample First effluent After 24 h exposure After 48 h exposure After 72 h exposure

Carbohydrate concentration µg/ml 6.5 <0.5 <0.5 <0.5

41

chromatographic properties. Under oxidizing conditions, limited hydrolysis of the polysaccharide chains may occur. The data in Table 7 demonstrate the stability of Sepharose CL-4B in solutions of chaotropic ions. Sepharose CL-4B was packed in a Pharmacia Column K 16/40 (bed volume 57.4 ml) and equilibrated with sodium phosphate buffer (0.02 M) containing NaCl (0.15 M). One bed volume of the same buffer containing the chaotropic salt KSCN (3 M) was ed through the column and the carbohydrate content of the effluent was estimated by the anthrone reaction. The column was allowed to stand overnight in the KSCN solution and the determination was repeated. After the initial elution the concentration of carbohydrate in the effluent was below the lower limit of sensitivity of the detection method. Stability in organic solvents

The gel structure of agarose differs significantly from that of Sephadex in that the gel-forming fibres are relatively stiff bundles of polysaccharide chains and not flexible single chains (38). Replacement of the water in the gel with other solvents has therefore a relatively small effect on pore size. Sepharose CL can be transferred from water to other solvents by the method described in the experimental section (see page 62). Solvents tested in our laboratories include ethanol, dimethylformamide, tetrahydrofuran, acetone, dimethylsulphoxide, chloroform, dichloromethane, dichloroethane, dichloroethane/pyridine (50:50), pyridine, triethyl phosphate and acetonitrile.

Physical stability Maximum flow rates obtainable with Sepharose CL are typically 50% higher than those obtainable with the corresponding types of Sepharose B. Practical details of flow rates obtainable with Sepharose CL are given on page 66. Because the gel structure of Sepharose CL is stabilized by crosslinking, it has comparable thermal stability to other cross-linked gels. Sepharose CL can be sterilized repeatedly by autoclaving at pH 7, 121 °C without significant changes in porosity or rigidity.

Chromatographic properties Selectivity The fractionation ranges and selectivity curves of the different types of Sepharose CL are not significantly different from those given for the equivalent Sepharose B in Table 6 and Figure 27.

42

Adsorption Sepharose CL shows even lower non-specific adsorption than the parent non cross-linked gel, in part due to its extremely low content of charged groups. Sepharose CL, for example, has been shown to separate nucleic acids in order of molecular size (39).

Availability Sepharose CL is supplied as a ready-to-use suspension in 1 litre packs containing 20% ethanol as a preservative.

Further information Further information on Sepharose CL with details of applications is given in the data sheet: “Sepharose and Sepharose CL gel filtration media”. For information on scale-up and the operation of large scale chromatography systems, please Pharmacia.

43

Experimental design Performance Resolution The principle objective of any fractionation experiment is to achieve adequate separation of the components of interest. In chromatography, the degree of separation, or resolution, is defined by Resolution (Rs)

=

distance between separated zones average zone width

Values of Rs greater than about 1.5 indicate baseline separation when the two components are present in approximately equal proportions. Greater resolution is needed if one of the components is present in considerable excess. Resolution can be increased in a number of ways (40) e.g. using a longer column, a gel medium with smaller particles and/or greater selectivity, a small sample volume, a low flow rate etc. However, the conditions which lead to the highest resolution are usually in conflict with other experimental objectives and a careful evaluation of the overall requirements is necessary. In particular, there is no need to design an experiment to give the ultimate in resolution unless the circumstances actually demand it. In many experiments adequate resolution is not difficult to achieve. More consideration can then be given to other objectives of a well designed experiment. In the case of preparative separations, these may be to achieve maximum recovery with least sample dilution in the shortest possible time. For analytical work we may be more interested in reducing sample consumption and maximizing run-to-run reproducibility.

Separation time Times for gel filtration separations range from a few minutes to many hours according to the difficulty of the separation, the gel medium, the column length and the flow rate. Since the factors which improve resolution also lead to longer separation times, there is always a compromise to be made between speed and resolution. Fortunately, the recent development of improved gel filtration media makes this choice less difficult and the vast majority of separations can be completed in a few hours at the most.

44

High performance gel filtration In high performance gel filtration, specialized media and equipment are employed to get the best possible combination of resolution and speed. Since zone broadening is a principle cause of loss of resolution, every effort is made to reduce dispersion. By using small bead sizes and columns packed to the highest possible efficiency, combined with small sample volumes, and detectors and other equipment designed for the purpose, excellent results can be obtained in the fractionation of complex samples in less than an hour.

Capacity The quantity of sample which can be fractionated successfully depends on its volume and concentration. These variables affect the resolution differently.

Sample concentration The concentration of protein in the sample has little direct result on resolution (Fig. 28). However, even moderately concentrated solutions of nucleic

Fig. 28. Influence of sample concentration on the resolution of transferrin and IgG on Superdex 200 prep grade.

45

acids and polysaccharides may be so viscous relative to the eluent that viscous fingering in the column leads to a catastrophic loss of resolution (5). Within the limits set by viscosity, see page 57, any sample concentration may be used. Using a concentrated sample is thus a simple and effective way of increasing the preparative capacity of a procedure.

Sample volume Resolution depends on the ratio of sample volume to column volume, large sample to column volume ratios giving lower resolution than smaller ones (Fig. 29). Recommended sample volumes for obtaining good resolution

Fig. 29. Influence of sample volume on the resolution of transferrin and IgG on Superdex 200 prep grade.

with different media are given in Table 8. The relationship between sample volume, bead size and resolution has been described by Hagel (41). The actual sample volume which can be applied for a given separation problem can only be found by experiment. 46

Table 8. Recommended sample volumes as a per cent of the total bed volume for good resolution. Many separations will show adequate resolution for larger sample volumes. Medium

Recommended sample volume %Vt

Sephadex Sepharose Sepharose CL Sephacryl HR Superdex prep grade Superose prep grade Superdex Superose

2 - 5% 2 - 5% 2 - 5% 1 - 2% 1 - 2% 1 - 2% 0.5% 0.5%

Process considerations In large scale applications of gel filtration it is not only important to obtain the desired separation result; the method used must be shown to perform to stringent demands for economy.

Productivity An important concept in discussing the performance of gel filtration in production processes is the productivity, or mass of material which can be processed per bed volume per hour (g . ml bed-1 . hour-1). For a given size of column, productivity is thus increased by increasing the sample concentration, the sample volume and the flow rate whilst maintaining adequate resolution (42). The exact conditions which lead to the highest productivity can only be ascertained by systematic experimentation.

Scale up Gel filtration is simple to scale up from the laboratory to process scale (43). Columns with volumes of 2 500 litres can be operated in favourable cases. The bed height, linear flow rate and sample concentration should be kept constant and the sample volume and volumetric flow rate increased in proportion to the increase in bed volume.

Choice of gel Choice of an appropriate gel depends on two main considerations, the purpose of the experiment and the sizes of the molecules to be separated. In some cases it may be important to consider other characteristics of the sample or the molecules to be separated. 47

The purpose of the experiment Analysis Analytical applications place special demands on the run-to-run reproducibility of the gel filtration column. It is thus specially important to choose a separation medium which gives a perfectly stable bed under the conditions of the analysis. Columns pre-packed with Sephacryl HR, Superdex or Superose are suitable, both for reproducibility and efficiency. Pre-packed columns are recommended to ensure the maximum column efficiency which is essential for the highest resolution. When the column is to be packed in the laboratory, special care should be taken to obtain a stable, well-packed bed and the packing recommendations should be followed in detail (see p 63–71).

Purity determination When gel filtration is used for purity determination, it is usually necessary to obtain the maximum resolution between the target protein and known contaminants. Small differences between the selectivities of different media can have a significant effect on the resolution and it may be necessary to run preliminary analyses with several different media since the optimum gel can only be ascertained by experiment.

Molecular weight determination and molecular weight distribution analysis For molecular weight determinations a gel should be chosen so that the sample’s expected molecular weight falls on the linear part of the selectivity curve and in the middle of a suitable range of calibration standards. Selectivity curves for the different media are shown on pages 25, 30, 33, 34, 37, 40. If the molecular weight is unknown, a gel with a wide fractionation range, e.g. Sephacryl HR, will be most suitable. A wide fractionation range is also recommended for molecular weight distribution analysis. If necessary, gels with different fractionation ranges can be used together in the same column or separately in two or more columns in series (42).

Binding equilibria Studies of binding equilibria require that the reacting species be separated. In many applications, one of the species is much larger than the other and choice of gel is simple. Sephadex G-25 is suitable for studies of binding equilibria between proteins and small molecules (20). 48

Preparative

Group separations and desalting The choice of gel is most easy for group separations such as desalting and buffer exchange, since there is a large difference in molecular weight between the two groups of components and complete resolution is not difficult to achieve. The gel is chosen so that the high molecular weight substances are eluted at the void volume (Kav = 0). This will give the minimum zone broadening and dilution and reduce the time the components of interest are on the column. The low molecular weight substances should be eluted near Vt (Kav = 1). Sephadex G-25 Fine is the recommended gel for the majority of desalting applications. It is easy to work with and has excellent separation capacity for desalting molecules down to about 5000 MW. Table 9. Pre-packed disposable columns for group separations Column designation HiTrap Desalting Fast Desalting PD-10 Columns NAP 5 Columns NAP 10 Columns NAP 25 Columns NICK Columns NICK Spin Columns cDNA Spun Columns Miniprep Spun Columns SizeSelect-400 Spun Columns

Medium Sephadex G-25 Superfine Sephadex G-25 Superfine Sephadex G-25 Medium Sephadex G-25 DNA grade Sephadex G-25 DNA grade Sephadex G-25 DNA grade Sephadex G-50 DNA grade Sephadex G-50 DNA grade Sephacryl S-300 HR Sephacryl S-400 HR Sephacryl S-400 HR

Max. sample volume 1.5 ml 0.5 ml 2.5 ml 500 µl 1.0 ml 2.5 ml 100 µl 150 µl 100 µl 50 µl 100 µl

Small disposable columns (Table 9) should be used if there is a risk of biological or radio-active contamination of the gel to reduce hazards in handling used materials. Disposable columns are also recommended when many small samples must be treated or when no risk of carry-over between one sample and another can be tolerated. The Fast Desalting Desalting Column HR 10/10 is recommended for rapid buffer exchange and other group separations of samples up to 2 ml in high performance separation schemes.

Group separations of DNA and oligonucleotides Sephadex DNA grade should be used for group separations of DNA or an oligonucleotide (greater than 10-mer) from salts, unincorporated 49

nucleotides etc. NAP-columns (Sephadex G-25 DNA grade) are recommended for desalting and buffer exchange and NICK-columns (Sephadex G-50 DNA grade) for removal of unincorporated nucleotides. cDNA or Size Select-400 spun columns should be used for group separation of cDNA from small molecules like linkers and adapters. Group separation of plasmids from proteins, RNA and NaOH in DNA sequencing may be carried out on Sephacryl S-400 packed in Miniprep spun columns thus eliminating the need for phenol extraction (30). Larger quantities of plasmids are quickly and conveniently prepared by gel filtration on Superose 6 prep grade (44).

Preparative fractionation Fractionation by size using gel filtration is a natural part of many procedures for purifying proteins and other biomacromolecules. The exact choice of gel will depend on the circumstances. The following guide-lines should be useful in most situations. Gel filtration is used in the early stages of a purification scheme to remove high molecular weight contaminants which may cause problems later on and to adjust the ionic composition of the sample to the pH and ionic strength required for a subsequent chromatographic step. The sample will be relatively crude and the ability to clean the gel from lipids and/or protein contamination is important. The sample will also be relatively large in volume and a large bed volume may be needed. The likely presence of proteases or other enzymes which might degrade the target molecules also makes it desirable to work with high flow rates. These considerations suggest that a gel from the Sephacryl HR series will generally be most suitable. Later on in the purification scheme, the sample will be simpler in composition and remaining impurities may require high performance gel filtration media to remove them. Pre-packed columns containing Superdex or Superose are available which ensure maximum resolution for particularly difficult fractionations.

Removal of dimers and aggregates Elimination of dimers and aggregates requires a gel with a selectivity such that the monomeric form of the molecule of interest is eluted in the latter part of the chromatogram. High resolution media, Superdex, Superose or Sephacryl HR, should be used for separation of dimers from monomers if baseline resolution is required. 50

Removal of degradation products and incomplete molecules When the impurities to be removed are known to be smaller than the target molecules, the gel should be chosen so that the target elutes early in the chromatogram. Sephacryl S-100 HR and Superdex 75 may be recommended when the protein of interest has a molecular weight in the range 50 000 – 100 000.

Solute characteristics Molecular weights of the components The molecular sizes of the molecules to be separated are the most important of the factors which govern the choice of separation medium. The medium must have a separation range which covers the molecular sizes of the target molecules. The selectivity curves of the different media are the best guide to the separation to be expected. Where there is a choice of matrix for a given molecular size range, the media with the steeper selectivity curves and the smaller bead sizes, e.g. Superdex, are to be preferred if high resolution is needed. Media with relatively shallow selectivity curves, e.g. Sephacryl HR or Sepharose CL, are preferable if fractionation over a wide range of molecular size is required.

Type of molecules to be separated The class of molecule, DNA, protein, peptide, polysaccharide etc, to be separated has an important bearing on the choice of gel since different classes of molecule may have very different shapes and thus very different exclusion properties for a given molecular weight (45–47). The selectivity curves on pages 25, 30, 33, 34, 37, 40 show this clearly and may be used as a guide to the selection of the correct medium for the main classes of biomacromolecules. Note that Sephacryl HR types are available with exclusion limits which make them useful for fractionation of even native nucleic acids and polysaccharides as well as membrane vesicles and multicomponent complexes.

Sample characteristics Sample size Sample size has an indirect influence on the choice of gel in that large sample volumes require large bed volumes; the medium chosen must therefore be capable of being run economically in a sufficiently large bed (see ‘Choice of column dimensions’ below).

Eluent Some samples contain proteins or other components which have a limited range of solubility or stability. There is thus always a risk that changing the 51

pH or other conditions of the sample will cause inactivation or even precipitation. If the sample precipitates in a gel filtration column, the column will be blocked, possibly irreversibly, and the sample may be lost. Care should always be taken to work within the solubility and stability range of the sample. The free choice of eluent in gel filtration enables this requirement to be met in almost every case. The composition of the eluent does not affect the choice of gel except when it can be expected to alter the conformation of the molecules to be separated or when it places special demands on the stability of the gel. Gel filtration in dissociating eluents, e.g. 6 M guanidine hydrochloride or urea, is extremely useful for molecular weight determination (14). However, because of the more extended configuration of proteins and polypeptides in these eluents, a more porous gel is usually required than predicted from selectivity curves prepared for native globular proteins. Thus, in many cases, Sephacryl HR or Sepharose CL will be found the most appropriate gels. Their high chemical and physical stability also makes them particularly suitable for use in these eluents. Detergents are particularly useful as solubilizing agents for proteins with low aqueous solubility, for example, membrane components (48–50). Once again the effect of these agents on protein conformation usually requires a more open gel to be used for gel filtration. Detergents do not appear to influence the pore structure of the gels (51). The more recently developed media, Sephacryl HR, Superdex and Superose, are in general more suitable than the traditional media for work in dissociating media, eluents containing organic solvents and eluents of extreme pH.

Choice of running conditions Choice of column To obtain the best results from gel filtration experiments care in the choice of column equipment is necessary.

Ideal features Gel filtration on a laboratory, pilot plant or industrial scale should be carried out on suitably designed columns. 52

The dead volumes at the outlet and inlet should always be as small as possible. Many simple columns have large dead volumes and should, therefore, be avoided. Bed s made from coarse sintered glass or glass wool cannot be recommended for long term use, because they soon become clogged, are difficult to clean and can give artifactual results (52). Pharmacia has developed a series of standard columns suitable for gel filtration. Their design is based on many years experience in the field of gel filtration. They are manufactured from materials which do not cause destruction of labile biological substances. All are easy to dismantle and reassemble to allow thorough cleaning, which is particularly important when handling biological samples. Other important characteristics include • Dead space at the outlet of less than 0.1% of the column volume. Minimizes dilution and prevents remixing of separated zones. • Advanced design bed s which give uniform flow. • All of the columns, except for K9 types, can be fitted with flow adaptors for easy sample application (p 73–77).

Pressure and solvent resistances Pharmacia columns, packing equipment and accessories are designed in several resistance classes. XK columns are recommended for use in all common buffer systems at pressures up to 5 bars. Columns in the C-column series are for laboratory applications at pressures of up to 1 bar. SR-columns are for use with organic solvents.

Column dimensions The resolution of two separated zones in gel filtration increases as the square root of column length. Long columns should, therefore, be used to obtain the best resolution in analytical fractionation. Bed heights of greater than 1 m are seldom required. If a very long bed is judged to be necessary, the effective bed height can often be increased simply by recycling (Fig. 30) or by using columns coupled in series. Both techniques require the use of adaptors or end pieces (p 70). For analytical purposes a column with an internal diameter of approximately 1.0 cm often proves satisfactory. In general the length of column is decided by the resolution required and the diameter by the sample volume (see “Sample size” page 55). 53

Rec102

Fig. 30. Increasing the effective column height by recycling. Eluent and sample are connected to the 3-way valve which can be closed during recycling. The 4-way valve connects the column outlet to the inlet, or the sample/eluent to the column and the column outlet to the fraction collector.

For group separations short columns often suffice and gel beds less than 50 cm can be used with the Coarse grade of Sephadex. With Fine and Superfine grades still shorter beds give satisfactory resolution for comparable separations.

Flow rate Resolution, under conditions which are usually encountered in gel filtration, decreases with increase in flow rate. The optimum flow rate for maximum resolution of proteins is of the order of 5 ml.cm-2.h-1 in laboratory columns, although flows up to 5 times faster can often be used without much deterioration. Maximum resolution is obtained with a long column and a low flow rate and the fastest run is obtained with a short column and a high flow rate. Good resolution and short running times may thus appear to be basically incompatible. 54

Using media with small particles will give increased efficiency and higher resolution. This may allow higher flow rates to be used, but usually at the expense of sample capacity. If peaks are well separated at a low flow rate using a long column, the excess resolution may be traded off for speed. The flow rate can be increased and a shorter column can be used, or alternatively more sample can be applied. For preparative purposes the advantage of a higher flow rate (and consequently a faster separation) often outweighs the loss of resolution in the chromatographic run. Full details of the maximum permissible flow rates of the various gel types, governed by their mechanical properties, are given on page 66.

Sample characteristics Sample size For analytical purposes and difficult fractionation experiments where maximum resolution is required the starting zone must be narrow relative to the length of the column. A sample volume of 0.5–5% of the bed volume is recommended, see Table 8. Smaller volumes do not normally improve resolution. In group separations and some fractionation experiments, where peaks are well resolved, it is often appropriate to improve the experimental design by increasing the sample size. In order to minimize sample dilution, which is an inevitable consequence of gel filtration, a maximum sample volume should be used within the limits set by the separation distance. Figure 31 shows how, if no zone-broadening were to occur during age down a column, the maximum sample volume could be as great as the separation volume (VSep). VSep = VeB - VeA However, due to eddy diffusion, non-equilibrium between the stationary phase and the mobile phase, and longitudinal diffusion in the bed, the zones will always be broadened (40). The sample size must, therefore, always be smaller than the separation volume. In desalting and buffer exchange volumes up to approximately 30% of the total bed volume (Vt) 55

Fig. 31. Elution curves for different sample sizes. The top diagram corresponds to the application of a small sample. The centre diagram corresponds to the maximum sample size giving complete separation if no zone broadening were to take place. The bottom diagram corresponds to the maximum sample volume to be applied to obtain complete separation in the conditions of the experiment. The shaded areas correspond to the elution profiles that would be obtained if no zone broadening were to occur.

can be used to minimize dilution and still retain good separation. Where complete recovery of desalted sample is the major requirement, sample volumes of between 15 and 20% of Vt are recommended. For routine small scale desalting with the PD-10 columns (p 49) use of the maximum recommended sample volume of 2.5 ml results in the effectively complete recovery of desalted material in a volume of 3.5 ml, a dilution factor of only 1.4 (Fig. 32).

Fig. 32. Desalting of albumin solution. A column PD-10 was equilibrated with distilled water. The sample contained human serum albumin (25 mg) dissolved in NaCl (0.5 M, 2.5 ml). Yield of albumin in 3.5 ml after sample application (between arrows) 95.3%; salt content, 2.0% of total salt originally present.

56

When deg an optimized desalting system, the volume of gel required should be packed in a short, wide column rather than a long, narrow one. This allows more rapid recovery of desalted materials since higher volume flow rates can be achieved with the shorter column.

Sample composition Due to the linear partition isotherms, gel filtration is to a large extent independent of mass of solute in the sample. However, in addition to the solubility of the solutes, the viscosity of the sample often limits the concentration that can be used. A high sample viscosity causes instability of the zone and an irregular flow pattern. This leads to very broad and skew zones. The critical variable is the viscosity of the sample relative to the eluent. This is illustrated in Figure 33 which shows the elution profiles of haemoglobin and NaCl at different sample viscosities.

Fig. 33. Elution diagrams obtained when haemoglobin and NaCl were separated. Experimental conditions were identical except that the viscosities were altered by the addition of increasing amounts of dextran. An increasing deterioration of the separation becomes strikingly apparent. (A lower flow rate will not improve the separation.)

57

In practice the relative viscosities of sample and eluent should not differ by more than a factor of about 2, corresponding to a protein concentration in the sample of about 70 mg/ml when a dilute aqueous buffer is used as the eluent. Approximate relative viscosities can be quickly estimated by comparing emptying times from a pipette.

Eluent Eluent composition does not directly influence the resolution which can be obtained in gel filtration. Completely uncharged substances may be eluted with distilled water. For chromatography of substances carrying charged groups an eluent containing a buffer, e.g. Tris-HCl, sodium phosphate etc., is often used to control pH, and an ionic strength of at least 0.02 for Sephadex and Sepharose or 0.15 for Sephacryl HR is recommended to safeguard against possible ionic interactions with the gel matrix. Sodium chloride can be used for this purpose. It should also be noted that some proteins may precipitate in solutions of low ionic strength. If the product is to be lyophilized, volatile buffers such as ammonium acetate, ammonium bicarbonate or ethylenediamine acetate can be used. In desalting, the separation volume is so large that, in general, charged substances can also be treated with distilled water as eluent. Complete removal of salt is not possible, but the amount of ions excluded and, therefore, eluted with the HMW fraction is so small it can be neglected in most cases. The most important consideration in the choice of eluent in gel filtration is its effect on the sample molecules. The pH and ionic composition of the buffer, and the presence of dissociating media or detergents can cause conformation changes, dissociation of proteins into subunits, dissociation of enzymes and cofactors, dissociation of hormones and carrier proteins etc. which must be taken into when choosing the gel and when interpreting the results of an experiment.

Optimization Optimization of a separation can only be carried out by careful experiment, since each separation problem is different. The behaviour of a three component model system of proteins A, B and C (Fig. 34) is used here to illustrate some of the factors which might need to be considered. Components A and B are separated at the lowest flow rate. However, this separation can not be speeded up significantly without serious loss of 58

Fig. 34. A model gel filtration experiment to demonstrate optimization of the separation of model proteins A, B and C.

resolution. Protein C is well separated from protein B and this particular separation can be speeded up by a factor of 50. (An alternative view of optimization could be to retain the flow rate and increase sample size to utilise more completely the separation volume between proteins C and B). In the first stage of the optimization, increasing the flow rate, the time for separating protein C from B is reduced from about 30 hours to about 2.5 hours. The resolution (Rs) is still 2.25. The bed height (L2) required to give Rs = 1 can be calculated from L2 =

L1 Rs1

59

where Rs1 is the resolution obtained with a column length L1. In this example L2=17 cm. Reducing the bed height to about 20 cm should still give adequate resolution of protein C and protein B. The result obtained with a bed height of 19.5 cm at 24.5 ml.cm-2.h-1 is shown in Figure 34. The separation time for protein C and protein B has been reduced from 30 hours to 35 minutes. For a full discussion of the optimization of gel filtration see (40).

60

Performing a gel filtration experiment Gel filtration is essentially simple to perform once a well packed column has been obtained. Providing that a column is used and maintained carefully it can be expected to give many months of reliable service.

Preparing the gel Pre-swollen media (Sephacryl HR, Superdex prep grade, Superose prep grade, Sepharose CL and Sepharose) These gels are supplied swollen and ready to use as a suspension containing 20% ethanol as a preservative. The suspension is too thick to be poured directly into a chromatography column (except for Superose prep grade) and must first be diluted with eluent to the required consistency.

Media which require swelling (Sephadex G-types) Sephadex is supplied as a dry powder and must be allowed to swell in excess solvent before use. During swelling excessive stirring should be avoided as it may break the beads. Do not use magnetic stirrers. For all Sephadex G-types, the process of swelling can be accelerated by using a boiling water bath which also serves to deaereate the buffer (Table 10). Table 10. Bed volume and swellings times for Sephadex G-types. Gel type Sephadex G-10 Sephadex G-15 Sephadex G-25 (all grades) Sephadex G-50 (all grades) Sephadex G-75 (all grades) Sephadex G-100 (all grades) Sephadex G-150 Sephadex G-150 Superfine Sephadex G-200 Sephadex G-200 Superfine

Approx. bed volume (ml/g) 2 – 3 2.5 – 3.5 4 – 6 9 – 11 12 – 15 15 – 20 20 – 30 18 – 22 30 – 40 20 – 25

Swelling time (h) 20 °C 3 3 3 3 24 72 72 72 72 72

90 °C 1 1 1 1 3 5 5 5 5 5

61

Preparation of Sephacryl HR, Superdex prep grade, Superose prep grade or Sepharose CL for use in organic solvents The aqueous medium in which a gel is swollen can be exchanged for a variety of organic solvents. (Note: Because the gel structure of Sepharose is only stabilized by hydrogen bonding, it is not suitable for gel filtration in many organic solvents. However, Sepharose CL can be used quite safely in a wide range of organic solvents.) To ensure efficient replacement of the water by the required solvent, the transfer must be made through a graded series of solvent mixtures. Thus to transfer from one pure solvent (A) to another pure solvent (B), the gel is transferred first to 70% A/30% B then to 30% A/70% B and finally to pure B. If A and B are not mutually

Fig. 35. Suggested routes for changing to organic solvents.

miscible, the transfer is made via an intermediate solvent e.g. from water to chloroform via acetone (Fig. 35). Transfer the required amount of gel to a sintered glass Buchner funnel and remove the excess aqueous medium by gentle suction. Add the next solvent (see Fig. 35) and resuspend the gel by gentle stirring. Suck off the excess solvent and resuspend in the same solvent. Repeat the process with the next solvent of the series, allowing two resuspensions and proceed until the required solvent composition is reached. Note that the gel volume of Sephacryl HR may be reduced by up to 15% on transfer to organic solvents (Table 2).

62

Gel filtration in organic solvents After transferring the gel to the solvent of choice it may be packed in the usual way (see below). Sepharose CL floats in dense solvents, e.g. chloroform, and in these cases packing should be carried out as described in the booklet “Sephadex LH-20. Chromatography in organic solvents”. Pharmacia columns SR 10/50, SR 25/45 or SR 25/100 should be used. Flow rates will depend on the viscosity of the eluent. Certain solvents which disrupt hydrogen bonds, e.g. DMSO, may cause softening of Sepharose CL so that flow rates become impracticably low.

Packing a column (Fig. 36-43) This is a very critical stage in any gel filtration experiment. A poorly packed column will give rise to uneven flow, zone broadening and loss of resolution. The flow rates obtainable are also affected. It is important to note that Sephacryl HR, Superdex prep grade and Superose prep grade, which have a relatively rigid bead form, are packed in a rather different manner than the Sephadex G-types and Sepharose CL or B types.

Packing Sephadex G types, Sepharose and Sepharose CL Filling the column 1. Prepare the gel as described above. The suspension of gel should be adjusted so that it is a fairly thick slurry. It should not be so thick it retains air bubbles. Usually about 75% settled gel is suitable. Fine particles can be removed at this stage by decantation, if desired.

Fig. 36.

63

The suspension should be de-gassed under vacuum (Fig. 36) This is not necessary for Sephadex which has been swollen on a boiling water bath. The gel suspension should reach the temperature of column operation before packing is begun. 2. Mount the column on a stable laboratory stand. It is important to avoid locations which are exposed to draughts or direct sunlight and which can cause temperature changes and the formation of bubbles in a packed column. Use of degassed buffers and a filled water-jacket will also help safeguard against the effects of rapid temperature changes. 3. Ensure that there are no air bubbles trapped in the dead space under the net by drawing water (or 20% ethanol) through it. This is best done by submerging the plunger in a beaker of water (or 20% ethanol) and attaching the tubing to a pump or a syringe. Alternatively, inject eluent into the outlet tubing until it es through the bed net (Fig. 37). When the dead space is properly filled, close the outlet tubing.

Fig. 37.

4. Tilt the column and pour the well-mixed gel suspension down the inside wall of the column. Immediately readjust the column to a vertical position. Alternatively the gel can be poured directly into the vertically mounted column using a glass rod. If the slurry volume is greater than the volume of the column, a gel reservoir (XK, C, K series columns) or a column extension (SR-10, HR 10 and HR 16 columns) should be attached (Fig. 38 and Fig. 39). All the gel required should be poured in a single operation. Preparing the gel from too thin a suspension or, for other reasons, packing the column in stages, often results in a badly packed bed. 64

Fig. 38.

Fig. 39.

5. The next step involves closing the column without trapping air inside. If an adaptor is used, follow the instructions given below (p 70–71). With C or K columns, a top piece can be used in the following way. Close the column outlet and fill the space above the gel with eluent to about 1 cm from the top of the glass tube. Fit the top piece, removing air via the air vent valve in the top piece This step is unnecessary if a column packing reservoir or extension is being used. 6. The flow should be started as soon as possible after filling the column to obtain even sedimentation. The top piece air vent should be closed and the column outlet opened to allow the packing to continue. Check for air bubbles and repeat steps if necessary. Do not exceed the maximum operating pressures given in Table 11. 65

Table 11. Approx maximum flow rates and pressures. Data has been obtained from columns 2.5 cm diameter with a bed height of 30 cm. Gel type

Sephacryl S-100 HR Sephacryl S-200 HR Sephacryl S-300 HR Sephacryl S-400 HR Sephacryl S-500 HR Sepharose CL-6B Sepharose CL-4B Sepharose CL-2B Sepharose 6B Sepharose 4B Sepharose 2B Sephadex G-10 - G-50 Sephadex G-75 Sephadex G-75 Superfine Sephadex G-100 Sephadex G-100 Superfine Sephadex G-100 Sephadex G-150 Superfine Sephadex G-200 Sephadex G-200 Superfine

Max. operating pressure cm H2O

Approx. max. flow rate ml/min

ml.cm.–2 h–1

500 500 500 500 500

2.5 2.5 2.5 2.5 2.5

30* 30* 30* 30* 30*

>200 120 50

2.5 2.17 1.25

30 26 15

200 80 40

1.16 0.96 0.83

14 11.5 10

6.4 1.5 4.2 1.0 1.9 0.5 1.0 0.25

77 18 50 12 23 6 12 3

These gels obey Darcy’s Law (see p 78) 160 160 96 96 36 36 16 16

* For flow rates used during packing please see Table 12.

Equilibrating the bed 1. Before opening the column outlet tubing, check the operating pressure. This depends on the difference between the free surface of eluent in the reservoir and the outlet as illustrated in Figure 40. With the softer gels the operating pressure must not exceed the limits given in Table 11. With rigid gels such as Sephadex G-10 or G-50 such precautions are unnecessary and packing can be rapidly achieved with a pump or gravity feed. 66

Fig. 40. Definition of operating pressure (A-D) and sample application from a sample reservoir (D). A and B. Pressure (cm water) is measured as the distance between the free surface in the column or reservoir and the end of the outlet tubing. C and D. Pressure (cm water) is measured from the bottom of the air inlet tube in the Mariotte flask to the end of the outlet tubing, no matter whether the flow through the column is downward (C) or upward (D).

67

2. Open the column outlet and start the flow. Two or three column volumes of eluent should be ed through the column in order to stabilize the bed and equilibrate with eluent buffer. A slightly higher flow rate than is to be used in the experiments should be used for packing.

Packing Sephacryl HR, Superdex prep grade and Superose prep grade Sephacryl HR, Superdex prep grade and Superose prep grade should be packed and equilibrated at a high flow rate in a column in the XK-series using a suitable pump (e.g. P-l, P-50, P-500). The packing method described below ensures that the gel is optimally packed at the point of entry of the sample, the most critical area, and it is highly recommended that this method be followed. Pack the column at the temperature at which it will be used. 1. Make sure the column is not damaged and that all parts are really clean. It is of special importance that the nets, net fasteners and glass tube are not damaged. 2. Attach the packing reservoir tightly and mount the column vertically on a stand. 3. Wet the adaptor by drawing water (or 20% ethanol) through it, making sure no air bubbles are trapped under the net. This is best done by submerging the plunger in a beaker of water (or 20% ethanol) and attaching the tubing to a pump or a syringe. Close the tubing with a stopper when all air bubbles have been removed. 4. Insert the adaptor at the bottom of the column far enough to give the desired bed height. 5. Wet the column glass tube with eluent leaving a few centimetres of fluid in the bottom making sure that the net is completely free from air bubbles. 6. Prepare the gel as described above. The suspension of gel should be adjusted so that it is a fairly thick slurry. It should not be so thick it retains air bubbles. Usually about 75% settled gel is suitable. Resuspend the gel and pour the well-mixed gel suspension carefully down the wall of the column using a glass rod. Pour all the gel in a single operation and fill the reservoir to the top with eluent. 7. Screw on the reservoir cap tightly, connect it to the pump and open the outlet. 8. Pack the column in two steps using the flow rates given in Table 12, 68

Table 12. Recommended flow rates for packing Sephacryl HR Column

Flow rates (ml/h) Step 1 Step 2

XK 26/40 XK 26/70 XK 26/100

240 240 180

490 360 320

XK 50/60 XK 50/100

600 600

1 400 950

9.

10.

11. 12. 13. 14.

10.

11. 12. 13.

pack the gel in STEP 1 for 2 hours or until the gel bed has reached a constant height, then increase the flow rate to the value listed for STEP 2 and pack for 60 minutes. Stop the pump, close the column outlet and remove the packing reservoir. This is most easily done by first removing the column from the stand and then unscrewing the reservoir over a sink. It may be easier to use a siphon when working with a large column. Using one adaptor and a bottom piece: Remove excess gel carefully with a small spoon or a plastic spatula until the bed surface is about 2 mm below the end of the glass tube. When the bottom piece is inserted in point 14 it will be pressed about 5 mm into the gel. If there is not enough gel in the column, it will be necessary to use a second adaptor or to repack the column with more gel. Mount the column vertically on the stand and fill the column to the top with buffer. Wet the bottom piece with eluent or 20% ethanol. Insert the bottom piece carefully so that no air bubbles are trapped under the net. Open the bottom piece outlet (NOT the outlet from the adaptor at the bottom of the column). Press down and tighten the bottom piece. This will cause eluent to flow out of the tubing attached to the bottom piece. Close the bottom piece outlet again. Using two adaptors: Remove excess gel by gently stirring the top of bed with a glass rod and removing the suspended gel with a Pasteur pipette. Remove enough gel so that the plunger will be visible below the end piece. Mount the column vertically on the stand and fill the column to the top with buffer. Wet the second adaptor with eluent or 20% ethanol. Remove the stopper from the second adaptor tubing (NOT from the adaptor at the bottom of the column) and insert the adaptor carefully so that no air bubbles are trapped under the net. 69

14. Bring the adaptor to the gel surface and then a further 5 mm into the gel bed. Liquid will flow out of the tubing attached to the adaptor during this process.

Adaptors Adaptors are adjustable column end pieces which the bed matrix. They allow automatic methods of sample application which eliminate disturbance of the bed surface and protect the bed from insoluble particles in the sample. After an extended period of use, flow rates through a column packed with Sephadex at a given hydrostatic pressure will be reduced. An upward flow arrangement using adaptors will discourage this process (Fig. 40 D). Adaptors are available for most Pharmacia columns. They should be fitted as follows. 1. The gel should be packed completely as described above, or alternatively should be allowed to settle sufficiently so that there are 2–3 cm of clear eluent at the top of the column. 2. Ensure the column outlet is closed otherwise the gel may be compressed when the adaptor is fitted. Carefully add more eluent to fill the column and form an upward meniscus (Fig. 41) 3. Slacken the adaptor tightening mechanism and insert it at an angle into the column so that no air is trapped under the net (Fig. 42).

Fig. 41.

70

Fig. 42.

Fig. 43.

4. Adjust the tightening mechanism to give a sliding seal between the column wall and O-ring. Screw the adaptor top piece onto the column end piece. 5. Make all tubing connections at this stage. Slide the plunger slowly down the column so that air in the adaptor above the net and in the capillary tubings is displaced by eluent. Valves on the inlet side of the column should be turned in all directions during this procedure to remove air from all connections. Lock the adaptor in position with the tightening mechanism and locking screw (Fig. 43), open the outlet and start the eluent flow. Readjust the position of the adaptor at the bed surface after packing has been completed (if the column has already been packed when the adaptor is fitted, only about 10 minutes further packing is required before re-adjustment).

Checking the packed bed Before starting any experiment, it is advisable to check the homogeneity of the bed by running a freshly prepared and filtered solution of a coloured substance. Blue Dextran 2000 at a concentration of 2 mg/ml can be used for this purpose and to determine the void volume of the bed. The quality of the packing can be checked by watching the progress of a zone of this substance through the bed. Visual inspection of the bed in transmitted light may also reveal heterogeneities and air-bubbles.

71

Using pre-packed columns The initial setting up of a gel filtration experiment is considerably simplified if a pre-packed column is used. 1. Connect the column to the sample application device (see below for alternatives) and to the monitor. 2. Follow the instructions supplied with the column to equilibrate it with the chosen eluent.

Sample application Sample application without an adaptor Note that these methods are not suitable for use with pre-packed columns since removing the upper adaptor to apply the sample would disturb the column packing. One of the methods described under ‘Sample application with an adaptor’ should be used.

Sample application on a drained bed surface Although this is the method which requires least equipment, it is not the simplest and considerable care must be taken to avoid disturbing the bed surface. An uneven bed surface leads to uneven separated bands and poor resolution. A sample applicator cup (available for columns series XK, C and K columns with diameters 16 and 26 mm) helps prevent disturbance of the bed surface. 1. Close the outlet and remove most of the eluent above the gel surface by suction. 2. Open the outlet and allow the remaining eluent to drain away. Under no circumstances should the bed be allowed to run dry. 3. Layer the sample on top of the bed. 4. Open the column outlet and allow the sample to drain into the bed. Do not allow the bed to run dry. 5. Wash the sample which remains on the bed surface and on the column wall into the bed with a small amount of eluent. 6. Refill the column with eluent and reconnect to a Mariotte flask or pump.

Sample application under the eluent (Fig. 44) The sample must be denser than the eluent. If not, it can be made so by the addition of a small amount of glucose, sodium chloride, buffer salt or another suitable inert material. Take care not to increase the sample viscosity too much. 72

Fig. 44.

1. Use a syringe fitted with a piece of fine capillary tubing or a pipette with a bent tip. Draw the sample into the syringe, then draw a little air into the capillary tubing to prevent mixture of sample with eluent. 2. Close the column outlet and place the tubing in the eluent so that its tip is held a few mm above the bed surface and dispense the sample slowly and evenly as a layer under the eluent. 3. After application draw a small amount of eluent into the tubing to prevent sample mixing with eluent when the tubing is withdrawn. No rinsing is required and eluent flow can be restarted directly.

Sample application with an adaptor These methods are always used with pre-packed columns and are also the most convenient for other columns if the column is to be used frequently. Samples must be applied by one of these methods if upward flow elution is used.

Syringe method. (Fig. 45). The valves LV-3 and LV-4 can be used as syringe holders to give a very simple method for the application of small samples .

Sample reservoir (Fig. 46). In a similar way, a sample reservoir (e.g. RK or R) can be connected via a 3-way valve to apply larger samples. Figure 46 also demonstrates sample application and elution with upward flow. 73

Fig. 45.

Fig. 46.

74

Sample applicators SA-5, SA-50 (Fig. 47). These are reservoirs which allow the sample to be introduced as a layer below the eluent using a syringe and needle without disturbing the chromatographic bed. They can also be used in a sample loop system (Fig. 47) where their large capacity (up to 5 ml for the SA-5 and 45 ml for the SA-50) and lack of tailing due to wall effects offer distinct advantages.

Fig. 47. A Sample Applicator SA-5 used as a sample loop.

Sample loops with valves LV-4 or SRV-4 (Fig. 48). This method is convenient for application of small samples. By using the same sample loop very reproducible sample volumes can be applied, although exact knowledge of the applied volume requires calibration of the capillary tubing loop. 75

Fig. 48. Sample application with a sample loop and two SRV-4 valves.

Sample loops or Superloop with valves V-7 or MV-7 (Fig. 49). This method is used for sample application when using high performance columns (e.g. Superdex HR 10/30 and Superose HR 10/30) and other

Fig. 49. Seven-port valves, V-7 and MV-7, have three operating positions which make sample application and changing eluents particularly convenient.

columns in FPLC System. Superloop (Fig. 50) is a unique sample application device from which a sample of any volume up to the capacity of the Superloop (10 ml or 50 ml) can be applied to a column without tailing. A movable seal separates the sample from the eluent. As eluent is pumped 76

Fig. 50. Principle of operation of Superloop. Sample is drawn into the space in front of the moving seal and applied to the column when eluent is pumped into the space behind the seal. When the seal reaches the end of its travel, eluent by-es the seal and washes the last of the sample quantitatively onto the column.

into the Superloop, the sample moves ahead of the seal and onto the column. When nearly all the sample has been applied, eluent flows round the seal to wash the remainder of the sample quantitatively onto the column. A Superloop should be used for applying sample volumes greater than approximately 1 ml. Very reproducible sample volumes can be applied, particularly when the system is operated automatically using LCC-501 Plus or FPLCdirector.

Elution The flow of liquid through the column can be controlled by difference in hydrostatic pressure or by a pump. Accurate and reproducible control of flow rate is particularly important when repeating experiments or performing routine preparative work. It is most easily achieved using a good peristaltic pump such as the P-l pump. Note that a pump should always be connected into the system so that it pumps the eluent onto the column rather than connecting it after the column. This reduces the risk of bubble formation in the column which can result from suction. When the flow is to be maintained by gravity feed a constant pressure flask, such as a 77

Gel and Eluent Reservoir used as a Mariotte flask, is recommended. Mention has already been made (see page 70) of the advantage of upward flow for long term use of a column. A problem which can occur when an experiment is continued without supervision, for example, overnight, is that of stopping the experiment before the eluent runs out. When using a pump this can be achieved by attaching it to the power supply via a timing/cut-off device or via a fraction collector which can control the pump. If gravity feed is used, a safety loop, of the type illustrated in Figure 51 can easily be arranged to prevent air from entering the column.

Fig. 51. Safety loop arrangements: A. The safety loop is placed after the column and the end of the outlet tubing is placed above the column. The flow stops when the eluent in the inlet tubing reaches the level of the outlet. B. The safety loop is placed before the column with the column outlet tubing in any position above the lower loop on the inlet side. The flow stops when the eluent in the inlet tubing reached the level of the outlet.

Flow rates Recommendations concerning flow rates for use in experiments are given on page 66. It is not possible to apply one set of rules for calculating flow rates over the whole range of gel types. The arguments, equations and data given here apply only to laboratory columns with diameters up to 5 cm.

Sephadex G-10, G-15, G-25 and G-50 These gels may be assumed to behave as rigid spheres in gel filtration and therefore obey Darcy’s Law, i.e. U = K ∆P L-1 78

(1)

where U is the linear flow rate expressed in cm/h (ml. cm-2.h-1), ∆p is the pressure drop over gel bed expressed in cm H2O, L is the bed height expressed in cm and K is a constant of proportionality depending on the properties of the bed material and the eluent. Assuming an eluent with viscosity of 1 one can write U = Ko ∆P L-1

(2)

where Ko is the “specific permeability” depending on the particle size of the gel beads and their water regain. Observe that the flow rate is proportional to the pressure drop over the bed and, assuming a constant pressure head, inversely proportional to the bed height. Notice that (to a good approximation) the flow rates are independent of the column diameter. Flow rates at viscosities greater than 1 can be obtained by using the relation: flow rate inversely proportional to viscosity. At first sight, it would appear that high eluent viscosities lead to poor flow rates but the operating pressure can be increased to compensate for the viscosity effect. Temperature influences the viscosity of the eluent. Lower flow rates are obtainable, for a given pressure head, in a cold room than at room temperature. Theoretical flow rates (not maximum) can be calculated from equation (2) by inserting values for ∆p and L. Specific permeabilities are given in Table 13. Table 13. Specific permeabilities of Sephadex G-types. Sephadex type

Permeability K

Sephadex G-10 Sephadex G-15 Sephadex G-25 Superfine Sephadex G-25 Fine Sephadex G-25 Medium Sephadex G-25 Coarse Sephadex G-50 Superfine Sephadex G-50 Fine Sephadex G-50 Medium Sephadex G-50 Coarse

19 18 9 30 80 290 13.5 36 145 400

Flow rates in columns packed with other media Calculation of flow rates in other less rigid gels is somewhat more complicated since Darcy’s Law is not applicable. Not only is the flow rate dependent upon the factors already mentioned but also on the column diameter. Wider columns do not allow as high a pressure and linear flow 79

rate (ml.cm-2.h-1) as narrower ones. These gels do not have a linear relationship between pressure and flow, exceeding the maximum recommended values can lead to gel compression, reduction in flow rate and loss of resolution. Recommended maximum operating pressures and corresponding approximate flow rates at room temperatures are given in Table 11. A bed diameter of 2.6 cm (column XK 26) and bed height of 30 cm have been assumed. Maximum operating pressures are independent of bed height but flow rates decrease with bed height. To calculate the flow rate for a different bed height it may be assumed to be inversely proportional to the bed height. Slightly reduced maximum operating pressures must be used with columns wider than 2.6 cm. To utilize fully the high rigidity and the excellent flow properties of Sephacryl HR a liquid delivery system including a pump should be used. A peristaltic pump can still be used with the other softer gels provided care is taken. It is recommended to determine the maximum flow rate for the column in question with gravity feed, taking care not to exceed the pressures given in Table 11, and then use flow rates not exceeding 75 % of this value with the pump. The maximum flow rates which are given in Table 11 serve as a guide only and may vary depending upon column packing or eluent viscosity (Note especially the effect of temperature on viscosity).

Cleaning gels and packed columns General cleaning procedures When a column has been in use for some time, it may be necessary to remove precipitated proteins or other contaminants which have built up on the gel bed. The need for cleaning may show itself as the appearance of a coloured band at top of the column, as a space between the upper adaptor and the bed surface, as a loss in resolution or as a significant increase in back-pressure. General procedures are given below for different media followed by special procedures for removing specific contaminants. In all cases, prevention is better than cure. The use of filtered eluents and samples is essential for high performance columns and will greatly reduce the number and severity of the problems encountered with any medium. Similarly, only fresh buffer solutions should be used. Many buffer substances are excellent ers of microbial growth. See also the section on ‘Prevention of microbial growth’ (p 84). 80

If an increase in back-pressure is observed, for example by the level of the gel falling, make sure that the high back-pressure is in fact caused by the column before starting the cleaning procedure. Disconnect one piece of equipment at a time (starting at the fraction collector) with the pump working and check the pressure as each piece is disconnected. A dirty filter is often the cause of increased back-pressure. The pressure should be checked at the same stage during each run, since the back-pressure can vary within a run during sample injection or when changing to a different eluent. Note that cleaning solutions should also be filtered before use. After cleaning, the column must be carefully re-equilibrated with 2–3 column volumes of eluent buffer before it is used again.

Cleaning Sephadex G-types Packed columns may be cleaned with 2 column volumes of a non-ionic detergent solution. Sephadex may also be washed with NaOH (0.2 M) on a Buchner funnel.

Cleaning Sepharose Wash with a non-ionic detergent.

Cleaning Sepharose CL Treat the gel with one bed volume of 0.5 M NaOH or a non-ionic detergent solution (1%) either in the column or on a Buchner funnel.

Cleaning Sephacryl HR Sephacryl HR may be cleaned and sanitized in the column with 1–2 column volumes of NaOH (0.2–0.5 M) at a flow rate which gives a time between the gel and the cleaning solution of at least 1 hour. This time is sufficient to solubilize most protein precipitates. Sephacryl HR may also be washed with a solution of a non-ionic detergent. Use the same method if the gel is severely contaminated, but reverse the direction of flow in the column during treatment with NaOH.

Cleaning Superose prep grade a. Cleaning a packed column:

Do NOT reverse the flow during cleaning since this may cause a loss of efficiency. 1. Change the filter on the top of the gel bed and check the bed , changing it if necessary. 81

2. Wash with at least 1/3 of the column volume NaOH (0.1 M-0.2 M). 3. Rinse with high purity water or buffer. Small aliquots (3 x 100 or 200 µl) of acetic acid (50%) may be added during this rinse. 4. Equilibrate with buffer until the baseline is stable. b. Cleaning the gel before repacking:

1. Put a large enough beaker under the column to collect the gel, remove the bottom piece and empty the column by pumping high purity water or buffer through it. Clean all column parts with soapy water or laboratory detergents. Inspect the top and bottom filters and change them. 2. Wash the gel on a glass filter with NaOH (0.1-0.2 M), then water and lastly ethanol (20%). 3. Re-suspend the gel in at least 5 times the gel volume of high purity water in a beaker. 4. Allow the gel to sediment and pour off the supernatant. 5. Repeat the washing procedure once more before re-packing the column.

Cleaning Superose HR 10/30 pre-packed columns Regular cleaning

The following procedure will help prevent contaminants from building up on the column. 1. Wash with 5 ml 0.1 M NaOH 2. Wash with 5 ml 50% acetic acid 3. Re-equilibrate with buffer until the base line is stable Rigourous cleaning to remove contamination

Do NOT reverse the flow during cleaning since this may cause a loss of efficiency and NEVER exceed the maximum pressure for the column. 1. Change the filter at the column inlet. Since the contaminants are introduced with the liquid flow, many of them are caught by the filter. 2. Set the pressure limit to the maximum for the column. 3. Wash in sequence with 25 ml acetic acid (50%), 25 ml water, 25 ml ethanol (20%, run at a low flow rate), 25 ml NaOH (0.1 M), 25 ml water and three aliquots of 100 or 200 ml of acetic acid (50%). This procedure is most conveniently carried under automatic control from a Liquid Chromatography Controller LCC-501 Plus. 4. Equilibrate with buffer until the baseline and the pH of the eluent leaving the column are stable. 82

The suggested cleaning volume of 25 ml is only a guide-line; the practical requirements are best determined by monitoring the baseline, which should be stable after each step in the sequence. In special cases only, it may be necessary to change the bottom filter or to remove and discard the top 2-3 mm of the gel. These operations must be carried out extremely carefully to avoid serious loss of resolution.

Cleaning Superdex pre-packed columns Superdex columns may be cleaned with one bed volume of NaOH (0.5 M). This treatment will remove most proteins from the gel. The frequency of cleaning depends on the degree of contamination, but a cleaning cycle at least every 10–20 separation cycles is recommended. Re-equilibrate the column with two bed volumes of buffer immediately after cleaning. Wait until the UV baseline is steady before applying the next sample. If the column still is contaminated, it may be washed with 0.5 bed volume of 1 M NaOH solution and 0.5 bed volume of 30% isopropanol or 0.1 M HCl. Replace the filters if necessary.

Procedures to remove specific contaminants If the general cleaning methods fail to give the desired result, the following methods may be used to remove specific contaminants. Various alternatives are given for each type of contaminant – choose the most convenient according to the reagents you have available; if this does not work, try one of the alternatives. Since some of the cleaning solutions are more viscous than normal buffer solutions, care must be taken not to exceed the maximum operating pressure which the gel can sustain.

Hydrophilic proteins and peptides Wash the column with the solution which previously dissolved the material during sample preparation e.g. an extraction solution, detergent, etc. (overnight, at low flow rate). Wash overnight in 30–50% acetic acid at 0.1 ml/min. Fill the column with 1 mg/ml pepsin in 0.1 M acetic acid and 0.5 NaCl and leave overnight at room temp. or 1 hour at 37 °C. Note: after enzymatic digestion, careful rinsing is required to remove trace amounts of enzyme remaining in the system. 83

Hydrophobic proteins and peptides These are usually soluble in polar organic solvents e.g. ethanol (24%), acetonitrile (30%). If the organic solvent which best dissolves the contaminant is known, run this overnight at a slow flow rate.

Nucleic acids RNA and DNA are very soluble in solutions of low ionic strength and may be re-dissolved by running low ionic strength buffer (e.g. 10 mM Tris-HCl, 1 mM EDTA, pH 8.0) through the column at low flow rate for 24 hours. RNA

Hydrolyse the RNA with NaOH (0.1–2 M, 1 hour) and rinse with water or with ribonuclease I solution (2 ml, 1 mg/ml in 0.1 M NaCl, 50 mM Tris-HCl, pH 7.5, 37 °C, 2 hours) and rinse with 10 mM Tris-HCl, 1 mM EDTA, pH 8.0. DNA

Hydrolyse the DNA with deoxyribonuclease I solution (2 ml, 1 mg/ml in 0.1 M NaCl,10 mM MgCl2, 50 mM Tris-HCl, pH 7.5, 37 C, 2 hours) and rinse with 10 mM Tris-HCl, 1 mM EDTA, pH 8.0. Note: after enzymatic digestion, careful rinsing is required to remove trace amounts of enzyme remaining in the system; special caution is recommended if subsequent separations of RNA or DNA are planned.

Lipids Wash the gel overnight at 0.1 ml/min with a non-ionic detergent (e.g. 0.2–1% NP-40 or Lubrol) in a basic or acidic solution and remove the detergent by washing with methanol or ethanol.

Storage of gels and columns Prevention of microbial growth Microbial growth rarely occurs in columns during use but steps should always be taken to prevent infection of packed columns, buffers and gel suspension. Antimicrobial agents may be eluted from columns before chromatographic runs or they may be present in the eluent during chromatography. Antimicrobial agents which interact with sample substances must be avoided if they are to be used in eluents, otherwise any agent which does not interact with the gel may be used. Some of the more commonly used antimicrobial agents are described below. 84

Antimicrobial agents Chlorhexidine Chlorhexidine (e.g. Hibitane®), 0.002%, is a very effective antimicrobial agent. It is incompatible with only a very few substances but is not recommended for use with Sepharose. Precipitation may occur on storage of Hibitane with appreciable concentrations of chloride or sulphate ions.

Chloroform, butanol and toluene These organic solvents are not recommended as antimicrobial agents for Sephadex G-100, G-150 or G-200, as they cause the gel particles to shrink slightly. In addition they are effective only in very concentrated solutions. They penetrate plastic parts of chromatographic equipment, softening the plastic and leaving the liquid without antimicrobial activity. Oxidizing substances should not be used as antimicrobial agents. For sterilization of gels by autoclaving of the gel see pages 24, 28, 31, 39, 41.

Ethanol 20% Superose, Superdex, Sephacryl HR, Sepharose CL and Sepharose are supplied in 20% ethanol. This solution can also be used as a bacteriostatic agent for storage of used gel.

Phenyl mercuric salts Phenyl mercuric salts (acetate, borate, nitrate), 0.001–0.01%, are effective only in weakly alkaline solutions.

Sodium azide Note. The use of sodium azide is discouraged in many countries. It can lead to explosions when disposed of via lead pipe waste disposal systems and is believed to be a mutagen. Sodium azide, NaN3, 0.02–0.05%, is very widely used. It does not interact notably with proteins or carbohydrates or change their chromatographic behaviour. Sodium azide interferes with fluorescent marking of proteins, the anthrone reaction and inhibits certain enzymes.

Sodium hydroxide Sodium hydroxide, 0.01 M, is an effective bacteriostatic agent. At higher concentrations (0.1–0.5 M) it is an effective disinfectant even for resistant bacteria such as Pseudomonas. Treatment with sodium hydroxide inactivates endotoxins and will, in many cases, solubilize substances 85

precipitated on the column. Its low toxicity is an advantage that reduces the risk of sample contamination. Sodium hydroxide is not recommended for use with Sepharose and for storage of Sephacryl HR.

Thimerosal Thimerosal (ethylmercurithiosalicylate e.g. Merthiolate®), 0.005%, is most effective in weakly acidic solutions. It is bound to and inactivated by substances containing thiol groups. It is not recommended for use with Sephacryl HR.

Trichlorbutanol Trichlorbutanol (e.g. Chloretone®), 0.05%, is effective only in weakly acidic solutions.

Storage of unused media Unused media should be kept at +4–25 °C. Note that it is important that swollen media are not allowed to freeze as the beads may be disrupted by ice crystals leading to the generation of fines.

Storage of used media Used media should be stored at +4–8 °C, pH 6-8 in the presence of a suitable bacteriostatic, e.g. sodium azide (0.05%) or ethanol (20%). Note that it is important that swollen media are not allowed to freeze as the beads may be disrupted by ice crystals leading to the generation of fines. Thimerosal should not be used with Sephacryl. Sephadex can also be stored partially shrunk in, for example, 60–70% alcohol. For special purposes Sephadex can be dried and restored to its original state by the following procedure. The swollen gel is thoroughly washed with water to remove salts and contaminants. After removal of excess water, the gel can be shrunk by successive addition of alcohol solutions of increasing percentage alcohol. The gel should be allowed to equilibrate in between each addition. Final shrinking should be with 96% alcohol. The gel is then sucked dry on a Büchner funnel and finally dried at 60–80 °C. A last wash with diethyl ether reduces the drying time. Clumps may appear during the shrinking process but they disperse on re-swelling. Risk for clump formation is reduced by slow shrinking and ether washing. 86

Storage of packed columns Packed columns should be stored at +4–8 °C in the presence of a suitable bacteriostat. Sodium azide (0.05%) or ethanol (20%) may be used for Superdex, Superose, Sephacryl, Sepharose and Sepharose CL, but only sodium azide (0.05%) is recommended for columns packed with Sephadex G-types. For short-term storage, e.g. overnight, the column should be left connected to the system; a low flow rate through the column will prevent bacterial growth. For long-term storage, the gel bed should be thoroughly cleaned before re-equilibration with the storage buffer. If ethanol (20%) is used in the storage buffer, de-gas the ethanol/water mixture well, start the equilibration at a low flow rate and check the back-pressure while equilibrating the column (water/ethanol mixtures are more viscous than normal aqueous buffer solutions which will increase the back-pressure). Disconnect the column from the system, close the bottom tubing of the column and insert the tubing from the top of the column in a Parafilm® covered vessel (e.g. test tube) containing ethanol (20%). Pre-packed HR columns are supplied with a rubber tubing between the inlet and the outlet of the column; this tubing may be filled with buffer and re-connected to prevent the column drying out. Note that columns may need to be re-packed if they are exposed to temperatures widely different from the temperature at which they were packed.

87

88

Fault finding chart Problem

Cause

Remedy

Column is clogged

Presence of lipoproteins or protein aggregates.

Prior to chromatography, precipitate with 10% Dextran Sulphate or 3% polyvinylpyrrolidone. See cleaning procedures p 80–84.

Precipitation of proteins Modify the eluent to in the column caused by maintain stability. removal of stabilizing agents during fractionation.

The sample substance is poorly resolved from other major peaks

Filter is clogged.

Replace the filter. Always filter samples and buffer before use.

Microbial growth has occurred in the column.

Microbial growth rarely occurs in columns during use, but steps should always be taken to prevent infection of packed columns, buffers and gel suspensions. Store gel in the presence of 20% ethanol or 0.05% sodium azide. See p 86–87.

Microbial growth has occurred in the column.

See above

89

Problem

Cause

The sample substance is poorly resolved from other major peaks

Sample volume is too Decrease sample volume large or the sample has and apply the sample been improperly applied. carefully. For maximum resolution do not exceed the sample volumes given in Table 10. Contaminated gel surface or top net will spoil sample application. Sample is too viscous.

Remedy

Dilute the sample with the elution buffer. Keep protein concentration below 30 mg/ ml.

Improper filtration of the Regenerate the column, sample before application filter the sample and to the column. repeat the chromatography step. Column is not mounted vertically.

Try again with the column mounted in a vertical position. You may need to repack the column.

Column is poorly packed. Check the packing by running a coloured compound, e.g. Blue Dextran and observing the band. Repack the column if necessary. Too much sample mass Decrease the sample load. has been loaded onto the column. The column is dirty.

90

Clean and regenerate the column.

Problem

Leading or very rounded peaks observed in the chromatogram

Cause

Remedy

Detector cell volume is too big.

Change the flow cell.

Wrong gel type.

Substances which are relatively close in molecular weight require Superose or Superdex HR. Check selectivity curve. Check for possibility of adsorption effects. Consider the effects of dissociating agents or detergents if present.

Large mixing spaces in or after column.

See p 53.

Column too short.

See p 53.

Flow rate too high.

See p 54.

Proteins or lipids precipitated on column.

Choose elution conditions which stabilize the sample. See p 80–84 for cleaning procedures.

Uneven temperature in the bed.

Use a column with a water-jacket.

Overloaded column.

Decrease the sample load and repeat the run.

Column is poorly packed. Check the packing by running a coloured compound, e.g. Blue Dextran and observing the band. Repack the column if necessary.

91

Problem

Cause

Remedy

Earlier elution profile can not be reproduced

The sample has altered during storage.

Prepare fresh sample.

Larger sample mass load Keep volume and mass of applied compared to sample constant when earlier run. High protein repeating runs. concentration can cause protein protein interaction resulting in a change in elution profile. The sample volume is different from earlier runs.

Resolution is dependent on the sample volume. Keep sample volume constant when repeating runs.

Proteins or lipids have precipitated on the column.

Clean and regenerate the column.

Sample has not been filtered properly.

Regenerate the column, filter the sample carefully and repeat this step.

Sample substance The molecular weight elutes at an or shape is other than unexpected elution expected. position Ionic interactions between the protein and the matrix.

–

Keep the ionic strength above 0.05 M to minimize ionic interactions.

Hydrophobic interactions Reduce the salt between the protein and concentration to minimize the matrix. hydrophobic interactions. Add a suitable detergent.

92

Problem

Cause

Remedy

Sample substance elutes later than expected or even after the total column volume

Retardation is probably due to hydrophobic interactions between protein and the matrix.

If possible decrease the ionic strength, increase pH or introduce organic solvent in the elution buffer, e.g. 5% isopropanol, in order to minimize hydrophobic interactions.

Late elution and broad peaks observed in the chromatogram

The column has become dirty.

Clean and regenerate the column.

Elution times are Leakage before the gel too long, but bed. elution volumes are correct

Eliminate the leak.

Pump wrongly calibrated. Re-calibrate the pump. More sample substance is recovered than expected

Sample substance co-elutes with other substances.

Try to optimize your separation in order to resolve the peak. Alternatively, combine several techniques to remove contaminants completely.

Low recovery of activity while normal recovery of protein

Sample substance may not be stable in the chosen eluent and is therefore inactivated.

Change the eluent.

Enzyme separated from co-factor or similar.

Test by pooling fractions and repeating the assay.

93

Problem

Cause

Remedy

Microbial growth.

See p 84.

Protein amount in The protein may have the eluted fractions been degraded by is much less than proteases. expected Microbial growth has occurred in the column.

Add protease inhibitors to the buffers to prevent proteolytic digestion.

See p 84.

Non-specific adsorption. Try adding ethylene glycol (e.g. 10%) to the buffers to prevent any hydrophobic interactions. Elution conditions too harsh.

Choose elution conditions which stabilize the sample.

Specific adsorption.

Lectins may bind to sugar residues in the matrix. Try specific elution with an analogous sugar.

Sample precipitates.

May be caused by removal of salts or sample dilution.

More activity is recovered than was applied to the column

Different assay conditions Use the same assay have been used before conditions for all the assays and after the in your purification scheme. chromatographic step.

Peaks too small

Wrong sensitivity range on detector.

Adjust.

Sample absorbs poorly at Use a different wavelength. the chosen wavelength. Recorder range incorrectly set.

94

Adjust.

Problem

No peaks

No flow through the column

Reduced or poor flow through the column

Cause

Remedy

Sample size is smaller than intended.

–

Excessive zone broadening

Check the column packing and re-pack if necessary.

Monitor switched off.

Switch it on.

Monitor not connected to recorder.

Check the electrical connections and cables.

UV-lamp not working.

Replace the lamp.

Sample not applied.

Check the operation of the sample application device.

Range button depressed or no sensitivity set on recorder.

Check the recorder settings.

Outlet closed.

–

No flow from pump.

With peristaltic pumps check the condition of the tubings. Check for leaks at all connections.

Air-lock in outlet tubing or bottom-piece.

See page 69.

Clogged end-piece or adaptor or tubing.

Bed s of porous glass or polythene are prone to clogging by gel particles.

See above.

–

95

Problem

Cause

Remedy

Bed surface blocked by precipitated proteins.

Remove contaminated gel from the bed surface. Stir the top 1–2 cm and allow to re-settle as an even layer.

Bed compressed.

Special care is needed when packing Sephadex G-75 – G-200 (see Table 13). Can also occur after prolonged use. Upward elution retards the process. Repacking the column may be necessary.

Microbial growth.

For prevention see page 84.

Gel not fully swollen (Sephadex G-types).

See page 61.

Fines (Sephadex G-types). Do not use a magnetic stirrer; it can break the beads. Fine particles can be removed by decanting from a settling suspension. Bubbles in the bed

96

Column packed or stored Small bubbles can often be at cool temperature and removed by ing well then warmed up. de-gassed buffer upwards through the column. Column may need to be repacked. Take special care if buffers are used after storage in a fridge or cold-room. Do not allow column to warm up due to sunshine or heating system. A waterjacket is a good safeguard. Use de-gassed buffers.

Problem

Cracks in the bed

Cause

Remedy

Eluent not properly de-gassed.

De-gas the eluent thoroughly.

Large air leak in column. Check all connections for leaks. Repack the column. Column violently mishandled.

Distorted bands as Air bubble at the top of sample runs into the column or in the the bed adaptor.

–

Re-install the adaptor taking care to avoid air bubbles. See p 70–71.

Particles in eluent or sample.

Filter or centrifuge the sample. Protect eluents from dust.

Particles on the bed surface or uneven bed surface.

Remove small amount of gel and stir up the top 1–2 cm, allowing it to re-settle as an even layer.

Clogged or damaged net in upper adaptor.

Dismantle the adaptor, clean or replace the net. Keep particles out of samples and eluents.

Distorted bands as Column poorly packed. sample es down bed

Gel suspension too thick or too thin. Bed packed at a temperature different from run. Bed insufficiently packed (too low packing pressure, too short equilibration). Column packed at too high pressure.

97

Problem

Cause

Gel beads in eluent Bed loose or broken. Column operated at too high pressure.

98

Remedy Replace or re-fasten. Do not exceed the recommended operating pressure for the gel medium.

References 1.

Gel filtration: A method for desalting and group separation. Nature 183 (1959) 1657–1659, Porath, J., Flodin, P.

2.

On the history of the development of Sephadex. Chromatographia 23 (1987) 361–369, Janson, J.-C.

3.

Gel filtration. In Protein Purification. Principles, high resolution methods, and applications, pp 63–106. (ed. J.-C. Janson, L.Rydén), VCH Publishers Inc., New York, Weinheim, Cambridge 1989 Hagel, L.

4.

The gel filtration behaviour of proteins related to their molecular weights over a wide range. Biochem. J. 96 (1965) 595–606, Andrews, P.

5.

Dextran gels and their applications in gel filtration. Dissertation 85 pp., AB Pharmacia, Uppsala, Sweden, 1962 Flodin, P.

6.

Some recently developed fractionation procedures and their application to peptide and protein hormones J. Appl. Chem. 6 (1963) 233–244, Porath, J.

7.

A relationship between the molecular weights of macromolecules and their elution volumes based on a model for Sephadex gel filtration. Arch. Biochem. Biophys. 107 (1964) 471–478, Squire, P.G.

8.

A theory of gel filtration and its experimental verification. J. Chromatogr. 14 (1964) 317–330, Laurent, T.C., Killander, J.

9.

A new calibration procedure for gel filtration columns. J. Biol. Chem. 242 (1967) 3237–3238, Ackers, G.K.

10. Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductase. Biochim. Biophys. Acta 112 (1966) 346–362, Siegel, L.M., Monty, K.J. 11. The correlation between molecular weight and elution behaviour in the gel chromatography of proteins. J. Chromatogr. 25 (1966) 303–313, Determann, H., Michel, W. 12. Estimation of molecular weights of proteins by agarose gel filtration. J. Chromatogr. 40 (1969) 453–457, Locascio, G.A., Tigier, H.A., Batlle, A.M. del C. 13. Estimation of molecular size and molecular weights of biological compounds by gel filtration. In Methods of Biochemical Analysis vol. 18 pp. 1–53 (ed. Glick, D.) Interscience Publishers, New York, London 1970 Andrews, P. 14. Molecular-weight estimation of proteins using Sepharose CL-6B in guanidine hydrochloride. J. Chromatogr. 140 (1977) 98–102, Ansari, A.A., Mage, R.G. 15. Fractionation of dextran by the gel filtration method. Makromolekulare Chem. 48 (1961) 160–71, Granath, K.A., Flodin, P. 16. Molecular-weight distribution determination of clinical dextran by gel permeation chromatography. J. Chromatogr. 101 (1974) 137–153, Nilsson, G., Nilsson, K. 17. Drug-plasma binding measured by Sephadex. J. Pharm. Pharmacol. 9 (1962) 550–555, Barlow, C.F., Firemark, H., Roth, L.J. 18. Separation of human heme- and hemoglobin-binding plasma proteins, ceruloplasmin and albumin by gel filtration. Biochim. Biophys. Acta 93 (1964) 1–14, Killander, J. 19. Estimation of serum haemoglobin-binding capacity (haptoglobin) on Sephadex G-100. J. Clin. Pathol. 17 (1964) 676–679, Ratcliff, A.P., Hardwicke, J. 20. Measurement of protein-binding phenomena by gel filtration. Biochim. Biophys. Acta 63 (1962) 530–532, Hummel, J.P., Dreyer, W.J. 21. Studies of chemically reacting systems on Sephadex. 1. Chromatographic demonstration of the Gilbert Theory. Biochemistry 2 (1963) 1263–1267, Winzor, D.J., Scheraga, H.A.

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22. Peptide-protein interaction as studied by gel filtration. Biochemistry 5 (1966) 673–683, Fairclough, Jr., G.F., Fruton, J.S. 23. Binding of sulfonamides to serum proteins: physico-chemical and immuno-chemical studies. J. Pharmacol. Exp. Therap. 153 (1966) 167–175, Clausen, J. 24. Protein-binding of small molecules. New gel filtration method. J. Pharm. Pharmacol. 20 (Suppl.) (1968) 1505– 1565, Cooper, P.F., Wood, G.C. 25. The application of gel filtration to the study of protein-binding of small molecules. Chromatogr. Rev. 12 (1970) 88–107, Wood, G.C., Cooper, P.F. 26. Determination by gel filtration of association constants for metal-nucleotide interaction. Anal. Biochem. 46 (1972) 358-363, Colman, R.F. 27. A hydrogen exchange method using tritium and Sephadex: its application to ribonuclease. Biochemistry 2 (1963) 798–807, Englander, S.W. 28. Determination of stoichiometry and equilibrium constants for reversibly associating systems by molecular sieve chromatography. Proc. Nat. Acad. Sci. USA 53 (1965) 342–349, Ackers, G.K., Thompson, T.E. 29. Labeling deoxyribonucleic acid to high specific activty in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113 (1977) 237–251, Rigby, P.W.J., Dieckmann, M., Rhodes, C. et al. 30. “Molecular Cloning. A laboratory manual”, T. Maniatis, E.F. Fritsch, J. Sambrook (Cold Spring Harbor 1982) 31. Properties, in theory and practice, of novel gel filtration media for standard liquid chromatography. J. Chromatogr. 476 (1989) 329–344, Hagel, L., Lundström, H., Andersson, T. et al. 32. Agarose-based media for high-resolution gel filtration of biopolymers. J. Chromatogr. 326 (1985) 33–44, Andersson, T., Carlsson, M., Hagel, L. et al. 33. Column lifetime of a new agarose medium for high-performance gel filtration chromatography at basic pH. J. Chromatogr. 330 (1985) 360–364, Johansson, B-L., Ellstrom, C. 34. Column lifetime of Superose 6 at 37 Celsius and basic pH. J. Chromatogr. 351 (1986) 136–139, Johansson, B-L., AAhsberg, L. 35. Separation of transfer ribonucleic acid by Sepharose chromatography using reverse salt gradients. Proc. Nat. Acad. Sci. USA 72 (1975) 1068–1071, Holmes, W.M., Hurd, R.E., Reid, B.R. et al. 36. A new general method for separation of nucleic acids. Prep. Biochem. 4 (1974) 509–522, Petrovic, S.L., Petrovic, J.S., Markovic, R.A. et al. 37. Agar derivatives for chromatography, electrophoresis and gel-bound enzymes. 1. Desulphated and reduced crosslinked agar and agarose in spherical bead form. J. Chromatogr. 60 (1971) 167–177, Porath, J., Janson, J.-C., Låås, T. 38. The agarose double helix and its function in agarose gel structure. J. Mol. Biol. 90 (1974) 269-284, Arnott, S., Fulmer, A., Scott, W.E. et al. 39. Resolution of ribonucleic acids by Sepharose 4B column chromatography. Biochemistry 16 (1977) 1378–1382, Zeichner, M., Stern, R. 40. “The Dynamics of Chromatography”, J.C. Giddings (Marcel Dekker, N.Y. 1965) Part 1, Principles and theory. 41. Effect of sample volume on peak width in high-performance gel filtration chromatography. J. Chromatogr. 324 (1985) 422–427, Hagel. L. 42. “Process chromatography. A practical guide”, G.K. Sofer and L.-E. Nyström (eds) (Academic Press, London 1989) pp 36–41. 43. “Process chromatography. A practical guide”, G.K. Sofer and L.-E. Nyström (eds) (Academic Press, London 1989) pp 55–66. 44. Purification of plasmid DNA by fast protein liquid chromatography on Superose 6 preparative grade. Anal. Biochem. 177 (1989) 378-382, McClung, J.K., Gonzales, R.A.

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45. Universal calibration of gel permeation chromatography and determination of molecular shape in solution. Anal. Biochem. 162 (1987) 47–64, Potschka, M. 46. Size-exclusion chromatography of DNA restriction fragments. Fragment length determinations and a comparison with the behaviour of proteins in size-exclusion chromatography. J. Chromatogr. 467 (1989) 217–226, Ellegren, H., Laas, T. 47. Size-exclusion chromatography and universal calibration of gel columns. Anal. Biochem. 177 (1989) 50–56, Le Maire, M., Viel, A., Møller, J. 48. Improved preparation of the integral membrane proteins of human red cells, with special reference to the glucose transporter. Biochim. Biophys. Acta 855 (1986) 345–356, Lundahl, P., Griejer, E., Cardell, S. et al. 49. Purification and characterization of membrane-bound phospholipase C specific for phosphoinositides from human platelets. J. Biol. Chem. 263 (1988) 11459–11465, Banno, Y., Yada, Y., Nozawa, Y. 50. Sodium dodecyl sulphate-protein complexes. Changes in size or shape below the critical micelle concentration, as monitored by high-performance agarose gel chromatography. J. Chromatogr. 476 (1989) 147–158, Mascher, E., Lundahl, P. 51. Molecular characterization of proteins in detergent solutions. Biochemistry 13 (1974) 2369–2376, Tanford, C., Nozaki, Y., Reynolds, J.A. et al. 52. Glass wool as a potential source of artifacts in chromatography. J. Chromatogr. 152 (1978) 514–516, Schwartz, D.P.

BioPilot, FPLC, FPLCmanager, HiLoad, HiPrep, HiTrap, LKB, MicroPerpex, NAP, NICK, Pharmacia, Sephacryl, Sephadex, Sepharose, Superdex, Superloop, Superose and Uvicord are trademarks of Amersham Pharmacia Biotech Limited or its subsidiaries Amersham is a trademark of Nycomed Amersham plc Pharmacia and Drop Design are trademarks of Pharmacia & Upjohn Inc All goods and services are sold subject to the and conditions of sale of the company within the Amersham Pharmacia Biotech group which supplies them. A copy of these and conditions of sale is available on request. © Amersham Pharmacia Biotech AB 1998 - All rights reserved Amersham Pharmacia Biotech UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Pharmacia Biotech AB SE-751 84 Uppsala Sweden Amersham Pharmacia Biotech Inc 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA

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Ordering information Column

Code No.

Media

Pack size

Superdex Peptide HR 10/30 Superdex Peptide PE 7.5/300 Superdex Peptide PC 3.2/30 Superdex 75 PC 3.2/30 Superdex 200 PC 3.2/30

17-1453-01 17-5003-01 17-1458-01 17-0771-01 17-1089-01

Superose 6 prep grade Superose 12 prep grade

125 ml 17-0489-01 125 ml 17-0536-01

Superose 6 PC 3.2/30 Superose 12 PC 3.2/30 Superose 6 HR 10/30 Superose 12 HR 10/30

17-0673-01 17-0674-01 17-0537-01 17-0538-01

Superdex 30 prep grade Superdex 30 prep grade Superdex 30 prep grade Superdex 30 prep grade

25 ml 150 ml 1 litre 5 litres

17-0905-10 17-0905-01 17-0905-03 17-0905-04

Superdex 75 HR 10/30 Superdex 200 HR 10/30

17-1047-01 17-1088-01

Superdex 75 prep grade Superdex 75 prep grade Superdex 75 prep grade Superdex 75 prep grade

25 ml 150 ml 1 litre 5 litres

17-1044-10 17-1044-01 17-1044-02 17-1044-04

HiLoad 16/60 Superdex 30 pg HiLoad 26/60 Superdex 30 pg HiLoad 16/60 Superdex 75 pg HiLoad 26/60 Superdex 75 pg HiLoad 16/60 Superdex 200 pg HiLoad 26/60 Superdex 200 pg

17-1139-01 17-1140-01 17-1068-01 17-1070-01 17-1069-01 17-1071-01

Superdex 200 prep grade Superdex 200 prep grade Superdex 200 prep grade Superdex 200 prep grade

25 ml 150 ml 1 litre 5 litres

17-1043-10 17-1043-01 17-1043-02 17-1043-04

HiPrep 16/60 Sephacryl S-100 HR HiPrep 26/60 Sephacryl S-100 HR HiPrep 16/60 Sephacryl S-200 HR HiPrep 26/60 Sephacryl S-200 HR HiPrep 16/60 Sephacryl S-300 HR HiPrep 26/60 Sephacryl S-300 HR

17-1165-01 17-1194-01 17-1166-01 17-1195-01 17-1167-01 17-1196-01

HiTrap Desalting (5 columns) Fast Desalting Column HR 10/10

17-1408-01 17-0591-01

Sephacryl S-100 HR Sephacryl S-100 HR Sephacryl S-100 HR Sephacryl S-200 HR Sephacryl S-200 HR Sephacryl S-200 HR Sephacryl S-300 HR Sephacryl S-300 HR Sephacryl S-300 HR Sephacryl S-400 HR Sephacryl S-400 HR Sephacryl S-400 HR Sephacryl S-500 HR Sephacryl S-500 HR Sephacryl S-500 HR Sephacryl S-1000 SF

150 ml 750 ml 10 litres 150 ml 750 ml 10 litres 150 ml 750 ml 10 litres 150 ml 750 ml 10 litres 150 ml 750 ml 10 litres 750 ml

17-0612-10 17-0612-01 17-0612-05 17-0584-10 17-0584-01 17-0584-05 17-0599-10 17-0599-01 17-0599-05 17-0609-10 17-0609-01 17-0609-05 17-0613-10 17-0613-01 17-0613-05 17-0476-01

Sepharose 6B Sepharose 6B Sepharose 4B Sepharose 4B Sepharose 2B Sepharose 2B

1 litre 10 litres 1 litre 10 litres 1 litre 10 litres

17-0110-01 17-0110-05 17-0120-01 17-0120-05 17-0130-01 17-0130-05

Sepharose CL-6B Sepharose CL-6B Sepharose CL-4B Sepharose CL-4B Sepharose CL-2B Sepharose CL-2B

1 litre 10 litres 1 litre 10 litres 1 litre 10 litres

17-0160-01 17-0160-05 17-0150-01 17-0150-05 17-0140-01 17-0140-05

Sephadex G-10 Sephadex G-10 Sephadex G-10

100 g 500 g 5 kg

17-0010-01 17-0010-02 17-0010-03

Column

Qty. Code No.

PD-10 Prepacked Disposable NAP-5 NAP 5 NAP-10 NAP 10 NAP-25 NAP 25 NICK Column NICK Column NICK Spin Columns NICK Spin Columns

30 20 50 20 50 20 50 20 50 20 50

17-0851-01 17-0853-01 17-0853-02 17-0854-01 17-0854-02 17-0852-01 17-0852-02 17-0855-01 17-0855-02 17-0862-01 17-0862-02

Sephadex G-15 Sephadex G-15 Sephadex G-15

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Code No.

500 g 17-0020-01 5 kg 17-0020-02 500 g 17-0020-02

Media

Pack Code No. size

Media

Pack Code No. size

Sephadex G-25 Fine Sephadex G-25 Fine Sephadex G-25 Fine Sephadex G-25 Medium Sephadex G-25 Medium Sephadex G-25 Medium Sephadex G-25 Coarse Sephadex G-25 Coarse Sephadex G-25 Coarse Sephadex G-25 Superfine Sephadex G-25 Superfine

100 g 500 g 5 kg 100 g 500 g 5 kg 100 g 500 g 5 kg 100 g 5 kg

17-0032-01 17-0032-02 17-0032-03 17-0033-01 17-0033-02 17-0033-03 17-0034-01 17-0034-02 17-0034-03 17-0031-01 17-0031-03

Sephadex G-25 DNA Grade SF

25 g 17-0572-01 100 g 17-0572-02

Sephadex G-50 DNA Grade F

25 g 17-0573-01 100 g 17-0573-02

Sephadex G-100 DNA Grade M

25 g 17-0045-01 100 g 17-0045-02

Sephadex G-100 DNA Grade SF

25 g 17-0574-01 100 g 17-0574-02

Sephadex G-50 Fine Sephadex G-50 Fine Sephadex G-50 Fine Sephadex G-50 Medium Sephadex G-50 Medium Sephadex G-50 Medium Sephadex G-50 Coarse Sephadex G-50 Coarse Sephadex G-50 Coarse Sephadex G-50 Superfine Sephadex G-50 Superfine

100 g 500 g 5 kg 100 g 500 g 5 kg 100 g 500 g 5 kg 100 g 5 kg

17-0042-01 17-0042-02 17-0042-03 17-0043-01 17-0043-02 17-0043-03 17-0044-01 17-0044-02 17-0044-03 17-0041-01 17-0041-03

Handbook. Gel filtration, Principles and Methods

Sephadex G-75 Sephadex G-75 Sephadex G-75 Sephadex G-75 Superfine Sephadex G-75 Superfine

100 g 500 g 5 kg 100 g 5 kg

17-0050-01 17-0050-02 17-0050-03 17-0051-01 17-0051-03

Sephadex G-100 Sephadex G-100 Sephadex G-100 Sephadex G-100 Superfine Sephadex G-100 Superfine

100 g 500 g 5 kg 100 g 5 kg

17-0060-01 17-0060-02 17-0060-03 17-0061-01 17-0061-03

18-1022-18

Standards

Pack Code No. size

Gel Filtration LMW Calibration kit Gel Filtration HMW Calibration kit Blue Dextran 2000

1 kit

17-0442-01

1 kit 10 g

17-0441-01 17-0360-01

Contents of the gel filtration calibration kits. Low Molecular Weight Gel Filtration Calibration Kit Protein ribonuclease A chymotrypsinogen A ovalbumin albumin Blue Dextran 2000

M Weight

Stokes’ Radius Å

13 700 25 000 43 000 67 000

Source

16.4 20.9 30.5 35.5

bovine pancreas bovine pancreas hen egg bovine serum

48.1 52.2 61.0 85.0

rabbit muscle bovine liver horse spleen bovine thyroid

High Molecular Weight Gel Filtration Calibration Kit Aldolase* catalase ferritin* thyroglobulin Blue Dextran 2000

158 000 232 000 440 000 669 000

Each Kit contains 50 mg of each protein and 50 mg of Blue Dextran 2000.

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