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

Availability ................. Separation time ...... Choice of column .................... Chromatographic properties ............................................. Optimization ................................. Sample characteristics ............................. Sepharose CL ....................................... Gel filtration in organic solvetns ........................................................................ Choice of running conditions ......................... Preparation of Sephacryl HR............................................................... Performance ...................................................................................................................... Sepharose CL and Sepharose) ...................................... Eluent ........................................................................................................................................................................ 35 35 36 37 38 38 38 40 41 41 41 41 42 43 43 44 44 44 44 45 45 47 47 48 51 51 52 52 54 55 58 58 Performing a gel filtration experiment .................................................................................................................................................................................... The purpose of the experiment ................................................. Sample characteristics .................................. Sepharose ........................ Chromatographic properties ................ Chemical and physical properties . Further information ...................................................................................... Experimental design ........................................................................................................................................................ High performance gel filtration ............................Sephadex ...................................................................................................................................................... Capacity ......................... 61 Preparing the gel .................................................................................... 61 Pre-swollen media (Sephacryl HR.......................................................................................................................................... Further information ........................................................................................... Solute characteristics .................... Availability ....... Superose prep grade or Sepharose CL for use in organic solvents ....... Flow rate ........ 61 61 62 63 Packing a column ...................................... Further information ............................................................................................... Chemical and physical properties ...................................................................................................................... Chromatographic properties ................................................................................... Superose prep grade............................ Chemical and physical properties ................. Resolution ................................. 63 2 ........................ Availability ............................................................... Choice of gel ...................................................................................................................................................................................................................................................................... Process considerations ....... Media which require swelling (Sephadex G-types) ...........................................................................

................................................................................................................................ 80 General cleaning procedures ............................. 72 Sample application with an adaptor ......................... 89 References .............. 63 68 70 71 Using pre-packed columns ...... 73 Elution ............... Storage of packed columns ........................................... Prevention of microbial growth ................................................................................................................................... 102 3 ............................. 72 Sample application withour an adaptor ............................................ 78 Cleaning gels and packed columns ........................................... 72 Sample application ............................................................................... 98 Ordering information .......... Packing Sephacryl HR and Superose prep grade ......................................................................................................................................................... 80 Procedures to remove specific contaminants . 84 84 85 86 86 87 Fault finding chart ............................ Storage of unused media ............................................................... Antimicrobial agents ...................................................................... 77 Flow rates .................................................... Checking the packed bed ......... 83 Storage of gels and columns ........... Storage of used media ....................................Packing Sephadex G types Sepharose and Sepharose CL ......................................................................................................................................................................................................................................................................... Adaptors ........................................................

Little equipment is required. 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. The reliability of gel filtration stems from the reliability of Sephadex and other gel filtration media from Amersham Pharmacia Biotech.2). polysaccharides. 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. backed by over 30 years of experience in developing biochemical separation techniques and by thousands of publications describing their use. the procedure is straightforward and good separations and yields are usually obtained even in the first 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. Among the new developments described in this handbook are Sephacryl HR for standard chromatography and Superdex and Superose for high speed gel filtration. 4 .Introduction For more than thirty years since the introduction of Sephadex (l. detergents. A gel filtration separation can be performed in the presence of essential ions or cofactors. concentrations of metal ions. at 37 °C or in the cold room according to the requirements of the experiment. urea. Gel filtration is one of these techniques. These new media are the result of continuing efforts of Pharmacia to improve the separation techniques which you need for your work. it is essential to refer to the original literature. nucleic acids. For specific separation problems where the substances in question may have special properties. No less valuable than its widespread usefulness. Biological macromolecules form a class of substances with special functions which are controlled in vivo by small changes in the environment. This handbook is designed as a laboratory aid in the selection and practical use of gel filtration media from Amersham Pharmacia Biotech. is the reliability and simplicity of gel filtration as an experimental procedure. proteins and other biological macromolecules. Changes in the pH. or for theoretical aspects where ideas are undergoing continual evolution. at high or low ionic strength. gel filtration has occupied a key position in the purification of thousands of enzymes.

Should we at any time be able to give you further information. We hope that this handbook will aid our cooperation and that you will find the information helpful. please contact us.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 user and manufacturer. 5 .

Dextran chains are covalently bonded to a highly cross-linked agarose gel matrix. for example Sephadex which is formed by cross-linking dextran. Composite gels may be prepared by. the eluent. like agarose. the pores have a carefully controlled range of sizes. and the matrix is chosen for its chemical and physical stability. Sufficiently large molecules are completely unable to diffuse into the gel and are thus confined to the solution outside. the gel matrix. usually aqueous. Composite gels are of interest since they can combine valuable properties from more than one gel-forming system. 6 . grafting a second polymer onto a pre-formed matrix. is contained within the pores of a continuous solid phase. for example.Principles Gel filtration In gel filtration molecules in solution are separated according to differences in their sizes as they pass through a column packed with a chromatographic medium which is a gel. 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. Some polymers. Relatively small molecules can diffuse into the gel from a surrounding solution. form gels spontaneously under the appropriate conditions. Superdex is such a gel. and inertness (lack of adsorptive properties). whereas relatively large molecules will be prevented by their size from diffusing into the gel to the same degree. is passed. Gels may be formed from polymers by cross-linking to form a three-dimensional network. Gel media A gel is a heterogeneous phase system in which a continuous liquid phase. In a gel filtration column. gel particles in bead form are packed to form a separation bed through which a buffer solution. In gels made for gel filtration.

is a direct measure of its loadability under otherwise comparable conditions and is chosen depending on the sample volume. If the column is evenly packed so the sample zone is not unnecessarily broadened as it passes down the column then good results can be obtained. is significant since it affects both the resolution and the time taken to elute it. cm. include its volume and viscosity. stability range of the gel filtration medium. apart from the solutes which are to be separated. Resolution and Elution time α column length α √column length The volume of the column. Note that the pH. ml. Column parameters The most important characteristic of a gel filtration column is the way in which the gel filtration medium has been packed.Sample molecules which are to be separated are added in solution as a zone to the top of the bed. The volume of the sample will influence the size of column which will be needed. The length of the column. 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. The small molecules which diffuse into the gel beads are delayed in their passage down the column compared with the large molecules which cannot diffuse into the gel and move continuously down the column in the flowing eluent. 7 . The sample zone moves down the bed as eluent is added to the top. The large molecules thus leave the column first followed by the smaller molecules in the order of their sizes. If the column is packed unevenly then good results will never be obtained from it. It is the viscosity which places an upper limit on the sample concentration which is permissible. Experimental parameters Sample concepts Characteristics of the sample which are important for the result. and the viscosity must not be so large as to cause hydrodynamic instability (see below).

Different criteria are used for the determination of elution volume (Fig. the lower the flow rate the better the resolution. but when comparing results between columns of different sizes it is useful to use the linear superficial flow rate. e. Pharmacia Monitor UV-l. e. 1). Results obtained at the same linear flow rate will be comparable as far as the effects of flow rate are concerned. ml/min. Generally speaking. The eluent can thus be whatever is convenient with regard to the overall requirements of the experiment. Flow rates are measured in simple volume terms. 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. the composition of the eluent is unimportant for the separation mechanism.Eluent parameters Since the separation depends only on the sizes of the molecules being separated. a chromatogram.g. An ionic strength of 0. 2). For protein and nucleic acid work and in many other applications continuous detection using a UV-monitor (e. From this diagram the elution volume (Ve ) of a given solute can be obtained. Running conditions The experimental variable of significance which remains to be considered is the rate at which the eluent flows through the column. cm/ hour.15 or greater is generally used to avoid any unwanted ionic interactions between the solute molecules and the gel matrix.g. at least for large molecules.g. 8 . UV-M II or Uvicord S II) and a recorder gives an immediate permanent record. 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. 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 passed through the column (Fig. This affects not only the speed at which the separation is obtained but also the resolution which can be achieved.

C. Reproduced by kind permission of the Authors and the Publisher). Sample size not negligible compared with bed volume.5x150 cm. Aspberg. Biological Structure and Function 1 (1961) 345-349. K.. Sample size negligible compared with bed volume. eluent. flow rate..9 ml. 1. Column. The numbers above the peaks indicate the degree of polymerisation. Measurement of elution volume. P. B.cm-2. 2. water.h-1 (Flodin. Sample giving plateau elution curve. Ve A.Fig. Fig. 1. Gel filtration of an oligosaccharide mixture from the acetolysis and hydrolysis of cellulose on Sephadex G-25. 4. 9 .

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

L. Fig. An introduction to Gel Chromatography. 3. 1 part II. Relationship between several expressions used for normalizing elution behaviour. 4. Reproduced by kind permission of the Authors and the Publisher). (Fischer. Note that Vt-Vo will include the volume of the solid material which forms the matrix. Amsterdam. North Holland Publishing Company. Diagrammatic representation of Vt and Vo.Fig. Vol. 11 . Laboratory Techniques in Biochemistry and Molecular Biology.

All of these models have been successfully applied to predict the elution behaviour of solutes. 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. An interesting approach by Laurent and Killander (8) assumes that the gel network is composed of rigid rods randomly arranged. Porath (6) derived a theoretical relationship between Kd and Stokes radius assuming that the pores in Sephadex are conical. The larger the molecular dimensions of the solute the greater is the proportion of the gel which is forbidden. none of them may be accurate in a structural sense. Most have regarded the partition of solute molecules between the gel particles and surrounding fluid as an entirely steric effect. 37. If the Kav is greater than 1. 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). 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. From a practical stand-point. In the permitted region the concentration of solute is assumed to be identical to that in the surrounding liquid. 12 . 34. 40). 30. Good correlation was found between Kav and molecular radius with this model. In another treatment Squire (7) considered pores and crevices as well as cones. If the Kav is less than 0 after calibration then channelling in the chromatographic bed is indicated and the column must be repacked. 4). However. it is still most common to construct a calibration curve and perform the estimations graphically. for molecular weight determination. 33. as has been pointed out by Ackers (9). particularly of proteins. Several models have been proposed to describe the behaviour of solutes during gel filtration (3. The steric approach has been extended in various ways. Thus Flodin (5) divided the gel into permitted and forbidden regions. some kind of adsorption is indicated. Calibration curves constructed in this way for a particular gel type are often termed selectivity curves (see p 25. is well documented (3).Theoretical considerations The use of gel filtration for the determination of molecular weight or size.

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. 13 . Under certain conditions. Further details of these effects can be found under the chromatographic properties of the different gels (see pages 22–43). However. Often they have proved highly advantageous. 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. factors other than the size and shape of the molecules being studied can influence the separation. 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. 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.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.

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. 10 % formic acid... Gel filtration of a tryptic digest of citraconylated totally reduced and 14C alkylated Fd' fragments of IgG3 on Sephadex G-50 Fine. E. (Michaelsen. J. eluent. Frangione.T. Chem.C.. 6). The peptide separation shown in Figure 5 Fig. Here the correct choice of gel and operating conditions are critical if good results are to be obtained. free choice of elution conditions and straight forward interpreta14 . Column. a protein can be separated from other species differing in molecular weight by a factor of two or slightly less (Fig. T. 5. Practical simplicity. B. 1x96 cm. Biol. Franklin. excellent recovery. 252 (1977) 883-889: Reproduced by kind permission of the Authors and the Publisher). illustrates this type of experiment. With the proper experimental design.

Sample concentration. Stockholm.5 ml/min. pH 9. in collaboration with KabiGen. Details about experimental design are given on p 44– 60. was initially purified by affinity chromatography on IgG-Sepharose Fast Flow and subsequently cleaved with hydroxylamine (2 M. Uppsala. 5 mg/ml. ZZ-IGF-1. 100 µl. The secreted fusion protein. sample volume.tion of results make fractionation by gel filtration an invaluable part of any purification scheme. 6. eluent. Practical details are given on p 61–87.25 M. pH 6. flow rate. 0. Sweden. 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.) 15 .0). Sweden. Fig. (Work by Pharmacia LKB Biotechnology.2). ammonium acetate (0.

flow rate. 50 ml. 2. sample concentration. 14 ml/min.05 %). 11. High resolution gel filtration provides an excellent and gentle means for separating the monomer from the aggregates. 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. eluent. 7) where protein aggregates must be reduced to below a specified level in the final product. Final purification of a mouse monoclonal antibody. sample volume. 1. Uppsala. The concentrate was desalted and purified initially by cation exchange on BioPilot Column S Sepharose High Performance 60/100. Fig. 7. Sweden. 3. column. transferrin. 16 .37 mg/ml. IgG2a. IgG dimers. Hybridoma cell culture supernatant was concentrated by ultrafiltration and clarified by centrifugation and passage through a 0. BioPilot Superdex 200 prep grade 60/600. IgG monomers. Work by Pharmacia LKB Biotechnology. on Superdex 200 prep grade. PBS (pH 7. Peaks. either as part of an analysis or for their final purification.5) containing sodium azide (0.22 µm filter.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.

Gel filtration of proteins on Sepharose CL-6B under denaturing conditions. Ansari and Mage (14) used the same column packed with Sepharose CL-6B for 17 . ionic strength.cm-2. 7. In the experiment depicted in Figure 8. 4. α-chymotrypsinogen. J. temperature etc. has proved particularly useful for molecular weight determinations. 8. Chromatogr. Superdex. (Ansari. bovine serum albumin. A. 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). R. 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. 140 (1977) 98-102. 3. 1. 2. Reproduced by kind permission of the Authors and the Publisher). native and other forms A calibrated column of Sephacryl HR. 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. 6 M guanidine-HCI.A.. dissociating agents which transform polypeptides and proteins to a random coil configuration reducing structural differences. Figure 8 illustrates this kind of application. 5. cytochrome c.0. 6. flow rate. Blue Dextran. 0. Gel filtration in the presence of urea or guanidine hydrochloride.G. insulin. both for molecular weight determination and for routine separations. Mage. Peaks. B chain of insulin. 8. rabbit IgG H chain. Superose.h-1..8 ml. DNP-ala: Column. freeing the researcher from the constraints imposed by the charge state of the molecules.. 6. for example.MW estimation.1 m sodium phosphate. Fig. Unlike electrophoretic techniques. pH 8.5x90 cm: Eluent. 1.

Uppsala. 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. ovalbumin. aldolase. 6. Calibration proteins being used to calibrate Sephadex G-200 Superfine. 3. bovine serum albumin.molecular weight determination in 6M guanidine hydrochloride over a period of 10 months. Peaks. 0. Fig. chymotrypsinogen A. 4. 18 . Determination of molecular weight distribution of polymers The molecular weight distribution is very important for characterization of natural and synthetic polymers. (Work by Pharmacia LKB Biotechnology. An elution profile obtained with Sephadex G-200 Superfine is shown in Figure 9. K 26/70: eluent.05 M potassium phosphate. catalase. 9. containing 0.1 M NaCI and 2 % sodium azide. without any change in the elution pattern. 2. 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. ribonuclease A: column. Sweden. 5. 1.8. pH 6. 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).

gel filtration is particularly efficient for many everyday laboratory operations including: • buffer exchange. 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. where the reactants and the products can be separated on a gel filtration column. From the elution curve of the reactants the complex formation can be studied. In this case. Gel filtration has also been applied to the estimation of reaction rates (27. It is unnecessary to determine molecular weights of the polymers in the effluent fractions of individual experiments. competition of two molecules for the same site (22). hydrophobic interaction chromatography. polyvinylpyrrolidones. this is most efficiently done by gel filtration. Gel filtration can also be used to determine the position of equilibrium of complex formation where the reactions are rapid. as the chromatographic behaviour of the columns is very reproducible.g. one of the reactants is chromatographed in an eluent containing the other reactant. affinity 19 . assays and other analytical procedures require that the sample be adjusted to the proper ionic conditions. 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. gelatin preparations and other water-soluble polymers (3).The ability of gel filtration to fractionate molecules according to molecular size offers an improved method for distribution analysis of many polymers (15). Desalting Since proteins and other biomacromolecules differ greatly in size from salts and other small molecules. thereby establishing the position of the equilibrium (17–19). 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. Determination of equilibrium constants Gel filtration has proved to be a valuable technique for the study of chemical equilibria. The elution curve can be recorded continuously or determined by investigating individual fractions. In the case of slow reactions. 28). A calibration curve once determined for the column can be applied to a large number of runs. dextrans. these substances can be quantitatively determined in the effluent. Enzymic reactions. for instance.

Removal of free 125l and Chloramine T from labelled albumin. (Work by Pharmacia LKB Biotechnology. Column. 0. • removal of products. 4 ml/min. 125I. sample concentration. 30). 10). from solutions of labelled proteins (Fig. 10. cofactors.4) containing Tween 20 (0. 20 . • removal of free low molecular weight labels. Sweden). Fig.05 M. • termination of reactions between macromolecules and low molecular weight reactants.05 %). FITC. • phenol removal from preparations of nucleic acids. Fast Desalting Column HR 10/10. Uppsala.5 mg/ml human serum albumin. eluent. e.chromatography and other LC techniques which use an aqueous mobile phase.g. 350 µl. sample volume. from enzymes. pH 7. • removal of unincorporated nucleotides during DNA sequencing (29. flow rate. inhibitors etc. • phenol red removal from culture fluids prior to anion exchange chromatography. sodium phosphate (0.

Pharmacia has built up considerable experience in this area and you are invited to contact us directly should you require further information. In particular. 21 .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. scale-up from pilot-plant to production capacity has proved a straightforward and trouble-free operation.

Fig. 11.N'-methylene bisacrylamide to form a hydrophilic matrix of high mechanical strength (Fig. 11).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. Hypothetical partial structure of Sephacryl HR. 22 .

The wet bead diameter is between 25–75 µm. 1% SDS. Short term exposure. The separation properties of the gel are not affected by detergents e. Table 1 summarizes some of the properties of Sephacryl HR. with an average bead diameter of approximately 50 µm. e. for cleaning. Table 2 lists the gel volumes in various organic solvents for an original 100 ml of gel sedimented in water. Sephacryl HR types are supplied pre-swollen and ready to use. Bed volumes of Sephacryl HR-types in common organic solvents starting from 100 ml sedimented gel. Table 2.g. chaotropic salts or dissociating agents e. 8 M urea and 6 M guanidine HCI. Properties of Sephacryl HR Gel type 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 Sephacryl S-100 HR Sephacryl S-200 HR Sephacryl S-300 HR Sephacryl S-400 HR Sephacryl S-500 HR Chemical stability Sephacryl HR is stable in all aqueous buffers commonly used in biochemistry within the pH range 3–11. Sephacryl HR can also be used with organic solvents.g.5M) can be used for cleaning if the gel is washed immediately afterwards with buffer or water.g.The porosity of the gel is controlled by the dextran component to give five types with different fractionation ranges. Strong solutions of NaOH (0. A method for transferring Sephacryl HR from water to organic solvents is described on page 62. Table 1. to pH´s in the range 2-13 has no adverse effect on subsequent chromatographic properties. Gel type Sephacryl S-100 HR Sephacryl S-200 HR Sephacryl S-300 HR Sephacryl S-400 HR Sephacryl S-500 HR Formamide 110 115 100 100 100 DMSO 100 110 90 90 95 Methanol 100 100 100 100 100 Ethanol 100 100 95 95 100 Acetone 85 85 85 85 90 23 .

12. temperature 25 °C. eluent distilled water. pH 7 for 30 minutes without significantly affecting its chromatographic properties.3 cm2 for XK 26). the mechanical rigidity of Sephacryl HR allows even relatively viscous eluents. To calculate the volumetric flow rate. to be run at practicable flow rates. including wide diameter production columns with total bed heights of 60-100 cm.5 x 106. Physical stability Fig. When fractionating proteins in the molecular weight range 5 x 103 –250 x 103 or 10 x 103 –1. 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. resulting in short overall separation times (31). 12. such as 8 M urea.Sephacryl HR may be autoclaved repeatedly at 121 °C. multiply the linear flow rate by the cross-sectional area of the column (2 cm2 for XK 16 or 5. 24 . Pressure drop as a function of flow rate for Sephacryl HR. Typical flow rates for a 60 cm long bed can be seen in the pressure/flow diagram in Fig. can be packed and equilibrated successfully at high flow rates. Likewise. Bed height approximately 60 cm. Chromatographic properties Selectivity The five Sephacryl HR types have different fractionation ranges. Columns of all dimensions.

Figure 13 shows the selectivity curves for globular proteins and dextrans respectively. 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. Selectivity curves for Sephacryl HR in phosphate buffer (0. A typical example of results obtainable with Sephacryl HR is shown in Figure 14. 13. plasmids. Sephacryl S-400 HR and S-500 HR are the choice for separation of larger proteins.0) containing NaCl (0. pH 7. DNA fragments and small particles e.including monoclonal antibodies and serum proteins. Table 1 gives the fractionation ranges for Sephacryl HR.15 M).g. 25 . Fig. polysaccharides. nucleic acids. use Sephacryl S-200 HR or S-300 HR respectively.05 M.

aldolase. Sweden.15 M NaCl.05 M phosphate. monomer of the anion transporter protein. The gels provide rapid cycle times and can be sanitized in place which is of particular advantage. (Work by E. pH 7. Peaks.1 M.0) with the following model substances: Blue Dextran 2000.05 M). ribonuclease A and cytochrome c. pH 7. BSA. Iysozyme. For the best results eluents with an ionic strength of at least 0. ferritin. Lundahl. Human erythrocyte membrane were solubilized in phosphate buffer (0.1 M). Greijer and P. University of Uppsala. 1. Separation of integral membrane proteins from human erythrocytes on Sephacryl S-300 HR. Uppsala. dimeric anion transporter protein. 2.4) containing SDS (0. 5.) Adsorption Tests in our laboratories have shown that Sephacryl S-100 HR gives yields of at least 96% (UV absorption at 280 nm. dimeric glycophorin A. flow rate. ovalbumin. myoglobin. chymotrypsinogen A. Its high resolution and high chemical stability make Sephacryl HR ideally suited for use in industry. pH 7. 0.15 should be used. 2 mg/ml. 14. Institute of Biochemistry. 58 ml/hour. using 0. DTE (1 mM).1 M. glucose transporter protein. EDTA (1 mM). sample concentration. 26 . 3. sample volume. eluent. 4. EDTA (1 mM). 61 cm. bed height. DTE (1 mM). column. FPLC and industrial scale Columns packed with Sephacryl can be used with with standard chromatography instrumentation and with FPLC system.Fig. phosphate buffer (0. K 26/70. both in pilot and in process scale. 2 ml.4) containing SDS (0. possibly lipids and SDS. catalase. β-lactoglobulin A+B.

Structure of Superdex. 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. Superdex 30 prep grade. Superdex 75 prep grade and Superdex 200 prep grade have Fig. Sephacryl S-100. For information on scale-up and the operation of large scale chromatography systems. Further information Further information on Sephacryl HR is given in the data sheet: “Sephacryl High Resolution gel filtration media” with details of applications. Dextran chains are covalently linked to a highly cross-linked agarose matrix. Different types with different fractionation ranges are available. please contact Pharmacia.Availability Sephacryl HR types are supplied pre-swollen as a suspension containing 20% ethanol in packs of 750 ml. S-200 and S-300 are also supplied pre-packed in XK-columns with the designation HiLoad columns. 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. 27 .

Exposure to temperatures outside this range will destroy the efficiency of the packed bed and the column will need to be re-packed. e. e. Table 3 summarizes some of the properties of Superdex and Superdex prep grade. Typical flow rates for a bed 60 cm long packed with Superdex prep grade 28 . A method for transferring Superdex from water to organic solvents is described on page 62. chaotropic salts or dissociating agents. and acids.g. e. HiLoad columns can be used both with standard chromatography systems and with FPLC system. Strong solutions of NaOH (0.2 M NaOH.g. has no adverse effect on subsequent chromatographic properties.g. and withstands strong bases.1 M acetic acid. Superdex can also be used with organic solvents. Physical stability Columns pre-packed with Superdex may be used at temperatures in the range +4–40 °C. 8 M urea or 6 M guanidine HCI.g.an average wet bead diameter of 34 µm with a range of 24–44 µm. 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. 1% SDS. Superdex packed in HR columns is for use with FPLC system.g. The separation properties of the gel are not affected by detergents.5 M) can be used for cleaning if the gel is immediately washed with buffer or water. Superdex peptide. for cleaning.01 M HCI or 0. Short term exposure to extremes of pH (1-14) e. Superdex 75 and Superdex 200 for high performance gel filtration have a bead size of 13 µm (mean).5 M) or HCl (0. e. 0. 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. 0. Table 3.

such as 8 M urea. Pressure drop as a function of flow rate for HiLoad columns packed with Superdex prep grade. multiply linear flow rate by cross-sectional area of column (2 cm2 for XK16. can be seen in the pressure/flow diagrams in Figure 16. oligonucleotides and small proteins in the molecular weight range up to 10000. To calculate volumetric flow rate.Fig. Chromatographic properties Efficiency HiLoad Superdex prep grade columns are supplied packed to an efficiency of >13 000 plates/metre. its mechanical rigidity allows even relatively viscous eluents. 5. Superdex 30 prep grade is recommended for separations of peptides.3 cm2 for XK26). to be run at practicable flow rates. resulting in short overall cycle times. Bed height approximately 60 cm in distilled water at 25 °C. Similarly. Superdex can be equilibrated at flow rates which are much higher than those recommended for the best resolution. Superdex 75 is particularly suited for 29 . 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. Superdex 75 and Superdex 200 HR 10/30 columns are supplied with an efficiency of > 30 000 plates/metre. 17). 16.

Fig. For information on scale-up and the operation of large scale chromatography systems. Superdex 200 prep grade is a suitable choice when the molecular weight of the protein of interest is unknown. Superdex with a bead size of 13 µm is supplied pre-packed in HR 10/30 columns. the separation of a wide range of recombinant DNA products.15 to 1. 5 litres or prepacked in HiLoad and larger BioPilot columns. Selectivity curves for Superdex. Further information Further information on Superdex with details of applications is given in the technical brochure “HiLoad columns”. the presence of a small number of negatively charged groups leads to retardation of basic proteins and exclusion of acidic proteins. Adsorption Non-specific interactions between proteins and Superdex are negligible under normal chromatographic conditions using buffer solutions with ionic strengths in the range 0. Availability Superdex prep grade is supplied in packs of 150 ml. It is especially suitable for the separation of monoclonal antibodies from dimers and from contaminants of lower molecular weight. At very low ionic strengths.5. 30 . 17. 1 litre. for example albumin and transferrin. please contact Pharmacia.

g. 34) e. pH 7 for 30 minutes without significantly affecting its chromatographic properties. chaotropic salts or dissociating agents e. 8 M urea and 6 M guanidine HCl.g. Superose prep grade may be autoclaved repeatedly at 121 °C.g. Table 4. Physical stability 31 .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. Prepacked columns must not be exposed to temperatures outside the range +4 –40 °C. The average wet bead diameter is 10 ±2 µm or 13 ±2 µm for Superose 6 and Superose 12 respectively. 1% SDS. has no adverse effect on subsequent chromatographic properties and the media withstand strong bases (33. Strong solutions of NaOH (0. Exposure to temperatures outside this range will destroy the efficiency of the packed bed .g. Superose prep grade may be transferred from water to organic solvents by the method described on page 62. and 20–40 µm for the corresponding prep grades.g. for cleaning. Properties of Superose Gel type Superose 12 prep grade Superose 12 Superose 6 prep grade Superose 6 Bead size µm 20 – 40 8 – 12 20 – 40 11 – 15 Fractionation range Globular proteins 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. The separation properties of the gels are not affected by detergents e.5 M) can be used for cleaning if the gel is immediately washed with buffer or water.01 M HCI and 1 M acetic acid.2 M NaOH. Superose can also be used with organic solvents. Table 4 summarizes some of the properties of Superose. Short term exposure to extremes of pH (1–14) e. and acids e.5 M) or HCl (0. 0. 0.

Typical pressure-flow relationships for Superose 6 and Superose 12 HR 10/30 columns in aqueous buffer solutions at room temperature. 32 . such as 8 M urea.d..5 ml/min for a column 16 mm i. 20 and 21. Fig. Pressure/flow diagrams for Superose 6 HR 10/30 and Superose 12 HR 10/30 columns are shown in figure 18.Recommended flow rates for Superose prep grade are in the range 0. with selectivity curves as shown in figures 19.3–0. Superose 6 and Superose 12. to be run at practicable flow rates. 18. The mechanical rigidity of Superose prep grade allows even relatively viscous eluents. Selectivity Gels with two different fractionation ranges are available. The selectivities of the corresponding prep grades are the same. Chromatographic properties Efficiency Superose 6 HR10/30 and 12 HR10/30 columns are supplied packed to an efficiency of >30 000 plates/metre. 50 cm long.

dextrans. 33 . Fig. 20. Selectivity curves of Superose 6 and 12.Fig. globular proteins. 19. Selectivity curves of Superose 6 and 12.

some compounds (e. please contact Pharmacia Biotech. membrane proteins or lipoproteins) may be eluted later than predicted (these interactions can be of considerable value to the resolution).e. Selectivity curves of Superose 6 and 12.15 M. Superose 6 HR 10/30 and Superose 12 HR 10/30 columns are supplied prepacked. The degree of interaction is less for Superose prep grade than on prepacked Superose and Superose 6 shows weaker hydrophobic effects than Superose 12. 21. Some hydrophobic interactions have been recognized. i. Adsorption Ionic interactions between solutes and Superose are negligible at ionic strengths above 0.g. 34 .Fig. Availability Superose prep grades are available pre-swollen in packs of 125 ml. Further information Further information on Superose with details of applications is given in the data file: “Superose 6. DNA-fragments. smaller hydrophobic and/or aromatic peptides. For information on scale-up and the operation of large scale chromatography systems. Superose 12”.

It should be noted that the degree of swelling in organic solvents or their mixtures will not be the same as in water alone. Sephacryl HR and Sepharose CL are recommended for gel filtration in organic solvents. and in dimethylsulphoxide and formamide. G-15. Sephadex LH-20 (described in a separate booklet). G-25 and G-50. 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. 22).Sephadex Chemical and physical properties Sephadex is a bead-formed gel prepared by cross-linking dextran with epichlorohydrin (Fig. Partial structure of Sephadex. 22. 35 . Table 5 lists the different G-types of Sephadex and their physical properties. Sephadex swells in aqueous solutions. Fig.

The highest efficiencies. The Fine grade is recommended for preparative purposes. Sephadex G-10.5 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 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 – 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 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. The DNA grade of Sephadex G-25. Sephadex is available in different particle size grades. 23 and 24). G-25 and G-50 are recommended for separations of peptides and other small biomolecules. In addition the Coarse grade is suitable for batch procedures. Properties of Sephadex. The Coarse and Medium grades are intended for preparative chromatographic processes where a high flow rate at a low operating pressure is essential. G-15. G-50 or G-100 should be used for work with DNA or oligonucleotides. The different grades give chromatographic beds with different efficiencies and operating pressures. Gel type Dry bead size µm 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 Swelling factor ml/g 2– 3 2.5 – 3. Sephadex G-75. are obtained with the Superfine grade. G-100. The Superfine grade is also suitable for thin layer gel filtration. and operating pressures.Table 5. 36 . G-150 and G-200 are useful in work with proteins and other macromolecules where their relatively poor physical stability is not a hindrance.

Sephadex G-25 Medium is supplied pre-packed in disposable PD-10 Columns. globular proteins. Selectivity curves of Sephadex G-types Superfine. globular proteins. Selectivity curves of Sephadex G-types. 23. In addition: Sephadex G-25 Superfine is supplied pre-packed in Fast Desalting Column HR 10/10. 37 . and Sephadex G-50 DNA grade is supplied pre-packed in disposable NICK Columns and NICK Spin Columns. Availability All Sephadex G-types are supplied as dry powders in 100 g and 500 g packs. Fig. 24.Fig. Sephadex G-25 DNA grade is supplied pre-packed in disposable NAP Columns.

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. 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. (Låås. Fig. Doctoral thesis. please contact Pharmacia. Reproduced by kind permission of the Author.Further information Further information on Sephadex with details of applications is given in the data sheet: “Sephadex gel filtration media”. Sepharose Chemical and physical properties Sepharose is a bead-formed gel prepared from agarose. 25). 38 . In its natural state agarose occurs as part of the complex mixture of charged and neutral polysaccharides referred to as agar. 25.) The individual polysaccharide chains may be represented as polymers of the repeating unit shown in Figure 26. T. Acta Universitatis Upsaliensis 1975. Gel structure of agarose. For information on scale-up and the operation of large scale chromatography systems.

26. Table 6.Fig. Sepharose may be used for long periods in dissociating media. Sepharose is available in three types with different agarose concentrations and different fractionation ranges (Table 6). such as guanidine hydrochloride and urea. but for the best results Sepharose CL (cross-linked Sepharose. It is stable in water and salt solutions over the pH range 4–9 and in the absence of oxidizing agents. it can still be used under most of the conditions encountered in gel filtration. Consequently Sepharose cannot be sterilized by autoclaving. Structure of the repeating unit of agarose. 39 Physical stability . Properties of Sepharose. by treatment with diethylpyrocarbonate. 10 000 – 4 000 000 60 000 – 20 000 000 70 000 – 40 000 000 Fractionation range Dextrans 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.6-anhydro-L-galactose. page 41) is recommended for these applications. should be avoided. but it may be sterilized chemically. % agarose 6 4 2 Bead size µm 45 – 165 45 – 165 60 – 200 Fractionation range Globular proteins. Note the presence of the unusual sugar 3. such as KSCN. Sepharose melts on heating above 40 °C and the bead structure may be irreversibly damaged on freezing. Chaotropic salts. Gel type Sepharose 6B Sepharose 4B Sepharose 2B Approx. but may be used with Sepharose CL. for example.

40 .5 M NaCl (36). globular proteins. Similarly DNA. Adsorption Sepharose contains a very small number of sulphate and carboxyl groups which may cause adsorption of basic proteins at low ionic strengths. Selectivity curves for Sepharose and Sepharose CL. 27. Chromatographic properties Selectivity The fractionation ranges for the different types are shown in Table 6 and Figure 27 shows the selectivity curves. Thus Sepharose 6B. tRNA and high molecular weight RNA from a variety of species may be separated on Sepharose in 1. 6% agarose) is considerably stronger than Sepharose 2B (approx. 2% agarose). Certain nucleic acids can be separated on Sepharose and this forms the basis of a number of schemes for their purification. These effects can be eliminated by using eluents with an ionic strength exceeding 0. makes it difficult to estimate the exclusion limits for proteins with confidence and these figures should be taken as a guide only. (approx. For example tRNA species may be resolved on Sepharose 4B in high concentrations of ammonium sulphate (35). Practical details on flow rates obtainable with Sepharose are given on page 66. The absence of suitable standards Fig.The mechanical strength of Sepharose depends on the agarose concentration in the beads.15 and effects due to protein adsorption are seldom encountered in practice.

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 contact 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 passed 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 contact Pharmacia.

43

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. Since the factors which improve resolution also lead to longer separation times. However.5 indicate baseline separation when the two components are present in approximately equal proportions. these may be to achieve maximum recovery with least sample dilution in the shortest possible time. the degree of separation. In the case of preparative separations. In particular. Greater resolution is needed if one of the components is present in considerable excess. a low flow rate etc. 44 .Experimental design Performance Resolution The principle objective of any fractionation experiment is to achieve adequate separation of the components of interest. Resolution can be increased in a number of ways (40) e. In many experiments adequate resolution is not difficult to achieve. a gel medium with smaller particles and/or greater selectivity.g. is defined by Resolution (Rs) = distance between separated zones average zone width Values of Rs greater than about 1. Fortunately. a small sample volume. or resolution. using a longer column. the column length and the flow rate. there is no need to design an experiment to give the ultimate in resolution unless the circumstances actually demand it. More consideration can then be given to other objectives of a well designed experiment. the gel medium. Separation time Times for gel filtration separations range from a few minutes to many hours according to the difficulty of the separation. For analytical work we may be more interested in reducing sample consumption and maximizing run-to-run reproducibility. there is always a compromise to be made between speed and resolution. In chromatography. 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.

Sample concentration The concentration of protein in the sample has little direct result on resolution (Fig. combined with small sample volumes. Since zone broadening is a principle cause of loss of resolution. 28). 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. 28. 45 . Influence of sample concentration on the resolution of transferrin and IgG on Superdex 200 prep grade. By using small bead sizes and columns packed to the highest possible efficiency. Capacity The quantity of sample which can be fractionated successfully depends on its volume and concentration. specialized media and equipment are employed to get the best possible combination of resolution and speed. even moderately concentrated solutions of nucleic Fig. every effort is made to reduce dispersion.High performance gel filtration In high performance gel filtration. However. These variables affect the resolution differently.

The relationship between sample volume. Using a concentrated sample is thus a simple and effective way of increasing the preparative capacity of a procedure. 29). The actual sample volume which can be applied for a given separation problem can only be found by experiment. 29. large sample to column volume ratios giving lower resolution than smaller ones (Fig. see page 57. with different media are given in Table 8.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). 46 . any sample concentration may be used. Within the limits set by viscosity. Recommended sample volumes for obtaining good resolution Fig. bead size and resolution has been described by Hagel (41). Sample volume Resolution depends on the ratio of sample volume to column volume. Influence of sample volume on the resolution of transferrin and IgG on Superdex 200 prep grade.

2% 1 . Columns with volumes of 2 500 litres can be operated in favourable cases. 47 . the method used must be shown to perform to stringent demands for economy. Recommended sample volumes as a per cent of the total bed volume for good resolution.5% 0. In some cases it may be important to consider other characteristics of the sample or the molecules to be separated.5% 2 . Productivity An important concept in discussing the performance of gel filtration in production processes is the productivity. 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 bed height. Scale up Gel filtration is simple to scale up from the laboratory to process scale (43). Medium Recommended sample volume %Vt Sephadex Sepharose Sepharose CL Sephacryl HR Superdex prep grade Superose prep grade Superdex Superose 2 . For a given size of column. or mass of material which can be processed per bed volume per hour (g . hour-1). the sample volume and the flow rate whilst maintaining adequate resolution (42).Table 8. the purpose of the experiment and the sizes of the molecules to be separated. ml bed-1 .2% 0. Many separations will show adequate resolution for larger sample volumes.5% Process considerations In large scale applications of gel filtration it is not only important to obtain the desired separation result. The exact conditions which lead to the highest productivity can only be ascertained by systematic experimentation.5% 1 .2% 1 .5% 2 . productivity is thus increased by increasing the sample concentration.

In many applications. it is usually necessary to obtain the maximum resolution between the target protein and known contaminants. Columns pre-packed with Sephacryl HR. 37. Purity determination When gel filtration is used for purity determination. both for reproducibility and efficiency. 48 . If the molecular weight is unknown. 30. Sephadex G-25 is suitable for studies of binding equilibria between proteins and small molecules (20).g. Binding equilibria Studies of binding equilibria require that the reacting species be separated. 33. Pre-packed columns are recommended to ensure the maximum column efficiency which is essential for the highest resolution. It is thus specially important to choose a separation medium which gives a perfectly stable bed under the conditions of the analysis. will be most suitable. a gel with a wide fractionation range. special care should be taken to obtain a stable.The purpose of the experiment Analysis Analytical applications place special demands on the run-to-run reproducibility of the gel filtration column. 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. 34. one of the species is much larger than the other and choice of gel is simple. Selectivity curves for the different media are shown on pages 25. If necessary. 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. e. Sephacryl HR. A wide fractionation range is also recommended for molecular weight distribution analysis. When the column is to be packed in the laboratory. well-packed bed and the packing recommendations should be followed in detail (see p 63–71). Superdex or Superose are suitable. gels with different fractionation ranges can be used together in the same column or separately in two or more columns in series (42). 40.

5 ml 2.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. Sephadex G-25 Fine is the recommended gel for the majority of desalting applications. 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. 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.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.5 ml 500 µl 1. unincorporated 49 . 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.0 ml 2. The low molecular weight substances should be eluted near Vt (Kav = 1).5 ml 0. Table 9. 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 gel is chosen so that the high molecular weight substances are eluted at the void volume (Kav = 0). sample volume 1. It is easy to work with and has excellent separation capacity for desalting molecules down to about 5000 MW. This will give the minimum zone broadening and dilution and reduce the time the components of interest are on the column.

Group separation of plasmids from proteins. The exact choice of gel will depend on the circumstances. 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. 50 . 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. 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). These considerations suggest that a gel from the Sephacryl HR series will generally be most suitable. 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. Pre-packed columns containing Superdex or Superose are available which ensure maximum resolution for particularly difficult fractionations. The sample will be relatively crude and the ability to clean the gel from lipids and/or protein contamination is important. the sample will be simpler in composition and remaining impurities may require high performance gel filtration media to remove them. Larger quantities of plasmids are quickly and conveniently prepared by gel filtration on Superose 6 prep grade (44). should be used for separation of dimers from monomers if baseline resolution is required. cDNA or Size Select-400 spun columns should be used for group separation of cDNA from small molecules like linkers and adapters. The likely presence of proteases or other enzymes which might degrade the target molecules also makes it desirable to work with high flow rates. Later on in the purification scheme. The following guide-lines should be useful in most situations. High resolution media. Superose or Sephacryl HR. The sample will also be relatively large in volume and a large bed volume may be needed.nucleotides etc. Preparative fractionation Fractionation by size using gel filtration is a natural part of many procedures for purifying proteins and other biomacromolecules. Superdex.

g. The selectivity curves of the different media are the best guide to the separation to be expected. e.Removal of degradation products and incomplete molecules When the impurities to be removed are known to be smaller than the target molecules. 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. peptide. protein. are preferable if fractionation over a wide range of molecular size is required. are to be preferred if high resolution is needed. Sephacryl HR or Sepharose CL. Where there is a choice of matrix for a given molecular size range. 37. the gel should be chosen so that the target elutes early in the chromatogram. 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). 40 show this clearly and may be used as a guide to the selection of the correct medium for the main classes of biomacromolecules. Eluent Some samples contain proteins or other components which have a limited range of solubility or stability. 34. DNA. Superdex. 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. Media with relatively shallow selectivity curves. There is thus always a risk that changing the 51 . 33. The selectivity curves on pages 25. e. polysaccharide etc. Type of molecules to be separated The class of molecule. the media with the steeper selectivity curves and the smaller bead sizes. the medium chosen must therefore be capable of being run economically in a sufficiently large bed (see ‘Choice of column dimensions’ below).g. 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. The medium must have a separation range which covers the molecular sizes of the target molecules. 30. Sample characteristics Sample size Sample size has an indirect influence on the choice of gel in that large sample volumes require large bed volumes.

Sephacryl HR or Sepharose CL will be found the most appropriate gels. pilot plant or industrial scale should be carried out on suitably designed columns. in many cases. Ideal features Gel filtration on a laboratory. eluents containing organic solvents and eluents of extreme pH. The more recently developed media. If the sample precipitates in a gel filtration column. Sephacryl HR. Detergents do not appear to influence the pore structure of the gels (51). are in general more suitable than the traditional media for work in dissociating media. 6 M guanidine hydrochloride or urea. a more porous gel is usually required than predicted from selectivity curves prepared for native globular proteins. Their high chemical and physical stability also makes them particularly suitable for use in these eluents. Once again the effect of these agents on protein conformation usually requires a more open gel to be used for gel filtration. However. Thus. The free choice of eluent in gel filtration enables this requirement to be met in almost every case. possibly irreversibly. because of the more extended configuration of proteins and polypeptides in these eluents.pH or other conditions of the sample will cause inactivation or even precipitation. 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. membrane components (48–50).g. the column will be blocked. 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. is extremely useful for molecular weight determination (14). Superdex and Superose. e. 52 . Gel filtration in dissociating eluents. and the sample may be lost. for example. Detergents are particularly useful as solubilizing agents for proteins with low aqueous solubility. Care should always be taken to work within the solubility and stability range of the sample.

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. are difficult to clean and can give artifactual results (52). In general the length of column is decided by the resolution required and the diameter by the sample volume (see “Sample size” page 55). therefore. except for K9 types. • All of the columns. Long columns should. Columns in the C-column series are for laboratory applications at pressures of up to 1 bar. Pharmacia has developed a series of standard columns suitable for gel filtration. the effective bed height can often be increased simply by recycling (Fig. If a very long bed is judged to be necessary. be avoided. Both techniques require the use of adaptors or end pieces (p 70). 53 . can be fitted with flow adaptors for easy sample application (p 73–77). 30) or by using columns coupled in series. therefore. Column dimensions The resolution of two separated zones in gel filtration increases as the square root of column length. Bed heights of greater than 1 m are seldom required. They are manufactured from materials which do not cause destruction of labile biological substances. be used to obtain the best resolution in analytical fractionation. Bed supports made from coarse sintered glass or glass wool cannot be recommended for long term use. • Advanced design bed supports which give uniform flow. Other important characteristics include • Dead space at the outlet of less than 0. Many simple columns have large dead volumes and should.The dead volumes at the outlet and inlet should always be as small as possible. which is particularly important when handling biological samples. SR-columns are for use with organic solvents. All are easy to dismantle and reassemble to allow thorough cleaning. Minimizes dilution and prevents remixing of separated zones.1% of the column volume. Pressure and solvent resistances Pharmacia columns. Their design is based on many years experience in the field of gel filtration.0 cm often proves satisfactory. because they soon become clogged. For analytical purposes a column with an internal diameter of approximately 1.

h-1 in laboratory columns. 30. With Fine and Superfine grades still shorter beds give satisfactory resolution for comparable separations.cm-2. although flows up to 5 times faster can often be used without much deterioration. Good resolution and short running times may thus appear to be basically incompatible. Flow rate Resolution. The optimum flow rate for maximum resolution of proteins is of the order of 5 ml. or the sample/eluent to the column and the column outlet to the fraction collector. The 4-way valve connects the column outlet to the inlet. Increasing the effective column height by recycling. decreases with increase in flow rate. For group separations short columns often suffice and gel beds less than 50 cm can be used with the Coarse grade of Sephadex. 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. 54 . Eluent and sample are connected to the 3-way valve which can be closed during recycling.Rec102 Fig. under conditions which are usually encountered in gel filtration.

If peaks are well separated at a low flow rate using a long column.Using media with small particles will give increased efficiency and higher resolution. and longitudinal diffusion in the bed. always be smaller than the separation volume. but usually at the expense of sample capacity. where peaks are well resolved. In desalting and buffer exchange volumes up to approximately 30% of the total bed volume (Vt) 55 . Smaller volumes do not normally improve resolution. are given on page 66. which is an inevitable consequence of gel filtration. a maximum sample volume should be used within the limits set by the separation distance.5–5% of the bed volume is recommended. A sample volume of 0. In group separations and some fractionation experiments. In order to minimize sample dilution. 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. Full details of the maximum permissible flow rates of the various gel types. This may allow higher flow rates to be used. VSep = VeB . the maximum sample volume could be as great as the separation volume (VSep). The flow rate can be increased and a shorter column can be used. therefore. governed by their mechanical properties. non-equilibrium between the stationary phase and the mobile phase. Figure 31 shows how. due to eddy diffusion. if no zone-broadening were to occur during passage down a column.VeA However. see Table 8. The sample size must. 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. the zones will always be broadened (40). the excess resolution may be traded off for speed. it is often appropriate to improve the experimental design by increasing the sample size.

can be used to minimize dilution and still retain good separation.5 ml). 56 .3%. 31.5 M. a dilution factor of only 1. Yield of albumin in 3.Fig. sample volumes of between 15 and 20% of Vt are recommended.5 ml after sample application (between arrows) 95. 2. A column PD-10 was equilibrated with distilled water. The sample contained human serum albumin (25 mg) dissolved in NaCl (0.5 ml results in the effectively complete recovery of desalted material in a volume of 3. The centre diagram corresponds to the maximum sample size giving complete separation if no zone broadening were to take place. 32. salt content.0% of total salt originally present. Elution curves for different sample sizes.4 (Fig. 2. 32). The bottom diagram corresponds to the maximum sample volume to be applied to obtain complete separation in the conditions of the experiment. Fig. The shaded areas correspond to the elution profiles that would be obtained if no zone broadening were to occur. The top diagram corresponds to the application of a small sample.5 ml. Where complete recovery of desalted sample is the major requirement. For routine small scale desalting with the PD-10 columns (p 49) use of the maximum recommended sample volume of 2. Desalting of albumin solution.

the viscosity of the sample often limits the concentration that can be used. (A lower flow rate will not improve the separation. This allows more rapid recovery of desalted materials since higher volume flow rates can be achieved with the shorter column. 33. Fig.) 57 . narrow one. 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. An increasing deterioration of the separation becomes strikingly apparent. wide column rather than a long. Experimental conditions were identical except that the viscosities were altered by the addition of increasing amounts of dextran. A high sample viscosity causes instability of the zone and an irregular flow pattern.When designing an optimized desalting system. This leads to very broad and skew zones. in addition to the solubility of the solutes. Elution diagrams obtained when haemoglobin and NaCl were separated. Sample composition Due to the linear partition isotherms. However. the volume of gel required should be packed in a short. gel filtration is to a large extent independent of mass of solute in the sample.

e. Approximate relative viscosities can be quickly estimated by comparing emptying times from a pipette. However. It should also be noted that some proteins may precipitate in solutions of low ionic strength. eluted with the HMW fraction is so small it can be neglected in most cases. Eluent Eluent composition does not directly influence the resolution which can be obtained in gel filtration. charged substances can also be treated with distilled water as eluent.02 for Sephadex and Sepharose or 0. volatile buffers such as ammonium acetate.15 for Sephacryl HR is recommended to safeguard against possible ionic interactions with the gel matrix. dissociation of enzymes and cofactors. dissociation of proteins into subunits.g. 34) is used here to illustrate some of the factors which might need to be considered. and the presence of dissociating media or detergents can cause conformation changes. in general. In desalting. this separation can not be speeded up significantly without serious loss of 58 . is often used to control pH. the separation volume is so large that.. Sodium chloride can be used for this purpose. Completely uncharged substances may be eluted with distilled water. For chromatography of substances carrying charged groups an eluent containing a buffer. Components A and B are separated at the lowest flow rate. sodium phosphate etc. B and C (Fig. 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. but the amount of ions excluded and. The behaviour of a three component model system of proteins A. and an ionic strength of at least 0. therefore.In practice the relative viscosities of sample and eluent should not differ by more than a factor of about 2. Optimization Optimization of a separation can only be carried out by careful experiment. which must be taken into account when choosing the gel and when interpreting the results of an experiment. since each separation problem is different. dissociation of hormones and carrier proteins etc. corresponding to a protein concentration in the sample of about 70 mg/ml when a dilute aqueous buffer is used as the eluent. Tris-HCl. Complete removal of salt is not possible. If the product is to be lyophilized. ammonium bicarbonate or ethylenediamine acetate can be used.

Fig. the time for separating protein C from B is reduced from about 30 hours to about 2. (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). The resolution (Rs) is still 2. 34. The bed height (L2) required to give Rs = 1 can be calculated from L2 = L1 Rs1 59 . In the first stage of the optimization. B and C. Protein C is well separated from protein B and this particular separation can be speeded up by a factor of 50. resolution. A model gel filtration experiment to demonstrate optimization of the separation of model proteins A. increasing the flow rate.5 hours.25.

Reducing the bed height to about 20 cm should still give adequate resolution of protein C and protein B.where Rs1 is the resolution obtained with a column length L1. In this example L2=17 cm. The separation time for protein C and protein B has been reduced from 30 hours to 35 minutes. The result obtained with a bed height of 19.5 ml.cm-2.h-1 is shown in Figure 34. For a full discussion of the optimization of gel filtration see (40). 60 .5 cm at 24.

During swelling excessive stirring should be avoided as it may break the beads. Preparing the gel Pre-swollen media (Sephacryl HR. bed volume (ml/g) 2 – 3 2. 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. For all 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. Superdex prep grade. the process of swelling can be accelerated by using a boiling water bath which also serves to deaereate the buffer (Table 10). Providing that a column is used and maintained carefully it can be expected to give many months of reliable service. Do not use magnetic stirrers. Sepharose CL and Sepharose) These gels are supplied swollen and ready to use as a suspension containing 20% ethanol as a preservative.5 – 3. Bed volume and swellings times for Sephadex G-types. Table 10. Superose prep grade.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 .Performing a gel filtration experiment Gel filtration is essentially simple to perform once a well packed column has been obtained.

miscible. the gel is transferred first to 70% A/30% B then to 30% A/70% B and finally to pure B. Superdex prep grade. from water to chloroform via acetone (Fig. However. Thus to transfer from one pure solvent (A) to another pure solvent (B). Sepharose CL can be used quite safely in a wide range of organic solvents.Preparation of Sephacryl HR. the transfer is made via an intermediate solvent e. Note that the gel volume of Sephacryl HR may be reduced by up to 15% on transfer to organic solvents (Table 2). 62 . 35. 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. Suck off the excess solvent and resuspend in the same solvent. 35) and resuspend the gel by gentle stirring. Repeat the process with the next solvent of the series. it is not suitable for gel filtration in many organic solvents. If A and B are not mutually Fig. allowing two resuspensions and proceed until the required solvent composition is reached. Transfer the required amount of gel to a sintered glass Buchner funnel and remove the excess aqueous medium by gentle suction. 35). the transfer must be made through a graded series of solvent mixtures. Add the next solvent (see Fig.) To ensure efficient replacement of the water by the required solvent. Suggested routes for changing to organic solvents.g.

The flow rates obtainable are also affected. Fine particles can be removed at this stage by decantation.g. Packing Sephadex G types. It is important to note that Sephacryl HR.Gel filtration in organic solvents After transferring the gel to the solvent of choice it may be packed in the usual way (see below). 36.g. 63 . Pharmacia columns SR 10/50. Certain solvents which disrupt hydrogen bonds. chloroform. if desired. SR 25/45 or SR 25/100 should be used. Sepharose and Sepharose CL Filling the column 1. Superdex prep grade and Superose prep grade. Chromatography in organic solvents”. 36-43) This is a very critical stage in any gel filtration experiment. DMSO. may cause softening of Sepharose CL so that flow rates become impracticably low. A poorly packed column will give rise to uneven flow. e. Packing a column (Fig. are packed in a rather different manner than the Sephadex G-types and Sepharose CL or B types. Sepharose CL floats in dense solvents. The suspension of gel should be adjusted so that it is a fairly thick slurry. e. Usually about 75% settled gel is suitable. It should not be so thick it retains air bubbles. zone broadening and loss of resolution. and in these cases packing should be carried out as described in the booklet “Sephadex LH-20. Prepare the gel as described above. which have a relatively rigid bead form. Flow rates will depend on the viscosity of the eluent. Fig.

Alternatively the gel can be poured directly into the vertically mounted column using a glass rod. 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. C. Immediately readjust the column to a vertical position. packing the column in stages. Ensure that there are no air bubbles trapped in the dead space under the net by drawing water (or 20% ethanol) through it. The gel suspension should reach the temperature of column operation before packing is begun. Preparing the gel from too thin a suspension or. a gel reservoir (XK.The suspension should be de-gassed under vacuum (Fig. Tilt the column and pour the well-mixed gel suspension down the inside wall of the column. close the outlet tubing. Use of degassed buffers and a filled water-jacket will also help safeguard against the effects of rapid temperature changes. 2. 3. HR 10 and HR 16 columns) should be attached (Fig. 4. All the gel required should be poured in a single operation. for other reasons. 39). often results in a badly packed bed. Mount the column on a stable laboratory stand. inject eluent into the outlet tubing until it passes through the bed support net (Fig. When the dead space is properly filled. K series columns) or a column extension (SR-10. 37. 37). If the slurry volume is greater than the volume of the column. 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. 36) This is not necessary for Sephadex which has been swollen on a boiling water bath. 64 . 38 and Fig. Fig.

Do not exceed the maximum operating pressures given in Table 11. 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. 39. The top piece air vent should be closed and the column outlet opened to allow the packing to continue. With C or K columns. Close the column outlet and fill the space above the gel with eluent to about 1 cm from the top of the glass tube. Fig. a top piece can be used in the following way. 5. 6. The next step involves closing the column without trapping air inside. Fit the top piece.Fig. The flow should be started as soon as possible after filling the column to obtain even sedimentation. Check for air bubbles and repeat steps if necessary. 38. follow the instructions given below (p 70–71). If an adaptor is used. 65 .

0 1. flow rate ml/min 2.5 cm diameter with a bed height of 30 cm.9 0.5 1.5 2. Before opening the column outlet tubing.5 2.5 2. 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.17 1. Data has been obtained from columns 2.83 ml.25 77 18 50 12 23 6 12 3 * For flow rates used during packing please see Table 12. Approx maximum flow rates and pressures.5 4.96 0.0 0.5 2.Table 11.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 6.4 1. 66 .5 10 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 . operating pressure cm H2O 500 500 500 500 500 >200 120 50 200 80 40 These gels obey Darcy’s Law (see p 78) 160 160 96 96 36 36 16 16 Approx.16 0.5 2. Equilibrating the bed 1.2 1.cm. Gel type Max.–2 h–1 30* 30* 30* 30* 30* 30 26 15 14 11. With the softer gels the operating pressure must not exceed the limits given in Table 11. This depends on the difference between the free surface of eluent in the reservoir and the outlet as illustrated in Figure 40. max. check the operating pressure.25 1.5 2.

no matter whether the flow through the column is downward (C) or upward (D). 40.Fig. 67 . Definition of operating pressure (A-D) and sample application from a sample reservoir (D). A and B. 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. Pressure (cm water) is measured as the distance between the free surface in the column or reservoir and the end of the outlet tubing.

Wet the adaptor by drawing water (or 20% ethanol) through it. making sure no air bubbles are trapped under the net. Pack the column at the temperature at which it will be used. and it is highly recommended that this method be followed. 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. 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. 1. Two or three column volumes of eluent should be passed through the column in order to stabilize the bed and equilibrate with eluent buffer. 6. Close the tubing with a stopper when all air bubbles have been removed. It is of special importance that the nets. Pour all the gel in a single operation and fill the reservoir to the top with eluent. Insert the adaptor at the bottom of the column far enough to give the desired bed height. It should not be so thick it retains air bubbles. Superdex prep grade and Superose prep grade Sephacryl HR. Make sure the column is not damaged and that all parts are really clean. 68 . Usually about 75% settled gel is suitable. P-l. 2. Prepare the gel as described above. The suspension of gel should be adjusted so that it is a fairly thick slurry. A slightly higher flow rate than is to be used in the experiments should be used for packing. 7. Attach the packing reservoir tightly and mount the column vertically on a stand. Resuspend the gel and pour the well-mixed gel suspension carefully down the wall of the column using a glass rod. Pack the column in two steps using the flow rates given in Table 12.2. Open the column outlet and start the flow. 3. 5. 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. P-50. Packing Sephacryl HR. 4. connect it to the pump and open the outlet. The packing method described below ensures that the gel is optimally packed at the point of entry of the sample. P-500). Screw on the reservoir cap tightly. 8.g. the most critical area. net fasteners and glass tube are not damaged.

Table 12. close the column outlet and remove the packing reservoir. Open the bottom piece outlet (NOT the outlet from the adaptor at the bottom of the column). 13. It may be easier to use a siphon when working with a large 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. 12. This will cause eluent to flow out of the tubing attached to the bottom piece. 69 . 10. Stop the pump. 11. 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. Press down and tighten the bottom piece. 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. pack the gel in STEP 1 for 2 hours or until the gel bed has reached a constant height. Wet the second adaptor with eluent or 20% ethanol. 14. 11. When the bottom piece is inserted in point 14 it will be pressed about 5 mm into the gel. Insert the bottom piece carefully so that no air bubbles are trapped under the net. Recommended flow rates for packing Sephacryl HR Column XK 26/40 XK 26/70 XK 26/100 XK 50/60 XK 50/100 Flow rates (ml/h) Step 1 Step 2 240 240 180 600 600 490 360 320 1 400 950 9. 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. 10. 12. If there is not enough gel in the column. Remove enough gel so that the plunger will be visible below the end piece. then increase the flow rate to the value listed for STEP 2 and pack for 60 minutes. 13. 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. This is most easily done by first removing the column from the stand and then unscrewing the reservoir over a sink.

42. The gel should be packed completely as described above. 1. Adaptors Adaptors are adjustable column end pieces which support the bed matrix. Fig. They allow automatic methods of sample application which eliminate disturbance of the bed surface and protect the bed from insoluble particles in the sample. 2. Adaptors are available for most Pharmacia columns. After an extended period of use. Fig. 41) 3. Carefully add more eluent to fill the column and form an upward meniscus (Fig. An upward flow arrangement using adaptors will discourage this process (Fig. Bring the adaptor to the gel surface and then a further 5 mm into the gel bed.14. flow rates through a column packed with Sephadex at a given hydrostatic pressure will be reduced. 40 D). 70 . 42). Liquid will flow out of the tubing attached to the adaptor during this process. 41. They should be fitted as follows. Slacken the adaptor tightening mechanism and insert it at an angle into the column so that no air is trapped under the net (Fig. or alternatively should be allowed to settle sufficiently so that there are 2–3 cm of clear eluent at the top of the column. Ensure the column outlet is closed otherwise the gel may be compressed when the adaptor is fitted.

71 . 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.Fig. it is advisable to check the homogeneity of the bed by running a freshly prepared and filtered solution of a coloured substance. Visual inspection of the bed in transmitted light may also reveal heterogeneities and air-bubbles. 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. Screw the adaptor top piece onto the column end piece. The quality of the packing can be checked by watching the progress of a zone of this substance through the bed. 5. Valves on the inlet side of the column should be turned in all directions during this procedure to remove air from all connections. 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. 43. 43). 4. open the outlet and start the eluent flow. Lock the adaptor in position with the tightening mechanism and locking screw (Fig. only about 10 minutes further packing is required before re-adjustment). Make all tubing connections at this stage. Checking the packed bed Before starting any experiment. Adjust the tightening mechanism to give a sliding seal between the column wall and O-ring.

buffer salt or another suitable inert material.Using pre-packed columns The initial setting up of a gel filtration experiment is considerably simplified if a pre-packed column is used. C and K columns with diameters 16 and 26 mm) helps prevent disturbance of the bed surface. 5. Layer the sample on top of the bed. 3. Under no circumstances should the bed be allowed to run dry. A sample applicator cup (available for columns series XK. If not. it is not the simplest and considerable care must be taken to avoid disturbing the bed surface. 2. Do not allow the bed to run dry. 2. Open the outlet and allow the remaining eluent to drain away. 1. Connect the column to the sample application device (see below for alternatives) and to the monitor. Sample application under the eluent (Fig. 6. One of the methods described under ‘Sample application with an adaptor’ should be used. 44) The sample must be denser than the eluent. Open the column outlet and allow the sample to drain into the bed. Wash the sample which remains on the bed surface and on the column wall into the bed with a small amount of 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. 4. Refill the column with eluent and reconnect to a Mariotte flask or pump. An uneven bed surface leads to uneven separated bands and poor resolution. 72 . Follow the instructions supplied with the column to equilibrate it with the chosen eluent. Sample application on a drained bed surface Although this is the method which requires least equipment. Close the outlet and remove most of the eluent above the gel surface by suction. sodium chloride. it can be made so by the addition of a small amount of glucose. 1. Take care not to increase the sample viscosity too much.

Sample reservoir (Fig. 1. 3. Syringe method. After application draw a small amount of eluent into the tubing to prevent sample mixing with eluent when the tubing is withdrawn. 73 . 45).Fig. a sample reservoir (e. 46). RK or R) can be connected via a 3-way valve to apply larger samples. then draw a little air into the capillary tubing to prevent mixture of sample with eluent. 2. Samples must be applied by one of these methods if upward flow elution is used. Use a syringe fitted with a piece of fine capillary tubing or a pipette with a bent tip.g. (Fig. Draw the sample into the syringe. 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. 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 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. No rinsing is required and eluent flow can be restarted directly. In a similar way. Figure 46 also demonstrates sample application and elution with upward flow. 44.

45. 74 .Fig. Fig. 46.

Fig. 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. although exact knowledge of the applied volume requires calibration of the capillary tubing loop. 48). Sample loops with valves LV-4 or SRV-4 (Fig. By using the same sample loop very reproducible sample volumes can be applied. 75 . 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. This method is convenient for application of small samples. 47.Sample applicators SA-5. They can also be used in a sample loop system (Fig. 47). A Sample Applicator SA-5 used as a sample loop. SA-50 (Fig.

Fig. This method is used for sample application when using high performance columns (e. 49). Superloop (Fig. A movable seal separates the sample from the eluent. 48. Sample loops or Superloop with valves V-7 or MV-7 (Fig. Superdex HR 10/30 and Superose HR 10/30) and other 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. V-7 and MV-7. 49. As eluent is pumped 76 .g. Seven-port valves. columns in FPLC System. have three operating positions which make sample application and changing eluents particularly convenient. Sample application with a sample loop and two SRV-4 valves.

Very reproducible sample volumes can be applied. Elution The flow of liquid through the column can be controlled by difference in hydrostatic pressure or by a pump. such as a 77 . into the Superloop. This reduces the risk of bubble formation in the column which can result from suction. particularly when the system is operated automatically using LCC-501 Plus or FPLCdirector. A Superloop should be used for applying sample volumes greater than approximately 1 ml.Fig. eluent flows round the seal to wash the remainder of the sample quantitatively onto the column. 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. When nearly all the sample has been applied. 50. When the seal reaches the end of its travel. 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. It is most easily achieved using a good peristaltic pump such as the P-l pump. Principle of operation of Superloop. Accurate and reproducible control of flow rate is particularly important when repeating experiments or performing routine preparative work. When the flow is to be maintained by gravity feed a constant pressure flask. the sample moves ahead of the seal and onto the column. eluent by-passes the seal and washes the last of the sample quantitatively onto the column.

The flow stops when the eluent in the inlet tubing reached the level of the outlet. for example. The flow stops when the eluent in the inlet tubing reaches the level of the outlet. G-25 and G-50 These gels may be assumed to behave as rigid spheres in gel filtration and therefore obey Darcy’s Law. equations and data given here apply only to laboratory columns with diameters up to 5 cm. Fig. B. The safety loop is placed after the column and the end of the outlet tubing is placed above the column. Sephadex G-10.e. overnight. The arguments. Mention has already been made (see page 70) of the advantage of upward flow for long term use of a column. is recommended. is that of stopping the experiment before the eluent runs out. G-15. U = K ∆P L-1 78 (1) . of the type illustrated in Figure 51 can easily be arranged to prevent air from entering the column. 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. i. 51. Flow rates Recommendations concerning flow rates for use in experiments are given on page 66.Gel and Eluent Reservoir used as a Mariotte flask. a safety loop. Safety loop arrangements: A. If gravity feed is used. The safety loop is placed before the column with the column outlet tubing in any position above the lower loop on the inlet side. A problem which can occur when an experiment is continued without supervision. It is not possible to apply one set of rules for calculating flow rates over the whole range of gel types.

Flow rates at viscosities greater than 1 cP can be obtained by using the relation: flow rate inversely proportional to viscosity. Notice that (to a good approximation) the flow rates are independent of the column diameter. Specific permeabilities of Sephadex G-types. 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. At first sight. for a given pressure head. Observe that the flow rate is proportional to the pressure drop over the bed and. ∆p is the pressure drop over gel bed expressed in cm H2O. inversely proportional to the bed height. Sephadex type 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 Permeability K 19 18 9 30 80 290 13. Temperature influences the viscosity of the eluent. Lower flow rates are obtainable. Not only is the flow rate dependent upon the factors already mentioned but also on the column diameter. Specific permeabilities are given in Table 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. Assuming an eluent with viscosity of 1 cP 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.where U is the linear flow rate expressed in cm/h (ml. assuming a constant pressure head. in a cold room than at room temperature. Wider columns do not allow as high a pressure and linear flow 79 .h-1). Table 13. cm-2. 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. Theoretical flow rates (not maximum) can be calculated from equation (2) by inserting values for ∆p and L.

These gels do not have a linear relationship between pressure and flow. 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. as a loss in resolution or as a significant increase in back-pressure. exceeding the maximum recommended values can lead to gel compression. 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). Slightly reduced maximum operating pressures must be used with columns wider than 2. It is recommended to determine the maximum flow rate for the column in question with gravity feed. only fresh buffer solutions should be used. reduction in flow rate and loss of resolution. To utilize fully the high rigidity and the excellent flow properties of Sephacryl HR a liquid delivery system including a pump should be used.rate (ml. Similarly. 80 . taking care not to exceed the pressures given in Table 11.6 cm. To calculate the flow rate for a different bed height it may be assumed to be inversely proportional to the bed height. it may be necessary to remove precipitated proteins or other contaminants which have built up on the gel bed. and then use flow rates not exceeding 75 % of this value with the pump.6 cm (column XK 26) and bed height of 30 cm have been assumed. A peristaltic pump can still be used with the other softer gels provided care is taken. In all cases.h-1) as narrower ones. General procedures are given below for different media followed by special procedures for removing specific contaminants. prevention is better than cure. Maximum operating pressures are independent of bed height but flow rates decrease with bed height. The need for cleaning may show itself as the appearance of a coloured band at top of the column. Recommended maximum operating pressures and corresponding approximate flow rates at room temperatures are given in Table 11.cm-2. as a space between the upper adaptor and the bed surface. A bed diameter of 2. Cleaning gels and packed columns General cleaning procedures When a column has been in use for some time. See also the section on ‘Prevention of microbial growth’ (p 84). Many buffer substances are excellent supporters of microbial growth.

The pressure should be checked at the same stage during each run.5 M NaOH or a non-ionic detergent solution (1%) either in the column or on a Buchner funnel. This contact time is sufficient to solubilize most protein precipitates. Cleaning Sepharose Wash with a non-ionic detergent. Cleaning Sephacryl HR Sephacryl HR may be cleaned and sanitized in the column with 1–2 column volumes of NaOH (0.5 M) at a flow rate which gives a contact time between the gel and the cleaning solution of at least 1 hour. Note that cleaning solutions should also be filtered before use. Sephacryl HR may also be washed with a solution of a non-ionic detergent. A dirty filter is often the cause of increased back-pressure. 1.2 M) on a Buchner funnel. but reverse the direction of flow in the column during treatment with NaOH.If an increase in back-pressure is observed. the column must be carefully re-equilibrated with 2–3 column volumes of eluent buffer before it is used again. for example by the level of the gel falling. Cleaning a packed column: Do NOT reverse the flow during cleaning since this may cause a loss of efficiency. 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. After cleaning. Cleaning Superose prep grade a. 81 . make sure that the high back-pressure is in fact caused by the column before starting the cleaning procedure. Sephadex may also be washed with NaOH (0. Cleaning Sephadex G-types Packed columns may be cleaned with 2 column volumes of a non-ionic detergent solution. Cleaning Sepharose CL Treat the gel with one bed volume of 0. Change the filter on the top of the gel bed and check the bed support. since the back-pressure can vary within a run during sample injection or when changing to a different eluent. changing it if necessary. Use the same method if the gel is severely contaminated.2–0.

Wash in sequence with 25 ml acetic acid (50%). Wash with 5 ml 50% acetic acid 3. 2. run at a low flow rate). 3. Equilibrate with buffer until the baseline is stable. 4. 3.1 M). 2. 25 ml water and three aliquots of 100 or 200 ml of acetic acid (50%). Re-suspend the gel in at least 5 times the gel volume of high purity water in a beaker. Clean all column parts with soapy water or laboratory detergents.2 M). 82 . b. 5. Allow the gel to sediment and pour off the supernatant. Inspect the top and bottom filters and change them. Equilibrate with buffer until the baseline and the pH of the eluent leaving the column are stable. Change the filter at the column inlet. This procedure is most conveniently carried under automatic control from a Liquid Chromatography Controller LCC-501 Plus. 25 ml NaOH (0. 3. Small aliquots (3 x 100 or 200 µl) of acetic acid (50%) may be added during this rinse. 1. Repeat the washing procedure once more before re-packing the column. Cleaning the gel before repacking: 1. 4. Wash with 5 ml 0. Rinse with high purity water or buffer. Cleaning Superose HR 10/30 pre-packed columns Regular cleaning The following procedure will help prevent contaminants from building up on the column. Set the pressure limit to the maximum for the column. 4. Put a large enough beaker under the column to collect the gel. 25 ml ethanol (20%.2 M). 1. many of them are caught by the filter. remove the bottom piece and empty the column by pumping high purity water or buffer through it. then water and lastly ethanol (20%).1 M NaOH 2. 25 ml water. 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 M-0. Wash the gel on a glass filter with NaOH (0. Wash with at least 1/3 of the column volume NaOH (0.2. Since the contaminants are introduced with the liquid flow.1-0.

if this does not work. Hydrophilic proteins and peptides Wash the column with the solution which previously dissolved the material during sample preparation e. If the column still is contaminated. careful rinsing is required to remove trace amounts of enzyme remaining in the system.1 ml/min. The frequency of cleaning depends on the degree of contamination. care must be taken not to exceed the maximum operating pressure which the gel can sustain. the following methods may be used to remove specific contaminants. it may be necessary to change the bottom filter or to remove and discard the top 2-3 mm of the gel.5 NaCl and leave overnight at room temp. etc. (overnight. Since some of the cleaning solutions are more viscous than normal buffer solutions.The suggested cleaning volume of 25 ml is only a guide-line. In special cases only. an extraction solution. Re-equilibrate the column with two bed volumes of buffer immediately after cleaning. or 1 hour at 37 °C. but a cleaning cycle at least every 10–20 separation cycles is recommended. This treatment will remove most proteins from the gel. it may be washed with 0. detergent.5 M).5 bed volume of 30% isopropanol or 0.g.1 M acetic acid and 0. Procedures to remove specific contaminants If the general cleaning methods fail to give the desired result. at low flow rate). try one of the alternatives.5 bed volume of 1 M NaOH solution and 0. the practical requirements are best determined by monitoring the baseline. Fill the column with 1 mg/ml pepsin in 0. Replace the filters if necessary. which should be stable after each step in the sequence.1 M HCl. Cleaning Superdex pre-packed columns Superdex columns may be cleaned with one bed volume of NaOH (0. 83 . Note: after enzymatic digestion. Wait until the UV baseline is steady before applying the next sample. Various alternatives are given for each type of contaminant – choose the most convenient according to the reagents you have available. These operations must be carried out extremely carefully to avoid serious loss of resolution. Wash overnight in 30–50% acetic acid at 0.

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. 1 mM EDTA.1 ml/min with a non-ionic detergent (e. Antimicrobial agents which interact with sample substances must be avoided if they are to be used in eluents.1 M NaCl.g. 1 mg/ml in 0. 37 C.Hydrophobic proteins and peptides These are usually soluble in polar organic solvents e. pH 8.g.5.0. If the organic solvent which best dissolves the contaminant is known.0) through the column at low flow rate for 24 hours. 84 . buffers and gel suspension. 50 mM Tris-HCl. pH 7.0. otherwise any agent which does not interact with the gel may be used. special caution is recommended if subsequent separations of RNA or DNA are planned. 1 mM EDTA.5. pH 8. Note: after enzymatic digestion. DNA Hydrolyse the DNA with deoxyribonuclease I solution (2 ml. 2 hours) and rinse with 10 mM Tris-HCl.1 M NaCl. Some of the more commonly used antimicrobial agents are described below. careful rinsing is required to remove trace amounts of enzyme remaining in the system. pH 7.1–2 M. Antimicrobial agents may be eluted from columns before chromatographic runs or they may be present in the eluent during chromatography. 1 hour) and rinse with water or with ribonuclease I solution (2 ml. 10 mM Tris-HCl. 0.g. 50 mM Tris-HCl. ethanol (24%). 37 °C. acetonitrile (30%). run this overnight at a slow flow rate. RNA Hydrolyse the RNA with NaOH (0. 1 mg/ml in 0.10 mM MgCl2. 1 mM EDTA.2–1% NP-40 or Lubrol) in a basic or acidic solution and remove the detergent by washing with methanol or ethanol. pH 8. Lipids Wash the gel overnight at 0. 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. 2 hours) and rinse with 10 mM Tris-HCl.

is an effective bacteriostatic agent. For sterilization of gels by autoclaving of the gel see pages 24. 31. G-150 or G-200. 39. NaN3. Oxidizing substances should not be used as antimicrobial agents. Sodium azide interferes with fluorescent marking of proteins. Superdex. nitrate). The use of sodium azide is discouraged in many countries. solubilize substances 85 . Precipitation may occur on storage of Hibitane with appreciable concentrations of chloride or sulphate ions.01%. Sepharose CL and Sepharose are supplied in 20% ethanol. Ethanol 20% Superose. It does not interact notably with proteins or carbohydrates or change their chromatographic behaviour.g.001–0. At higher concentrations (0. 0.02–0. 41. borate. softening the plastic and leaving the liquid without antimicrobial activity. is a very effective antimicrobial agent. 0. are effective only in weakly alkaline solutions. It can lead to explosions when disposed of via lead pipe waste disposal systems and is believed to be a mutagen. 28. In addition they are effective only in very concentrated solutions. Sodium azide. in many cases.5 M) it is an effective disinfectant even for resistant bacteria such as Pseudomonas.Antimicrobial agents Chlorhexidine Chlorhexidine (e. is very widely used. This solution can also be used as a bacteriostatic agent for storage of used gel.01 M. 0. Treatment with sodium hydroxide inactivates endotoxins and will. as they cause the gel particles to shrink slightly. Chloroform.002%. It is incompatible with only a very few substances but is not recommended for use with Sepharose. 0. Phenyl mercuric salts Phenyl mercuric salts (acetate. Hibitane®). Sodium azide Note. the anthrone reaction and inhibits certain enzymes.05%. Sodium hydroxide Sodium hydroxide. butanol and toluene These organic solvents are not recommended as antimicrobial agents for Sephadex G-100. Sephacryl HR.1–0. They penetrate plastic parts of chromatographic equipment.

Clumps may appear during the shrinking process but they disperse on re-swelling. A last wash with diethyl ether reduces the drying time. 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. 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. 0.g. It is bound to and inactivated by substances containing thiol groups. The swollen gel is thoroughly washed with water to remove salts and contaminants. is effective only in weakly acidic solutions. 60–70% alcohol. Its low toxicity is an advantage that reduces the risk of sample contamination. Merthiolate®). For special purposes Sephadex can be dried and restored to its original state by the following procedure. pH 6-8 in the presence of a suitable bacteriostatic. 86 . Sephadex can also be stored partially shrunk in. The gel is then sucked dry on a Büchner funnel and finally dried at 60–80 °C. Storage of used media Used media should be stored at +4–8 °C.005%. is most effective in weakly acidic solutions. Risk for clump formation is reduced by slow shrinking and ether washing.05%) or ethanol (20%).g. sodium azide (0. It is not recommended for use with Sephacryl HR. the gel can be shrunk by successive addition of alcohol solutions of increasing percentage alcohol. Thimerosal Thimerosal (ethylmercurithiosalicylate e. 0. After removal of excess water. for example.g. e. Sodium hydroxide is not recommended for use with Sepharose and for storage of Sephacryl HR. Trichlorbutanol Trichlorbutanol (e. Thimerosal should not be used with Sephacryl. The gel should be allowed to equilibrate in between each addition. Storage of unused media Unused media should be kept at +4–25 °C.05%. Final shrinking should be with 96% alcohol.precipitated on the column. Chloretone®).

05%) is recommended for columns packed with Sephadex G-types. Sepharose and Sepharose CL. If ethanol (20%) is used in the storage buffer. the gel bed should be thoroughly cleaned before re-equilibration with the storage buffer. 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. but only sodium azide (0. de-gas the ethanol/water mixture well. Sodium azide (0.g. 87 . test tube) containing ethanol (20%). Superose.05%) or ethanol (20%) may be used for Superdex. this tubing may be filled with buffer and re-connected to prevent the column drying out. the column should be left connected to 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. Sephacryl. For long-term storage. Pre-packed HR columns are supplied with a rubber tubing between the inlet and the outlet of the column. Disconnect the column from the system. overnight. For short-term storage.g. 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).Storage of packed columns Packed columns should be stored at +4–8 °C in the presence of a suitable bacteriostat. e. a low flow rate through the column will prevent bacterial growth.

88 .

Remedy Prior to chromatography. Filter is clogged. 89 . buffers and gel suspensions. Precipitation of proteins Modify the eluent to in the column caused by maintain stability. See cleaning procedures p 80–84. but steps should always be taken to prevent infection of packed columns. Replace the filter. See above Microbial growth has occurred in the column. See p 86–87.Fault finding chart Problem Column is clogged Cause Presence of lipoproteins or protein aggregates. precipitate with 10% Dextran Sulphate or 3% polyvinylpyrrolidone.05% sodium azide. Store gel in the presence of 20% ethanol or 0. removal of stabilizing agents during fractionation. Microbial growth rarely occurs in columns during use. Always filter samples and buffer before use. The sample substance is poorly resolved from other major peaks Microbial growth has occurred in the column.

Contaminated gel surface or top net will spoil sample application. Sample is too viscous. Column is not mounted vertically. 90 . sample before application filter the sample and to the column. Check the packing by running a coloured compound. For maximum resolution do not exceed the sample volumes given in Table 10. e. Too much sample mass Decrease the sample load. Blue Dextran and observing the band. Repack the column if necessary. The column is dirty. carefully.Problem The sample substance is poorly resolved from other major peaks Cause Remedy Sample volume is too Decrease sample volume large or the sample has and apply the sample been improperly applied. Keep protein concentration below 30 mg/ ml. Improper filtration of the Regenerate the column. repeat the chromatography step. Dilute the sample with the elution buffer. You may need to repack the column. Column is poorly packed. Clean and regenerate the column. Try again with the column mounted in a vertical position. has been loaded onto the column.g.

Choose elution conditions which stabilize the sample. Blue Dextran and observing the band. See p 53. Check for possibility of adsorption effects. See p 80–84 for cleaning procedures. See p 54. Use a column with a water-jacket. Consider the effects of dissociating agents or detergents if present. Large mixing spaces in or after column. Substances which are relatively close in molecular weight require Superose or Superdex HR. Proteins or lipids precipitated on column. Column is poorly packed.g. Column too short. Check the packing by running a coloured compound.Problem Cause Detector cell volume is too big. See p 53. Remedy Change the flow cell. Wrong gel type. Decrease the sample load and repeat the run. Check selectivity curve. e. Repack the column if necessary. Leading or very rounded peaks observed in the chromatogram Overloaded column. Flow rate too high. 91 . Uneven temperature in the bed.

Problem Earlier elution profile can not be reproduced

Cause The sample has altered during storage.

Remedy 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. Clean and regenerate the column.

Proteins or lipids have precipitated on 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 Sample substance elutes later than expected or even after the total column volume

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

Remedy 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. Clean and regenerate the column.

Late elution and broad peaks observed in the chromatogram

The column has become dirty.

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. Enzyme separated from co-factor or similar.

Change the eluent.

Test by pooling fractions and repeating the assay.

93

Problem

Cause Microbial growth.

Remedy See p 84. Add protease inhibitors to the buffers to prevent proteolytic digestion.

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.

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. Specific adsorption. Choose elution conditions which stabilize the sample. Lectins may bind to sugar residues in the matrix. Try specific elution with an analogous sugar. May be caused by removal of salts or sample dilution.

Sample precipitates.

More activity is recovered than was applied to the column Peaks too small

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. Wrong sensitivity range on detector. Adjust.

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

94

– No flow from pump. Clogged end-piece or adaptor or tubing. Replace the lamp. Bed supports of porous glass or polythene are prone to clogging by gel particles. No flow through the column Outlet closed. No peaks Monitor switched off. Sample not applied. Check the recorder settings. 95 .Problem Cause Sample size is smaller than intended. UV-lamp not working. Check the electrical connections and cables. Switch it on. Air-lock in outlet tubing or bottom-piece. Check the operation of the sample application device. Range button depressed or no sensitivity set on recorder. Monitor not connected to recorder. With peristaltic pumps check the condition of the tubings. Excessive zone broadening Remedy – Check the column packing and re-pack if necessary. – Reduced or poor flow through the column See above. Check for leaks at all connections. See page 69.

See page 61. Stir the top 1–2 cm and allow to re-settle as an even layer. Can also occur after prolonged use. it can break the beads. Gel not fully swollen (Sephadex G-types). Fine particles can be removed by decanting from a settling suspension. For prevention see page 84. de-gassed buffer upwards through the column. Take special care if buffers are used after storage in a fridge or cold-room. Fines (Sephadex G-types). Upward elution retards the process. Column may need to be repacked. A waterjacket is a good safeguard. Use de-gassed buffers. Special care is needed when packing Sephadex G-75 – G-200 (see Table 13). Do not allow column to warm up due to sunshine or heating system.Problem Cause Bed surface blocked by precipitated proteins. Bed compressed. 96 . Remedy Remove contaminated gel from the bed surface. Repacking the column may be necessary. Do not use a magnetic stirrer. Microbial growth. Bubbles in the bed Column packed or stored Small bubbles can often be at cool temperature and removed by passing well then warmed up.

Protect eluents from dust. Particles on the bed surface or uneven bed surface. Column violently mishandled. clean or replace the net. Remove small amount of gel and stir up the top 1–2 cm.Problem Cause Eluent not properly de-gassed. Re-install the adaptor taking care to avoid air bubbles. Repack the column. See p 70–71. sample passes down bed 97 . allowing it to re-settle as an even layer. Keep particles out of samples and eluents. Bed packed at a temperature different from run. Particles in eluent or sample. Gel suspension too thick or too thin. Dismantle the adaptor. Bed insufficiently packed (too low packing pressure. Column packed at too high pressure. too short equilibration). Distorted bands as Column poorly packed. Cracks in the bed Large air leak in column. Clogged or damaged net in upper adaptor. Filter or centrifuge the sample. Check all connections for leaks. Remedy De-gas the eluent thoroughly. – Distorted bands as Air bubble at the top of sample runs into the column or in the the bed adaptor.

Problem Cause Remedy Replace or re-fasten. 98 . Do not exceed the recommended operating pressure for the gel medium. Gel beads in eluent Bed support loose or broken. Column operated at too high pressure.

Killander. P. 96 (1965) 595–606. J. Nature 183 (1959) 1657–1659. Makromolekulare Chem. 14. J. New York. Measurement of protein-binding phenomena by gel filtration. H. 17. Roth. Sweden. Hummel. Cambridge 1989 Hagel. 7. Pathol. G. W. 15. 48 (1961) 160–71.. Principles. Tigier. Biochim. Determann. Acta 93 (1964) 1–14.. 9 (1962) 550–555. J. 18. and applications. J. T. Weinheim. 101 (1974) 137–153. A. 12. Winzor.J. Mage. Acta 112 (1966) 346–362.. J. Ratcliff. Chem. P.A. Chromatographia 23 (1987) 361–369. L. 1–53 (ed. Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. J. Squire. J. Biochim. Acta 63 (1962) 530–532. Appl. Chromatogr. Biochim. AB Pharmacia. Chromatographic demonstration of the Gilbert Theory. Molecular-weight distribution determination of clinical dextran by gel permeation chromatography. 99 . Application to crude preparations of sulfite and hydroxylamine reductase. Biophys. Biochemistry 2 (1963) 1263–1267. 140 (1977) 98–102. Estimation of molecular weights of proteins by agarose gel filtration. The correlation between molecular weight and elution behaviour in the gel chromatography of proteins. 9. Chromatogr. Fractionation of dextran by the gel filtration method. L. H. Killander. Batlle. 10.) Interscience Publishers. Biophys. K. In Methods of Biochemical Analysis vol. L. Ackers.A. K.References 1. J.M. A. K. VCH Publishers Inc. 20.J. J. Laurent. Granath. Hardwicke. 19. Dissertation 85 pp. Biophys. 16.A.. W. D. J. Flodin. 40 (1969) 453–457. A relationship between the molecular weights of macromolecules and their elution volumes based on a model for Sephadex gel filtration. New York. Nilsson. P.. J. Dextran gels and their applications in gel filtration..K. P.M. Biochem. Firemark. Biophys.A. 18 pp. In Protein Purification.G. H. Biol. Flodin. Studies of chemically reacting systems on Sephadex.P.-C.. Siegel.J. 4. Nilsson. P. A. The gel filtration behaviour of proteins related to their molecular weights over a wide range. Chromatogr. 21. H..C.. Pharmacol. Glick. 107 (1964) 471–478. 3.G. R. J. del C. On the history of the development of Sephadex.Rydén). Clin. G. 5. J. G.. Chromatogr. 25 (1966) 303–313.. J.F. Michel. P. Ansari.. J. Separation of human heme. Chromatogr. Uppsala. Andrews. 14 (1964) 317–330. C. pp 63–106.and hemoglobin-binding plasma proteins.-C. 2. 6 (1963) 233–244. Dreyer. ceruloplasmin and albumin by gel filtration.J. A theory of gel filtration and its experimental verification. Estimation of serum haemoglobin-binding capacity (haptoglobin) on Sephadex G-100. D. 11. Barlow. Drug-plasma binding measured by Sephadex.. Porath. high resolution methods. Gel filtration: A method for desalting and group separation. Some recently developed fractionation procedures and their application to peptide and protein hormones J. 13. 6. J. (ed. 17 (1964) 676–679. Scheraga. 1962 Flodin.A.. Biochem. Monty. Chem. J.P.. 8. A new calibration procedure for gel filtration columns. 1. Molecular-weight estimation of proteins using Sepharose CL-6B in guanidine hydrochloride. Janson. Janson. Porath. Estimation of molecular size and molecular weights of biological compounds by gel filtration. Locascio. L. London 1970 Andrews.. Gel filtration. Pharm. Arch. 242 (1967) 3237–3238.

177 (1989) 378-382. 1.S. 60 (1971) 167–177. B-L. Ackers. T. Protein-binding of small molecules. T. Biochemistry 16 (1977) 1378–1382. Proc. T.K. Chromatogr.M. et al. Agarose-based media for high-resolution gel filtration of biopolymers. Nat. M. Determination of stoichiometry and equilibrium constants for reversibly associating systems by molecular sieve chromatography.W. Agar derivatives for chromatography. Acad. 113 (1977) 237–251.F. Fruton. Carlsson. Biochem. Fulmer. New gel filtration method. Prep. USA 72 (1975) 1068–1071. 25. Petrovic. The application of gel filtration to the study of protein-binding of small molecules. Giddings (Marcel Dekker.. Colman...E. Reid. McClung. Gonzales. Sci. Rhodes. 46 (1972) 358-363. Scott. Peptide-protein interaction as studied by gel filtration. et al. Effect of sample volume on peak width in high-performance gel filtration chromatography. L. 23.-E. S. J. 37.. Mol.K. Lundström. Hagel. Wood.. Acad. Cooper.F.C. Nyström (eds) (Academic Press.F. Chromatogr. L. M. J. Separation of transfer ribonucleic acid by Sepharose chromatography using reverse salt gradients.K. J. Pharm. Biochemistry 5 (1966) 673–683. G. Clausen. Column lifetime of a new agarose medium for high-performance gel filtration chromatography at basic pH. Binding of sulfonamides to serum proteins: physico-chemical and immuno-chemical studies.A. Janson.. P. A.-C..S. Porath. 330 (1985) 360–364. B-L.-E. A hydrogen exchange method using tritium and Sephadex: its application to ribonuclease.E. G. The agarose double helix and its function in agarose gel structure. H. 32. S. T. Maniatis... Låås. W. Chromatogr.W. Biochem. Desulphated and reduced crosslinked agar and agarose in spherical bead form. 1965) Part 1. R. 29... Properties. Therap. et al. 44. 34. USA 53 (1965) 342–349.A. W. 100 . J.J. 351 (1986) 136–139. Nyström (eds) (Academic Press. G. Wood. et al. Biol.L. Petrovic. 326 (1985) 33–44. 41. Johansson. Determination by gel filtration of association constants for metal-nucleotide interaction. 35. 30. 38. Biochem.C. R. et al. 324 (1985) 422–427. L.F. J. et al. L. C. electrophoresis and gel-bound enzymes. J. Rigby. J. J. Chromatogr. N. J.. G. G. J. Cooper. of novel gel filtration media for standard liquid chromatography. Johansson. Pharmacol. 4 (1974) 509–522.E. Resolution of ribonucleic acids by Sepharose 4B column chromatography. Holmes. 43. Arnott. 27.. M. 36. Sambrook (Cold Spring Harbor 1982) 31. “The Dynamics of Chromatography”. J. J.. 20 (Suppl. 12 (1970) 88–107. Chromatogr.. Biochemistry 2 (1963) 798–807. “Process chromatography. 90 (1974) 269-284.R. A laboratory manual”. Dieckmann.. Hagel. Mol. Englander. Hagel.. in theory and practice. Zeichner. Ellstrom. R. Exp. Sci. Andersson. London 1989) pp 55–66. J. 24. J.. Fritsch. Stern.. G. P. 40.K. Column lifetime of Superose 6 at 37 Celsius and basic pH. Rev. 26. Andersson. Sofer and L. Nat. P. 39. 153 (1966) 167–175. Purification of plasmid DNA by fast protein liquid chromatography on Superose 6 preparative grade. J. J. Thompson. London 1989) pp 36–41.C. Chromatogr.. 33. B...) (1968) 1505– 1565. Fairclough. “Process chromatography. Anal. Proc.F. 476 (1989) 329–344. R. T. “Molecular Cloning. Pharmacol. S. Chromatogr.22. 42. J. Biol. E. R.Y. Jr. A practical guide”. A practical guide”. 28. A new general method for separation of nucleic acids. C. Hurd. J. Anal.. Labeling deoxyribonucleic acid to high specific activty in vitro by nick translation with DNA polymerase I. AAhsberg. Markovic. Sofer and L. Principles and theory.

Laas. Anal. Superdex. C. Y. Le Maire. J. E. Sodium dodecyl sulphate-protein complexes. Schwartz. Universal calibration of gel permeation chromatography and determination of molecular shape in solution. Y. et al.. NICK. Møller.45. Acta 855 (1986) 345–356. MicroPerpex. Chromatogr. P. Biol. Improved preparation of the integral membrane proteins of human red cells. Potschka. Viel. 46. D.. 162 (1987) 47–64. Superloop. J. FPLCmanager. J. Chromatogr. Changes in size or shape below the critical micelle concentration. Pharmacia. Purification and characterization of membrane-bound phospholipase C specific for phosphoinositides from human platelets. Sephacryl. Glass wool as a potential source of artifacts in chromatography. Yada. A. Chem.. 52. Tanford. Size-exclusion chromatography of DNA restriction fragments..A. Y. Ellegren. Griejer. J. 152 (1978) 514–516. 476 (1989) 147–158. 47.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 101 . Fragment length determinations and a comparison with the behaviour of proteins in size-exclusion chromatography. 177 (1989) 50–56. Cardell. HiTrap. Sepharose. Biochem. Molecular characterization of proteins in detergent solutions. HiPrep. Biochem. Sephadex. 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 terms and conditions of sale of the company within the Amersham Pharmacia Biotech group which supplies them.. Chromatogr. Biophys. Biochemistry 13 (1974) 2369–2376. Nozaki. Mascher. S. Banno. Reynolds. T. Size-exclusion chromatography and universal calibration of gel columns. et al. HiLoad. as monitored by high-performance agarose gel chromatography.. Lundahl. E. H. 51. 48. with special reference to the glucose transporter.. BioPilot. J. Lundahl. A copy of these terms and conditions of sale is available on request. 49.. FPLC.. M.P. Nozawa. Anal. Y. J. © Amersham Pharmacia Biotech AB 1998 . NAP. LKB. 263 (1988) 11459–11465.. Biochim. 467 (1989) 217–226. P. M. 50.

17-1453-01 17-5003-01 17-1458-01 17-0771-01 17-1089-01 17-0673-01 17-0674-01 17-0537-01 17-0538-01 17-1047-01 17-1088-01 17-1139-01 17-1140-01 17-1068-01 17-1070-01 17-1069-01 17-1071-01 17-1165-01 17-1194-01 17-1166-01 17-1195-01 17-1167-01 17-1196-01 17-1408-01 17-0591-01 Media Superose 6 prep grade Superose 12 prep grade Superdex 30 prep grade Superdex 30 prep grade Superdex 30 prep grade Superdex 30 prep grade Superdex 75 prep grade Superdex 75 prep grade Superdex 75 prep grade Superdex 75 prep grade Superdex 200 prep grade Superdex 200 prep grade Superdex 200 prep grade Superdex 200 prep grade 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 Sepharose 6B Sepharose 6B Sepharose 4B Sepharose 4B Sepharose 2B Sepharose 2B Sepharose CL-6B Sepharose CL-6B Sepharose CL-4B Sepharose CL-4B Sepharose CL-2B Sepharose CL-2B Sephadex G-10 Sephadex G-10 Sephadex G-10 Sephadex G-15 Sephadex G-15 Sephadex G-15 Pack size Code No.5/300 Superdex Peptide PC 3. Code No.2/30 Superose 6 HR 10/30 Superose 12 HR 10/30 Superdex 75 HR 10/30 Superdex 200 HR 10/30 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 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 HiTrap Desalting (5 columns) Fast Desalting Column HR 10/10 Code No.2/30 Superdex 75 PC 3.2/30 Superdex 200 PC 3. 125 ml 17-0489-01 125 ml 17-0536-01 25 ml 150 ml 1 litre 5 litres 25 ml 150 ml 1 litre 5 litres 25 ml 150 ml 1 litre 5 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 150 ml 750 ml 10 litres 750 ml 1 litre 10 litres 1 litre 10 litres 1 litre 10 litres 1 litre 10 litres 1 litre 10 litres 1 litre 10 litres 100 g 500 g 5 kg 17-0905-10 17-0905-01 17-0905-03 17-0905-04 17-1044-10 17-1044-01 17-1044-02 17-1044-04 17-1043-10 17-1043-01 17-1043-02 17-1043-04 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 17-0110-01 17-0110-05 17-0120-01 17-0120-05 17-0130-01 17-0130-05 17-0160-01 17-0160-05 17-0150-01 17-0150-05 17-0140-01 17-0140-05 17-0010-01 17-0010-02 17-0010-03 Column 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 Qty.2/30 Superose 6 PC 3. 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 17-0862-02 500 g 17-0020-01 5 kg 17-0020-02 500 g 17-0020-02 102 .Ordering information Column Superdex Peptide HR 10/30 Superdex Peptide PE 7.2/30 Superose 12 PC 3.

size 25 g 17-0572-01 100 g 17-0572-02 25 g 17-0573-01 100 g 17-0573-02 25 g 17-0045-01 100 g 17-0045-02 25 g 17-0574-01 100 g 17-0574-02 18-1022-18 Standards Gel Filtration LMW Calibration kit Gel Filtration HMW Calibration kit Blue Dextran 2000 Pack Code No.5 35.1 52.4 20. Gel filtration.9 30.2 61.0 rabbit muscle bovine liver horse spleen bovine thyroid Each Kit contains 50 mg of each protein and 50 mg of Blue Dextran 2000. Low Molecular Weight Gel Filtration Calibration Kit Protein ribonuclease A chymotrypsinogen A ovalbumin albumin Blue Dextran 2000 M Weight 13 700 25 000 43 000 67 000 Stokes’ Radius Å 16. Principles and Methods Pack Code No. size 100 g 500 g 5 kg 100 g 500 g 5 kg 100 g 500 g 5 kg 100 g 5 kg 100 g 500 g 5 kg 100 g 500 g 5 kg 100 g 500 g 5 kg 100 g 5 kg 100 g 500 g 5 kg 100 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 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 17-0050-01 17-0050-02 17-0050-03 17-0051-01 17-0051-03 17-0060-01 17-0060-02 17-0060-03 17-0061-01 17-0061-03 Media Sephadex G-25 DNA Grade SF Sephadex G-50 DNA Grade F Sephadex G-100 DNA Grade M Sephadex G-100 DNA Grade SF Handbook. 103 .Media 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 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 Sephadex G-75 Sephadex G-75 Sephadex G-75 Sephadex G-75 Superfine Sephadex G-75 Superfine Sephadex G-100 Sephadex G-100 Sephadex G-100 Sephadex G-100 Superfine Sephadex G-100 Superfine Pack Code No.0 85.5 Source bovine pancreas bovine pancreas hen egg bovine serum High Molecular Weight Gel Filtration Calibration Kit Aldolase* catalase ferritin* thyroglobulin Blue Dextran 2000 158 000 232 000 440 000 669 000 48. size 1 kit 1 kit 10 g 17-0442-01 17-0441-01 17-0360-01 Contents of the gel filtration calibration kits.

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