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Gel filtration
Principles and Methods
8 th edition
18-1022-18
ISBN 91-97-0490-2-6
Gel filtrationPrinciples and Methods
ContentsIntroduction............................................................................................. 4
Principles ................................................................................................. 6Gel filtration ...................................................................................... 6Gel media .......................................................................................... 6Separation by size ............................................................................... 6Experimental parameters ..................................................................... 7
Sample concepts ........................................................................... 7Column parameters ...................................................................... 7Eluent parameters ......................................................................... 8Running conditions ...................................................................... 8
Characterization of solute behaviour ................................................ 8Theoretical considerations ........................................................... 12Deviations from ideal behaviour in gel filtration ............................. 13
Application examples ..................................................................... 14Fractionation by size ................................................................... 14Separation of monomers from dimers and higher aggregates ............ 16MW estimation, native and other forms ........................................ 17Determination of molecular weight distribution of polymers ............ 18Determination of equilibrium constants ........................................ 19Desalting ................................................................................... 19Industrial applications ................................................................... 21
Properties of gel filtration media ............................................................ 22Sephacryl HR ................................................................................. 22
Chemical and physical properties ................................................. 22Chromatographic properties ........................................................ 24Availability ................................................................................ 27Further information .................................................................... 27
Superdex ......................................................................................... 27Chemical and physical properties ................................................. 27Chromatographic properties ........................................................ 29Availability ................................................................................ 30Further information .................................................................... 30
Superose .......................................................................................... 31Chemical and physical properties ................................................. 31Chromatographic properties ........................................................ 32Availability ................................................................................ 34Further information .................................................................... 34
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Sephadex ......................................................................................... 35Chemical and physical properties ................................................. 35Chromatographic properties ........................................................ 36Availability ................................................................................ 37Further information .................................................................... 38
Sepharose ....................................................................................... 38Chemical and physical properties ................................................. 38Chromatographic properties ........................................................ 40Availability ................................................................................ 41Further information .................................................................... 41
Sepharose CL ................................................................................. 41Chemical and physical properties ................................................. 41Chromatographic properties ........................................................ 42Availability ................................................................................ 43Further information .................................................................... 43
Experimental design............................................................................... 44Performance ................................................................................... 44
Resolution ................................................................................. 44Separation time ........................................................................ 44High performance gel filtration .................................................... 45Capacity .................................................................................... 45Process considerations ................................................................. 47
Choice of gel .................................................................................. 47The purpose of the experiment ..................................................... 48Solute characteristics ................................................................... 51Sample characteristics ................................................................. 51
Choice of running conditions ......................................................... 52Choice of column ....................................................................... 52Flow rate ................................................................................... 54Sample characteristics ................................................................. 55Eluent ....................................................................................... 58Optimization ............................................................................. 58
Performing a gel filtration experiment ................................................... 61Preparing the gel ............................................................................. 61
Pre-swollen media (Sephacryl HR, Superose prep grade,Sepharose CL and Sepharose) ...................................................... 61Media which require swelling (Sephadex G-types) .......................... 61Preparation of Sephacryl HR, Superose prep grade orSepharose CL for use in organic solvents ....................................... 62Gel filtration in organic solvetns ................................................... 63
Packing a column ........................................................................... 63
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Packing Sephadex G types Sepharose and Sepharose CL.................. 63Packing Sephacryl HR and Superose prep grade ............................. 68Adaptors ................................................................................... 70Checking the packed bed ............................................................. 71
Using pre-packed columns .............................................................. 72
Sample application ......................................................................... 72Sample application withour an adaptor ......................................... 72Sample application with an adaptor .............................................. 73
Elution ........................................................................................... 77Flow rates .................................................................................. 78
Cleaning gels and packed columns ................................................. 80General cleaning procedures ........................................................ 80Procedures to remove specific contaminants .................................. 83
Storage of gels and columns ........................................................... 84Prevention of microbial growth .................................................... 84Antimicrobial agents ................................................................... 85Storage of unused media .............................................................. 86Storage of used media ................................................................. 86Storage of packed columns .......................................................... 87
Fault finding chart ................................................................................. 89
References .............................................................................................. 98
Ordering information ............................................................................ 102
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IntroductionFor more than thirty years since the introduction of Sephadex (l,2), gelfiltration has occupied a key position in the purification of thousands ofenzymes, polysaccharides, nucleic acids, proteins and other biologicalmacromolecules. Its continuing value depends partly on the special natureof the macromolecules studied by the biochemist and partly on thereliability and simplicity of gel filtration as a separation technique.
Biological macromolecules form a class of substances with special functionswhich are controlled in vivo by small changes in the environment. Changesin the pH, concentrations of metal ions, cofactors etc may have a profoundeffect on the molecules being studied and it is clearly necessary to haveavailable mild separation techniques which operate independently of thesefactors. Gel filtration is one of these techniques. A gel filtration separationcan be performed in the presence of essential ions or cofactors, detergents,urea, at high or low ionic strength, at 37 °C or in the cold room accordingto the requirements of the experiment. The stability of gel filtration mediafrom Amersham Pharmacia Biotech and their inertness towardsbiopolymers under a wide range of conditions have made them the stan-dard in practically every biochemistry laboratory. No less valuable than itswidespread usefulness, is the reliability and simplicity of gel filtration as anexperimental procedure. Little equipment is required, the procedure isstraightforward and good separations and yields are usually obtained evenin the first experiment. The reliability of gel filtration stems from thereliability of Sephadex and other gel filtration media from AmershamPharmacia Biotech, backed by over 30 years of experience in developingbiochemical separation techniques and by thousands of publicationsdescribing their use. Among the new developments described in this hand-book are Sephacryl HR for standard chromatography and Superdex andSuperose for high speed gel filtration. These new media are the result ofcontinuing efforts of Pharmacia to improve the separation techniqueswhich you need for your work.
This handbook is designed as a laboratory aid in the selection and practicaluse of gel filtration media from Amersham Pharmacia Biotech. For specificseparation problems where the substances in question may have specialproperties, or for theoretical aspects where ideas are undergoing continualevolution, it is essential to refer to the original literature.
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Our aim remains to provide better tools for the life sciences and we believethat to achieve this it is important to maintain close cooperation betweenuser and manufacturer. We hope that this handbook will aid ourcooperation and that you will find the information helpful. Should we atany time be able to give you further information, please contact us.
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Principles
Gel filtrationIn gel filtration molecules in solution are separated according to differencesin their sizes as they pass through a column packed with a chromatographicmedium which is a gel.
Gel mediaA gel is a heterogeneous phase system in which a continuous liquid phase,usually aqueous, is contained within the pores of a continuous solid phase,the gel matrix. In gels made for gel filtration, the pores have a carefullycontrolled range of sizes, and the matrix is chosen for its chemical andphysical stability, and inertness (lack of adsorptive properties).
Gels may be formed from polymers by cross-linking to form athree-dimensional network; for example Sephadex which is formed bycross-linking dextran. Some polymers, like agarose, form gels spontaneou-sly under the appropriate conditions.
Composite gels may be prepared by, for example, grafting a second poly-mer onto a pre-formed matrix. Superdex is such a gel. Dextran chains arecovalently bonded to a highly cross-linked agarose gel matrix. Compositegels are of interest since they can combine valuable properties from morethan one gel-forming system.
Separation by sizeThe pores in the gel matrix which are filled by the liquid phase arecomparable in size to the molecules we may wish to separate. Relativelysmall molecules can diffuse into the gel from a surrounding solution,whereas relatively large molecules will be prevented by their size fromdiffusing into the gel to the same degree. Sufficiently large molecules arecompletely unable to diffuse into the gel and are thus confined to thesolution outside.
In a gel filtration column, gel particles in bead form are packed to form aseparation bed through which a buffer solution, the eluent, is passed.
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Sample molecules which are to be separated are added in solution as a zoneto the top of the bed. The sample zone moves down the bed as eluent isadded to the top. The small molecules which diffuse into the gel beads aredelayed in their passage down the column compared with the largemolecules which cannot diffuse into the gel and move continuously downthe column in the flowing eluent. The large molecules thus leave thecolumn first followed by the smaller molecules in the order of their sizes.
Experimental parameters
Sample conceptsCharacteristics of the sample which are important for the result, apart fromthe solutes which are to be separated, include its volume and viscosity. Thevolume of the sample will influence the size of column which will beneeded, and the viscosity must not be so large as to cause hydrodynamicinstability (see below). It is the viscosity which places an upper limit on thesample concentration which is permissible. Note that the pH, ionic strengthand composition are not significant as long as they do not affect the sizes orstability of the molecules to be separated and are not outside the, wide,stability range of the gel filtration medium.
Column parametersThe most important characteristic of a gel filtration column is the way inwhich the gel filtration medium has been packed. If the column is evenlypacked so the sample zone is not unnecessarily broadened as it passes downthe column then good results can be obtained. If the column is packedunevenly then good results will never be obtained from it.
The length of the column, cm, is significant since it affects both the resolu-tion and the time taken to elute it.
Resolution α √column length
and
Elution time α column length
The volume of the column, ml, is a direct measure of its loadability underotherwise comparable conditions and is chosen depending on the samplevolume.
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Eluent parametersSince the separation depends only on the sizes of the molecules beingseparated, the composition of the eluent is unimportant for the separationmechanism. The eluent can thus be whatever is convenient with regard tothe overall requirements of the experiment. Usually it is a buffer solutionwith a well defined pH and ionic composition chosen to preserve thestructure and biological activity of the substances of interest. An ionicstrength of 0.15 or greater is generally used to avoid any unwanted ionicinteractions between the solute molecules and the gel matrix.
Running conditionsThe experimental variable of significance which remains to be considered isthe rate at which the eluent flows through the column. This affects not onlythe speed at which the separation is obtained but also the resolution whichcan be achieved. Generally speaking, the lower the flow rate the better theresolution, at least for large molecules. Flow rates are measured in simplevolume terms, e.g. ml/min, but when comparing results between columns ofdifferent sizes it is useful to use the linear superficial flow rate, e.g. cm/hour.
Linear superficial flow rate (cm/h) =
Volume flow rate (ml/min) x 60
Cross-sectional area of the column (cm2)
The flow rate defined in this was is usually simply referred to as the linearflow rate. Results obtained at the same linear flow rate will be comparableas far as the effects of flow rate are concerned.
Characterization of solute behaviourResults in gel filtration are typically expressed in the form of an elutiondiagram showing the variation of solute concentration in the eluent withthe volume of eluent passed through the column (Fig. 1). For protein andnucleic acid work and in many other applications continuous detectionusing a UV-monitor (e.g. Pharmacia Monitor UV-l, UV-M II or UvicordS II) and a recorder gives an immediate permanent record, a chromato-gram. From this diagram the elution volume (Ve ) of a given solute can beobtained. Different criteria are used for the determination of elutionvolume (Fig. 2).
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Fig. 1. Gel filtration of anoligosaccharide mixture fromthe acetolysis and hydrolysisof cellulose on SephadexG-25. The numbers abovethe peaks indicate the degreeof polymerisation. Column,4.5x150 cm; eluent, water;flow rate, 1.9 ml.cm-2.h-1
(Flodin, P., Aspberg, K.,Biological Structure andFunction 1 (1961) 345-349.Reproduced by kindpermission of the Authorsand the Publisher).
Fig. 2. Measurement ofelution volume, VeA. Sample size negligible
compared with bedvolume.
B. Sample size not negligiblecompared with bedvolume.
C. Sample giving plateauelution curve.
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• When very small samples are applied (small enough to be neglectedcompared with the elution volume), the position of the peak maximumin the elution diagram should be taken as Ve (Fig. 2A).
• If the sample volume cannot be neglected compared with the elutionvolume, the elution volume is measured from half the sample volume tothe position of the peak maximum (Fig. 2B).
• When very large sample volumes are used (giving a plateau region in theelution curve), the volume eluted from the start of sample application tothe inflexion point (or half height) of the rising part of the elution peakshould be taken as Ve. This criterion is often incorrectly applied tosamples not giving plateau regions. (Fig. 2C).
In gel filtration solutes normally give symmetrical peaks. Elution volumesare, therefore, easily determined by these methods. More sophisticatedcriteria are seldom useful in practice. Ve is not in itself sufficient to definethe behaviour of the sample substance, since this parameter varies with thetotal volume of the packed bed (Vt) and with the way the column has beenpacked. By analogy with other types of partition chromatography theelution of a solute is best characterized by a distribution coefficient (Kd)The volume of the mobile phase is equal to the void volume, Vo, the elutionvolume of molecules which are confined to the mobile phase because theyare larger than the largest pores in the gel. The volume of the stationaryphase, Vs, in gel filtration is equal to Vi, the volume of solvent inside the gelwhich is available to very small molecules, i.e. the elution volume of asolute which will distribute freely between the mobile and stationary sol-vent phases minus the void volume. Thus Kd represents the fraction of thestationary phase which is available for diffusion of a given solute species. Inpractice the volume of the stationary phase defined in this way is ratherdifficult to determine. Methods which can be used often involvemeasurement of the elution volumes of radioactive ions such as 23Na. It ismuch more convenient to substitute the term (Vt-Vo) for Vs, when weobtain
Kav = (Ve - Vo)/(Vt-Vo)
Since (Vt-Vo) includes the volume of the gel forming substance, which isinaccessible to all solute molecules, Kav is not a true partition coefficient(Fig. 3). However, for a given gel there is a constant ratio of Kav:Kd whichis independent of the nature of the solute or its concentration. Kav is easilydetermined and, like Kd, defines solute behaviour independently of the beddimensions and packing. Other methods of normalizing data give valueswhich vary depending upon how well the column is packed. Theapproximate relationships between some of these terms are shown inFigure 4.
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Fig. 3. Diagrammaticrepresentation of Vt and Vo.Note that Vt-Vo will includethe volume of the solidmaterial which forms thematrix. (Fischer, L.Laboratory Techniques inBiochemistry and MolecularBiology. Vol. 1 part II. Anintroduction to GelChromatography. NorthHolland PublishingCompany, Amsterdam.Reproduced by kindpermission of the Authorsand the Publisher).
Fig. 4. Relationship betweenseveral expressions used fornormalizing elutionbehaviour.
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Theoretical considerationsThe use of gel filtration for the determination of molecular weight or size,particularly of proteins, is well documented (3). In practice it is found thatfor a series of compounds of similar molecular shape and density asigmoidal relationship exists between their Kav values and the logarithmsof their molecular weights (MW). Calibration curves constructed in thisway for a particular gel type are often termed selectivity curves (see p 25,30, 33, 34, 37, 40), and over a considerable range there is a convenientlylinear relationship between Kav and log MW. In ideal gel filtrationbehaviour no molecules can be eluted with a Kav greater than 1 or less than0. If the Kav is greater than 1, some kind of adsorption is indicated. If theKav is less than 0 after calibration then channelling in the chromatographicbed is indicated and the column must be repacked.
Several models have been proposed to describe the behaviour of solutesduring gel filtration (3, 4). Most have regarded the partition of solutemolecules between the gel particles and surrounding fluid as an entirelysteric effect. Thus Flodin (5) divided the gel into permitted and forbiddenregions. The larger the molecular dimensions of the solute the greater is theproportion of the gel which is forbidden. In the permitted region the con-centration of solute is assumed to be identical to that in the surroundingliquid.
The steric approach has been extended in various ways. Porath (6) deriveda theoretical relationship between Kd and Stokes radius assuming that thepores in Sephadex are conical. In another treatment Squire (7) consideredpores and crevices as well as cones. An interesting approach by Laurent andKillander (8) assumes that the gel network is composed of rigid rodsrandomly arranged. Good correlation was found between Kav andmolecular radius with this model.
All of these models have been successfully applied to predict the elutionbehaviour of solutes. However, as has been pointed out by Ackers (9), noneof them may be accurate in a structural sense. Results are equally inaccordance with a formulation where the fractions of the stationary phaseavailable to molecules of different radii are defined by a Gaussianprobability curve and no assumption is made about the geometric shape ofthe pores. From a practical stand-point, for molecular weight determina-tion, it is still most common to construct a calibration curve and performthe estimations graphically.
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Deviations from ideal behaviour in gel filtrationIn ideal gel filtration the only effects which contribute to the behaviour ofsolute molecules are steric effects. Insensitivity of gel filtration to thecomposition of the eluent is a major advantage and is observed in the vastmajority of cases in aqueous systems at neutral pH in the presence ofelectrolyte. However, deviation from a Kav:log MW calibration curve maystill occur if a compound being studied does not have the same molecularshape as the standards. Under certain conditions, factors other than the sizeand shape of the molecules being studied can influence the separation.These effects can usually be avoided and are generally only significant whenchromatographing highly acidic or basic substances at low ionic strength oraromatic materials on those gels which have a high matrix content. Oftenthey have proved highly advantageous. Thus an important application ofSephadex types G-10, G-15 and G-25 is in the separation of aromaticpeptides and other substances which may differ only slightly in molecularweight. Further details of these effects can be found under thechromatographic properties of the different gels (see pages 22–43).
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Application examples
Fractionation by sizeThe special ability of gel filtration to separate macromolecules on the basisof small differences in size between them makes it an essential fractionationtool. Here the correct choice of gel and operating conditions are critical ifgood results are to be obtained. The peptide separation shown in Figure 5
Fig. 5. Gel filtration of a tryptic digest of citraconylatedtotally reduced and 14C alkylated Fd' fragments of IgG3 onSephadex G-50 Fine. Column, 1x96 cm; eluent, 10 % formicacid. (Michaelsen, T.T., Frangione, B., Franklin, E.C., J. Biol.Chem. 252 (1977) 883-889: Reproduced by kind permissionof the Authors and the Publisher).
illustrates this type of experiment. With the proper experimental design, aprotein can be separated from other species differing in molecular weightby a factor of two or slightly less (Fig. 6). Practical simplicity, excellentrecovery, free choice of elution conditions and straight forward interpreta-
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tion of results make fractionation by gel filtration an invaluable part of anypurification scheme. Details about experimental design are given on p 44–60. Practical details are given on p 61–87.
Fig. 6. Separation of IGF-1 (MW 7 600) from its fusion partner ZZ (MW 14 500) anduncleaved fusion protein on Superdex 75 HR 10/30. The secreted fusion protein, ZZ-IGF-1,was initially purified by affinity chromatography on IgG-Sepharose Fast Flow andsubsequently cleaved with hydroxylamine (2 M, pH 9.2).Sample concentration, 5 mg/ml; sample volume, 100 µl; eluent, ammonium acetate (0.25 M,pH 6.0); flow rate, 0.5 ml/min. (Work by Pharmacia LKB Biotechnology, Uppsala, Sweden,in collaboration with KabiGen, Stockholm, Sweden.)
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Separation of monomers from dimers andhigher aggregates
It is frequently found that proteins with a high degree of homogeneity withrespect to the protein species present contain dimers and higher aggregates.High resolution gel filtration provides an excellent and gentle means forseparating the monomer from the aggregates, either as part of an analysisor for their final purification. Gel filtration is thus especially useful as apolishing step in a more complex purification scheme and finds widespreaduse for this purpose in the purification of recombinant and other proteinsin industrial processes (Fig. 7) where protein aggregates must be reduced tobelow a specified level in the final product.
Fig. 7. Final purification of a mouse monoclonal antibody, IgG2a, on Superdex 200prep grade.Hybridoma cell culture supernatant was concentrated by ultrafiltration and clarified bycentrifugation and passage through a 0.22 µm filter. The concentrate was desalted andpurified initially by cation exchange on BioPilot Column S Sepharose HighPerformance 60/100.Peaks, 1. IgG dimers; 2. IgG monomers; 3. transferrin; column, BioPilot Superdex 200prep grade 60/600; sample concentration, 11.37 mg/ml; sample volume, 50 ml; eluent,PBS (pH 7.5) containing sodium azide (0.05 %); flow rate, 14 ml/min. Work byPharmacia LKB Biotechnology, Uppsala, Sweden.
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MW estimation, native and other formsA calibrated column of Sephacryl HR, Superdex, Superose, Sephadex orSepharose provides a simple and well-documented way of determining themolecular weights of proteins during a natural stage in their purification(10–14).
Unlike electrophoretic techniques, gel filtration provides a means ofdetermining the molecular weight or size (Stokes radius) of native ordenatured globular proteins under a wide variety of conditions of pH, ionicstrength, temperature etc., freeing the researcher from the constraintsimposed by the charge state of the molecules. Gel filtration in the presenceof urea or guanidine hydrochloride, dissociating agents which transformpolypeptides and proteins to a random coil configuration reducingstructural differences, has proved particularly useful for molecular weightdeterminations. Figure 8 illustrates this kind of application.
Fig. 8. Gel filtration of proteins on Sepharose CL-6B underdenaturing conditions.Peaks, 1. Blue Dextran; 2. bovine serum albumin; 3. rabbitIgG H chain; 4. α-chymotrypsinogen; 5. cytochrome c; 6.insulin; 7. B chain of insulin; 8. DNP-ala: Column, 1.5x90cm: Eluent, 6 M guanidine-HCI, 0.1 m sodium phosphate,pH 8.0; flow rate, 6.8 ml.cm-2.h-1. (Ansari, A.A., Mage,R.G., J. Chromatogr. 140 (1977) 98-102. Reproduced bykind permission of the Authors and the Publisher).
Gel filtration also eliminates the need to set up a separate experiment foreach determination as the calibrated column can be used for extendedperiods, both for molecular weight determination and for routineseparations. In the experiment depicted in Figure 8, for example, Ansariand Mage (14) used the same column packed with Sepharose CL-6B for
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molecular weight determination in 6M guanidine hydrochloride over aperiod of 10 months, without any change in the elution pattern.The only special requirements for molecular weight determination are acolumn packed with the appropriate gel and a series of protein standards tocalibrate it. Gel Filtration Calibration Kits HMW and LMW fromPharmacia provide a series of well characterized globular protein standardscarefully chosen to give reliable calibration points in the molecular weightrange 13 700 to 669 000 (see p 87–88). An elution profile obtained withSephadex G-200 Superfine is shown in Figure 9.
Fig. 9. Calibration proteins being used to calibrate SephadexG-200 Superfine.Peaks, 1. catalase; 2. aldolase; 3. bovine serum albumin; 4.ovalbumin; 5. chymotrypsinogen A; 6. ribonuclease A:column, K 26/70: eluent, 0.05 M potassium phosphate, pH6.8, containing 0.1 M NaCI and 2 % sodium azide. (Workby Pharmacia LKB Biotechnology, Uppsala, Sweden.
Determination of molecular weight distribu-tion of polymers
The molecular weight distribution is very important for characterization ofnatural and synthetic polymers. Distribution analysis by classical methodsis difficult and tedious as it involves fractionation of the macromolecules byprecipitation and determination of molecular weight and amount ofsubstance in every fraction.
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The ability of gel filtration to fractionate molecules according to molecularsize offers an improved method for distribution analysis of many polymers(15). The elution curve can be recorded continuously or determined byinvestigating individual fractions. It is unnecessary to determine molecularweights of the polymers in the effluent fractions of individual experiments,as the chromatographic behaviour of the columns is very reproducible. Acalibration curve once determined for the column can be applied to a largenumber of runs. The precision of this method of distribution analysis isvery high (16). The method is used for the determination of molecularweight distributions of e.g. dextrans, polyvinylpyrrolidones, gelatinpreparations and other water-soluble polymers (3).
Determination of equilibrium constantsGel filtration has proved to be a valuable technique for the study ofchemical equilibria. In the case of slow reactions, where the reactants andthe products can be separated on a gel filtration column, these substancescan be quantitatively determined in the effluent, thereby establishing theposition of the equilibrium (17–19).
Gel filtration can also be used to determine the position of equilibrium ofcomplex formation where the reactions are rapid. In this case, one of thereactants is chromatographed in an eluent containing the other reactant.From the elution curve of the reactants the complex formation can bestudied. This method is of great value in the study of protein binding of lowmolecular weight substances such as drugs (20–26) and can be extended tothe study of, for instance, competition of two molecules for the same site(22). Gel filtration has also been applied to the estimation of reaction rates(27, 28).
DesaltingSince proteins and other biomacromolecules differ greatly in size from saltsand other small molecules, gel filtration is particularly efficient for manyeveryday laboratory operations including:
• buffer exchange. Enzymic reactions, assays and other analyticalprocedures require that the sample be adjusted to the proper ionicconditions; this is most efficiently done by gel filtration. Gel filtration isthe most efficient way to adjust the ionic composition of a sample to therequired species and concentration prior to ion exchangechromatography, hydrophobic interaction chromatography, affinity
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chromatography and other LC techniques which use an aqueous mobilephase.
• phenol removal from preparations of nucleic acids.• removal of unincorporated nucleotides during DNA sequencing (29, 30).• removal of free low molecular weight labels, e.g. 125I, FITC, from
solutions of labelled proteins (Fig. 10).
• termination of reactions between macromolecules and low molecularweight reactants.
• removal of products, cofactors, inhibitors etc. from enzymes.• phenol red removal from culture fluids prior to anion exchange
chromatography.
Fig. 10. Removal of free 125land Chloramine T fromlabelled albumin.Column, Fast DesaltingColumn HR 10/10; sampleconcentration, 0.5 mg/mlhuman serum albumin;sample volume, 350 µl;eluent, sodium phosphate(0.05 M, pH 7.4) containingTween 20 (0.05 %); flowrate, 4 ml/min. (Work byPharmacia LKBBiotechnology, Uppsala,Sweden).
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Industrial applicationsThe advantages of gel filtration for the separation of biological moleculescan be equally well realized in large-scale applications as they are in theresearch laboratory. In particular, scale-up from pilot-plant to productioncapacity has proved a straightforward and trouble-free operation.Pharmacia has built up considerable experience in this area and you areinvited to contact us directly should you require further information.
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Properties of gel filtrationmedia
Sephacryl HRChemical and physical properties
Sephacryl High Resolution (HR) is a composite gel prepared by covalentlycross-linking allyl dextran with N,N'-methylene bisacrylamide to form ahydrophilic matrix of high mechanical strength (Fig. 11).
Fig. 11. Hypothetical partial structure of Sephacryl HR.
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The porosity of the gel is controlled by the dextran component to give fivetypes with different fractionation ranges. The wet bead diameter is between25–75 µm, with an average bead diameter of approximately 50 µm.Sephacryl HR types are supplied pre-swollen and ready to use. Table 1summarizes some of the properties of Sephacryl HR.
Table 2. Bed volumes of Sephacryl HR-types in common organic solvents starting from100 ml sedimented gel.
Gel type Formamide DMSO Methanol Ethanol Acetone
Sephacryl S-100 HR 110 100 100 100 85Sephacryl S-200 HR 115 110 100 100 85Sephacryl S-300 HR 100 90 100 95 85Sephacryl S-400 HR 100 90 100 95 85Sephacryl S-500 HR 100 95 100 100 90
Table 1. Properties of Sephacryl HR
Gel type Bead size Fractionation range Fractionation range Exclusionµm Globular proteins Dextrans limit
DNA
Sephacryl S-100 HR 25 – 75 1 000 – 100 000 ND NDSephacryl S-200 HR 25 – 75 5 000 – 250 000 1 000 – 80 000 118Sephacryl S-300 HR 25 – 75 10 000 – 1 500 000 2 000 – 400 000 118Sephacryl S-400 HR 25 – 75 20 000 – 8 000 000 10 000 – 2 000 000 271Sephacryl S-500 HR 25 – 75 ND 40 000 – 20 000 000 1078
Chemical stabilitySephacryl HR is stable in all aqueous buffers commonly used inbiochemistry within the pH range 3–11. Short term exposure, e.g. forcleaning, to pH´s in the range 2-13 has no adverse effect on subsequentchromatographic properties. The separation properties of the gel are notaffected by detergents e.g. 1% SDS, chaotropic salts or dissociating agentse.g. 8 M urea and 6 M guanidine HCI.
Strong solutions of NaOH (0.5M) can be used for cleaning if the gel iswashed immediately afterwards with buffer or water. Sephacryl HR canalso be used with organic solvents. A method for transferring Sephacryl HRfrom water to organic solvents is described on page 62. Table 2 lists the gelvolumes in various organic solvents for an original 100 ml of gelsedimented in water.
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Physical stabilitySephacryl HR may be autoclaved repeatedly at 121 °C, pH 7 for 30 minu-tes without significantly affecting its chromatographic properties.
Typical flow rates for a 60 cm long bed can be seen in the pressure/flowdiagram in Fig. 12.
Fig. 12. Pressure drop as a function of flow rate for Sephacryl HR. Bed heightapproximately 60 cm; eluent distilled water; temperature 25 °C. To calculate the volumetricflow rate, multiply the linear flow rate by the cross-sectional area of the column (2 cm2 forXK 16 or 5.3 cm2 for XK 26).
Columns of all dimensions, including wide diameter production columnswith total bed heights of 60-100 cm, can be packed and equilibratedsuccessfully at high flow rates, resulting in short overall separation times(31). Likewise, the mechanical rigidity of Sephacryl HR allows evenrelatively viscous eluents, such as 8 M urea, to be run at practicable flowrates.
Chromatographic properties
SelectivityThe five Sephacryl HR types have different fractionation ranges. SephacrylS-100 HR is excellent for gel filtration of peptides and small proteins in themolecular weight range 1 x 103–100 x 103. When fractionating proteins inthe molecular weight range 5 x 103 –250 x 103 or 10 x 103 –1.5 x 106,
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including monoclonal antibodies and serum proteins, use Sephacryl S-200HR or S-300 HR respectively. Sephacryl S-400 HR and S-500 HR are thechoice for separation of larger proteins, polysaccharides, nucleic acids,DNA fragments and small particles e.g. plasmids.
Table 1 gives the fractionation ranges for Sephacryl HR. Figure 13 showsthe selectivity curves for globular proteins and dextrans respectively.
Fig. 13. Selectivity curves for Sephacryl HR in phosphate buffer (0.05 M, pH 7.0)containing NaCl (0.15 M).
The narrow particle size distribution of Sephacryl HR allows columns to bepacked easily and with high efficiency giving > 9000 plates per metre if thepacking instruction is followed.
A typical example of results obtainable with Sephacryl HR is shown inFigure 14.
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AdsorptionTests in our laboratories have shown that Sephacryl S-100 HR gives yieldsof at least 96% (UV absorption at 280 nm, using 0.05 M phosphate, 0.15 M NaCl, pH 7.0) with the following model substances: Blue Dextran2000, ferritin, catalase, aldolase, BSA, ovalbumin, β-lactoglobulin A+B,chymotrypsinogen A, myoglobin, Iysozyme, ribonuclease A andcytochrome c. For the best results eluents with an ionic strength of at least0.15 should be used.
FPLC and industrial scaleColumns packed with Sephacryl can be used with with standardchromatography instrumentation and with FPLC system. Its high resolutionand high chemical stability make Sephacryl HR ideally suited for use inindustry, both in pilot and in process scale. The gels provide rapid cycletimes and can be sanitized in place which is of particular advantage.
Fig. 14. Separation of integral membrane proteins fromhuman erythrocytes on Sephacryl S-300 HR.Human erythrocyte membrane were solubilized in phosphatebuffer (0.1 M, pH 7.4) containing SDS (0.1 M), EDTA (1mM), DTE (1 mM).Peaks, 1. dimeric anion transporter protein; 2. monomer ofthe anion transporter protein; 3. dimeric glycophorin A; 4.glucose transporter protein; 5. possibly lipids and SDS;column, K 26/70; bed height, 61 cm; sample concentration, 2mg/ml; sample volume, 2 ml; eluent, phosphate buffer (0.1 M,pH 7.4) containing SDS (0.05 M), EDTA (1 mM), DTE (1mM); flow rate, 58 ml/hour. (Work by E. Greijer and P.Lundahl, Institute of Biochemistry, University of Uppsala,Uppsala, Sweden.)
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AvailabilitySephacryl HR types are supplied pre-swollen as a suspension containing20% ethanol in packs of 750 ml. Sephacryl S-100, S-200 and S-300 arealso supplied pre-packed in XK-columns with the designation HiLoadcolumns.
Further informationFurther information on Sephacryl HR is given in the data sheet: “SephacrylHigh Resolution gel filtration media” with details of applications. Forinformation on scale-up and the operation of large scale chromatographysystems, please contact Pharmacia.
SuperdexChemical and physical properties
Superdex is based on highly cross-linked porous agarose beads to whichdextran has been covalently bonded. Superdex is thus a composite gel (Fig.15) in which the high physical and chemical stabilities are chiefly due to theagarose matrix and the gel filtration properties are principally determinedby the dextran chains.
Different types with different fractionation ranges are available. Superdex30 prep grade, Superdex 75 prep grade and Superdex 200 prep grade have
Fig. 15. Structure ofSuperdex. Dextran chains arecovalently linked to a highlycross-linked agarose matrix.
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an average wet bead diameter of 34 µm with a range of 24–44 µm.Superdex peptide, Superdex 75 and Superdex 200 for high performance gelfiltration have a bead size of 13 µm (mean). Table 3 summarizes some ofthe properties of Superdex and Superdex prep grade; HiLoad columns canbe used both with standard chromatography systems and with FPLC sys-tem. Superdex packed in HR columns is for use with FPLC system.
Chemical stabilitySuperdex can be used with all aqueous buffers commonly used inbiochemistry within the pH range 3–12, and withstands strong bases, e.g.0.2 M NaOH, and acids, e.g. 0.01 M HCI or 0.1 M acetic acid. Short termexposure to extremes of pH (1-14) e.g. for cleaning, has no adverse effecton subsequent chromatographic properties. The separation properties ofthe gel are not affected by detergents, e.g. 1% SDS, chaotropic salts ordissociating agents, e.g. 8 M urea or 6 M guanidine HCI.
Strong solutions of NaOH (0.5 M) or HCl (0.5 M) can be used for cleaningif the gel is immediately washed with buffer or water. The high stability ofSuperdex prep grade makes it very suitable for use in industrial processeswhere high flow rates and effective cleaning-in-place are required.
Superdex can also be used with organic solvents. A method for transferringSuperdex from water to organic solvents is described on page 62.
Physical stabilityColumns pre-packed with Superdex may be used at temperatures in therange +4–40 °C. Exposure to temperatures outside this range will destroythe efficiency of the packed bed and the column will need to be re-packed.
Typical flow rates for a bed 60 cm long packed with Superdex prep grade
Table 3. Properties of Superdex
Gel type Bead size µm Fractionation range Fractionation rangeGlobular proteins Dextrans
Superdex peptide 11 – 15 100 – 7 000 –Superdex 30 prep grade 24 – 44 – 10 000 –Superdex 75 prep grade 24 – 44 3 000 – 70 000 500 – 30 000Superdex 75 11 – 15 3 000 – 70 000 500 – 30 000Superdex 200 prep grade 24 – 44 10 000 – 600 000 1 000 – 100 000Superdex 200 11 – 15 10 000 – 600 000 1 000 – 100 000
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Fig. 16. Pressure drop as a function of flow rate for HiLoadcolumns packed with Superdex prep grade. Bed heightapproximately 60 cm in distilled water at 25 °C. To calculatevolumetric flow rate, multiply linear flow rate by cross-sectionalarea of column (2 cm2 for XK16, 5.3 cm2 for XK26).
can be seen in the pressure/flow diagrams in Figure 16.Superdex can be equilibrated at flow rates which are much higher thanthose recommended for the best resolution, resulting in short overall cycletimes. Similarly, its mechanical rigidity allows even relatively viscouseluents, such as 8 M urea, to be run at practicable flow rates.
Chromatographic properties
EfficiencyHiLoad Superdex prep grade columns are supplied packed to an efficiencyof >13 000 plates/metre. Superdex 75 and Superdex 200 HR 10/30columns are supplied with an efficiency of > 30 000 plates/metre.
SelectivityThe fractionation ranges of Superdex 75 and Superdex 200 correspondclosely to the fractionation ranges of Sephadex G-75 and Sephadex G-200respectively (Fig. 17). Superdex 30 prep grade is recommended forseparations of peptides, oligonucleotides and small proteins in themolecular weight range up to 10000. Superdex 75 is particularly suited for
30
Fig. 17. Selectivity curves for Superdex.
the separation of a wide range of recombinant DNA products. Superdex200 prep grade is a suitable choice when the molecular weight of the pro-tein of interest is unknown. It is especially suitable for the separation ofmonoclonal antibodies from dimers and from contaminants of lowermolecular weight, for example albumin and transferrin.
AdsorptionNon-specific interactions between proteins and Superdex are negligibleunder normal chromatographic conditions using buffer solutions with ionicstrengths in the range 0.15 to 1.5. At very low ionic strengths, the presenceof a small number of negatively charged groups leads to retardation ofbasic proteins and exclusion of acidic proteins.
AvailabilitySuperdex prep grade is supplied in packs of 150 ml, 1 litre, 5 litres or pre-packed in HiLoad and larger BioPilot columns. Superdex with a bead sizeof 13 µm is supplied pre-packed in HR 10/30 columns.
Further informationFurther information on Superdex with details of applications is given in thetechnical brochure “HiLoad columns”. For information on scale-up andthe operation of large scale chromatography systems, please contactPharmacia.
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SuperoseChemical and physical properties
Superose (32) is composed of highly cross-linked porous agarose beads intwo different particle sizes and two different fractionation ranges.The average wet bead diameter is 10 ±2 µm or 13 ±2 µm for Superose 6and Superose 12 respectively, and 20–40 µm for the corresponding prepgrades. Table 4 summarizes some of the properties of Superose.
Table 4. Properties of Superose
Gel type Bead size µm Fractionation range Fractionation rangeGlobular proteins Dextrans
Superose 12 prep grade 20 – 40 1 000 – 300 000 NDSuperose 12 8 – 12 1 000 – 300 000 NDSuperose 6 prep grade 20 – 40 5 000 – 5 000 000 NDSuperose 6 11 – 15 5 000 – 5 000 000 ND
Chemical stabilitySuperose can be used with all aqueous buffers commonly used inbiochemistry within the pH range 3–12. Short term exposure to extremesof pH (1–14) e.g. for cleaning, has no adverse effect on subsequentchromatographic properties and the media withstand strong bases (33, 34)e.g. 0.2 M NaOH, and acids e.g. 0.01 M HCI and 1 M acetic acid.
The separation properties of the gels are not affected by detergents e.g. 1%SDS, chaotropic salts or dissociating agents e.g. 8 M urea and 6 Mguanidine HCl.
Strong solutions of NaOH (0.5 M) or HCl (0.5 M) can be used for cleaningif the gel is immediately washed with buffer or water. Superose can also beused with organic solvents. Superose prep grade may be transferred fromwater to organic solvents by the method described on page 62.
Physical stabilitySuperose prep grade may be autoclaved repeatedly at 121 °C, pH 7 for 30minutes without significantly affecting its chromatographic properties. Pre-packed columns must not be exposed to temperatures outside the range+4 –40 °C. Exposure to temperatures outside this range will destroy theefficiency of the packed bed .
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Recommended flow rates for Superose prep grade are in the range 0.3–0.5ml/min for a column 16 mm i.d., 50 cm long. Pressure/flow diagrams forSuperose 6 HR 10/30 and Superose 12 HR 10/30 columns are shown infigure 18.
Fig. 18. Typical pressure-flow relationships for Superose 6 and Superose 12 HR 10/30columns in aqueous buffer solutions at room temperature.
The mechanical rigidity of Superose prep grade allows even relativelyviscous eluents, such as 8 M urea, to be run at practicable flow rates.
Chromatographic properties
EfficiencySuperose 6 HR10/30 and 12 HR10/30 columns are supplied packed to anefficiency of >30 000 plates/metre.
SelectivityGels with two different fractionation ranges are available, Superose 6 andSuperose 12, with selectivity curves as shown in figures 19, 20 and 21. Theselectivities of the corresponding prep grades are the same.
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Fig. 19. Selectivity curves of Superose 6 and 12, globularproteins.
Fig. 20. Selectivity curves of Superose 6 and 12, dextrans.
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Fig. 21. Selectivity curvesof Superose 6 and 12,DNA-fragments.
AdsorptionIonic interactions between solutes and Superose are negligible at ionicstrengths above 0.15 M. Some hydrophobic interactions have beenrecognized, i.e. some compounds (e.g. smaller hydrophobic and/or aromaticpeptides, membrane proteins or lipoproteins) may be eluted later thanpredicted (these interactions can be of considerable value to the resolution).The degree of interaction is less for Superose prep grade than on pre-packed Superose and Superose 6 shows weaker hydrophobic effects thanSuperose 12.
AvailabilitySuperose prep grades are available pre-swollen in packs of 125 ml. Supe-rose 6 HR 10/30 and Superose 12 HR 10/30 columns are supplied pre-packed.
Further informationFurther information on Superose with details of applications is given in thedata file: “Superose 6, Superose 12”. For information on scale-up and theoperation of large scale chromatography systems, please contact PharmaciaBiotech.
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Fig. 22. Partial structure of Sephadex.
Sephadex swells in aqueous solutions, and in dimethylsulphoxide andformamide. Dimethylformamide may be used with Sephadex G-10 and G-15 and mixtures of water with the lower alcohols may be used withSephadex G-10, G-15, G-25 and G-50. It should be noted that the degreeof swelling in organic solvents or their mixtures will not be the same as inwater alone. Sephadex LH-20 (described in a separate booklet), SephacrylHR and Sepharose CL are recommended for gel filtration in organicsolvents.
SephadexChemical and physical properties
Sephadex is a bead-formed gel prepared by cross-linking dextran withepichlorohydrin (Fig. 22). Table 5 lists the different G-types of Sephadexand their physical properties.
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Table 5. Properties of Sephadex.
Fractionation Fractionation rangeGel type Dry bead size range Dextrans Swelling
µm Globular factor ml/gproteins
Sephadex G-10 40 – 120 – 700 – 700 2 – 3Sephadex G-15 40 – 120 – 1 500 – 1 500 2.5 – 3.5Sephadex G-25 Coarse 100 – 300 1 000 – 5 000 100 – 5 000 4 – 6Sephadex G-25 Medium 50 – 150 1 000 – 5 000 100 – 5 000 4 – 6Sephadex G-25 Fine 20 – 80 1 000 – 5 000 100 – 5 000 4 – 6Sephadex G-25 Superfine 10 – 40 1 000 – 5 000 100 – 5 000 4 – 6Sephadex G-50 Coarse 100 – 300 1 500 – 30 000 500 – 10 000 9 – 11Sephadex G-50 Medium 50 – 150 1 500 – 30 000 500 – 10 000 9 – 11Sephadex G-50 Fine 20 – 80 1 500 – 30 000 500 – 10 000 9 – 11Sephadex G-50 Superfine 10 – 40 1 500 – 30 000 500 – 10 000 9 – 11Sephadex G-75 40 – 120 3 000 – 80 000 1 000 – 50 000 12 – 15Sephadex G-75 Superfine 10 – 40 3 000 – 70 000 1 000 – 50 000 12 – 15Sephadex G-100 40 – 120 4 000 – 150 000 1 000 – 100 000 15 – 20Sephadex G-100 Superfine 10 – 40 4 000 – 100 000 1 000 – 100 000 15 – 20Sephadex G-150 40 – 120 5 000 – 300 000 1 000 – 150 000 20 – 30Sephadex G-150 Superfine 10 – 40 5 000 – 150 000 1 000 – 150 000 18 – 22Sephadex G-200 40 – 120 5 000 – 600 000 1 000 – 200 000 30 – 40Sephadex G-200 Superfine 10 – 40 5 000 – 250 000 1 000 – 150 000 20 – 25
Chromatographic propertiesThe G-types of Sephadex differ in their degree of cross-linking and hence intheir degree of swelling and fractionation range (Fig. 23 and 24).
Sephadex is available in different particle size grades. The different gradesgive chromatographic beds with different efficiencies and operatingpressures. The highest efficiencies, and operating pressures, are obtainedwith the Superfine grade. The Superfine grade is also suitable for thin layergel filtration. The Fine grade is recommended for preparative purposes. TheCoarse and Medium grades are intended for preparative chromatographicprocesses where a high flow rate at a low operating pressure is essential. Inaddition the Coarse grade is suitable for batch procedures.Sephadex G-10, G-15, G-25 and G-50 are recommended for separations ofpeptides and other small biomolecules. Sephadex G-75, G-100, G-150 andG-200 are useful in work with proteins and other macromolecules wheretheir relatively poor physical stability is not a hindrance. The DNA grade ofSephadex G-25, G-50 or G-100 should be used for work with DNA oroligonucleotides.
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Fig. 23. Selectivity curves of Sephadex G-types Superfine,globular proteins.
Fig. 24. Selectivity curves of Sephadex G-types, globularproteins.
AvailabilityAll Sephadex G-types are supplied as dry powders in 100 g and 500 gpacks. In addition: Sephadex G-25 Superfine is supplied pre-packed in FastDesalting Column HR 10/10; Sephadex G-25 Medium is suppliedpre-packed in disposable PD-10 Columns; Sephadex G-25 DNA grade issupplied pre-packed in disposable NAP Columns; and Sephadex G-50DNA grade is supplied pre-packed in disposable NICK Columns and NICKSpin Columns.
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Further informationFurther information on Sephadex with details of applications is given in thedata sheet: “Sephadex gel filtration media”. For information on scale-upand the operation of large scale chromatography systems, please contactPharmacia.
SepharoseChemical and physical properties
Sepharose is a bead-formed gel prepared from agarose. In its natural stateagarose occurs as part of the complex mixture of charged and neutralpolysaccharides referred to as agar. The agarose used to make Sepharose isobtained by a purification process which removes the chargedpolysaccharides to give a gel with only a very small number of residualcharged groups.
A gel forms spontaneously as a hot solution of agarose is cooled. Theindividual polysaccharide chains form double helices which subsequentlyaggregate to form bundles during the formation of a stable gel (Fig. 25).
The individual polysaccharide chains may be represented as polymers of therepeating unit shown in Figure 26.
Fig. 25. Gel structure of agarose. (Låås, T. Doctoral thesis. Acta Universitatis Upsaliensis1975. Reproduced by kind permission of the Author.)
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Fig. 26. Structure of the repeating unit of agarose. Note the presence of the unusual sugar3,6-anhydro-L-galactose.
Chemical stabilityAlthough the gel structure of Sepharose is stabilized by hydrogen bondingand not by covalent cross-links, it can still be used under most of theconditions encountered in gel filtration. It is stable in water and saltsolutions over the pH range 4–9 and in the absence of oxidizing agents.Sepharose may be used for long periods in dissociating media, such asguanidine hydrochloride and urea, but for the best results Sepharose CL(cross-linked Sepharose, page 41) is recommended for these applications.Chaotropic salts, such as KSCN, should be avoided, but may be used withSepharose CL.
Physical stabilitySepharose melts on heating above 40 °C and the bead structure may beirreversibly damaged on freezing. Consequently Sepharose cannot besterilized by autoclaving, but it may be sterilized chemically, for example,by treatment with diethylpyrocarbonate.
Table 6. Properties of Sepharose.
Gel type Approx. % Bead size Fractionation range Fractionation rangeagarose µm Globular proteins. Dextrans
Sepharose 6B 6 45 – 165 10 000 – 4 000 000 10 000 – 1 000 000Sepharose 4B 4 45 – 165 60 000 – 20 000 000 30 000 – 5 000 000Sepharose 2B 2 60 – 200 70 000 – 40 000 000 100 000 – 20 000 000
Sepharose is available in three types with different agarose concentrationsand different fractionation ranges (Table 6).
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The mechanical strength of Sepharose depends on the agarose concent-ration in the beads. Thus Sepharose 6B, (approx. 6% agarose) is considera-bly stronger than Sepharose 2B (approx. 2% agarose). Practical details onflow rates obtainable with Sepharose are given on page 66.
Chromatographic properties
SelectivityThe fractionation ranges for the different types are shown in Table 6 andFigure 27 shows the selectivity curves. The absence of suitable standards
makes it difficult to estimate the exclusion limits for proteins withconfidence and these figures should be taken as a guide only.
AdsorptionSepharose contains a very small number of sulphate and carboxyl groupswhich may cause adsorption of basic proteins at low ionic strengths. Theseeffects can be eliminated by using eluents with an ionic strength exceeding0.15 and effects due to protein adsorption are seldom encountered inpractice. Certain nucleic acids can be separated on Sepharose and thisforms the basis of a number of schemes for their purification. For exampletRNA species may be resolved on Sepharose 4B in high concentrations ofammonium sulphate (35). Similarly DNA, tRNA and high molecularweight RNA from a variety of species may be separated on Sepharose in1.5 M NaCl (36).
Fig. 27. Selectivity curves for Sepharose and Sepharose CL,globular proteins.
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AvailabilitySepharose is supplied as a ready-to-use suspension in 1 litre packscontaining 20% ethanol as a preservative.
Further informationFurther information on Sepharose with details of applications is given inthe data sheet: “Sepharose and Sepharose CL gel filtration media”. Forinformation on scale-up and the operation of large scale chromatographysystems, please contact Pharmacia.
Sepharose CLChemical and physical properties
Sepharose CL is prepared from Sepharose by reaction with 2,3dibromopropanol under strongly alkaline conditions. This produces across-linked agarose gel with substantially the same porosity as the parentgel, but with greatly increased thermal and chemical stability. Aftercross-linking, the gel is desulphated by alkaline hydrolysis under reducingconditions yielding a gel with an extremely low content of ionizable groups(37). The resultant cross-links have the same structure as those present inSephadex (5) and in the epichlorohydrin cross-linked agarose gels describedby Porath, Janson and Låås (37).
Sepharose CL is available in three types corresponding to the parent gelsSepharose 2B, 4B and 6B (Table 6).
Chemical stabilityStability in aqueous media
Sepharose CL can be used in aqueous media in the range pH 3–13. Itsstability in alkaline media is particularly high (37). Short term exposure toextremes 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 Carbohydrate concentrationµg/ml
First effluent 6.5After 24 h exposure <0.5After 48 h exposure <0.5After 72 h exposure <0.5
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chromatographic properties. Under oxidizing conditions, limited hydrolysisof the polysaccharide chains may occur.The data in Table 7 demonstrate the stability of Sepharose CL-4B insolutions of chaotropic ions. Sepharose CL-4B was packed in a PharmaciaColumn K 16/40 (bed volume 57.4 ml) and equilibrated with sodiumphosphate buffer (0.02 M) containing NaCl (0.15 M). One bed volume ofthe same buffer containing the chaotropic salt KSCN (3 M) was passedthrough the column and the carbohydrate content of the effluent wasestimated by the anthrone reaction. The column was allowed to standovernight in the KSCN solution and the determination was repeated. Afterthe initial elution the concentration of carbohydrate in the effluent wasbelow the lower limit of sensitivity of the detection method.
Stability in organic solventsThe gel structure of agarose differs significantly from that of Sephadex inthat the gel-forming fibres are relatively stiff bundles of polysaccharidechains and not flexible single chains (38). Replacement of the water in thegel with other solvents has therefore a relatively small effect on pore size.Sepharose CL can be transferred from water to other solvents by themethod described in the experimental section (see page 62). Solvents testedin our laboratories include ethanol, dimethylformamide, tetrahydrofuran,acetone, dimethylsulphoxide, chloroform, dichloromethane,dichloroethane, dichloroethane/pyridine (50:50), pyridine, triethylphosphate and acetonitrile.
Physical stabilityMaximum 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 onpage 66. Because the gel structure of Sepharose CL is stabilized by cross-linking, it has comparable thermal stability to other cross-linked gels.Sepharose CL can be sterilized repeatedly by autoclaving at pH 7, 121 °Cwithout significant changes in porosity or rigidity.
Chromatographic properties
SelectivityThe fractionation ranges and selectivity curves of the different types ofSepharose CL are not significantly different from those given for theequivalent Sepharose B in Table 6 and Figure 27.
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AdsorptionSepharose CL shows even lower non-specific adsorption than the parentnon cross-linked gel, in part due to its extremely low content of chargedgroups. Sepharose CL, for example, has been shown to separate nucleicacids in order of molecular size (39).
AvailabilitySepharose CL is supplied as a ready-to-use suspension in 1 litre packscontaining 20% ethanol as a preservative.
Further informationFurther information on Sepharose CL with details of applications is givenin the data sheet: “Sepharose and Sepharose CL gel filtration media”. Forinformation on scale-up and the operation of large scale chromatographysystems, please contact Pharmacia.
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Experimental design
PerformanceResolution
The principle objective of any fractionation experiment is to achieveadequate separation of the components of interest. In chromatography, thedegree of separation, or resolution, is defined by
distance between separated zonesaverage zone width
Values of Rs greater than about 1.5 indicate baseline separation when thetwo components are present in approximately equal proportions. Greaterresolution is needed if one of the components is present in considerableexcess. Resolution can be increased in a number of ways (40) e.g. using alonger column, a gel medium with smaller particles and/or greaterselectivity, a small sample volume, a low flow rate etc. However, theconditions which lead to the highest resolution are usually in conflict withother experimental objectives and a careful evaluation of the overallrequirements is necessary. In particular, there is no need to design anexperiment to give the ultimate in resolution unless the circumstancesactually demand it.
In many experiments adequate resolution is not difficult to achieve. Moreconsideration can then be given to other objectives of a well designedexperiment. In the case of preparative separations, these may be to achievemaximum recovery with least sample dilution in the shortest possible time.For analytical work we may be more interested in reducing sampleconsumption and maximizing run-to-run reproducibility.
Separation timeTimes for gel filtration separations range from a few minutes to manyhours according to the difficulty of the separation, the gel medium, thecolumn length and the flow rate. Since the factors which improve resolu-tion also lead to longer separation times, there is always a compromise tobe made between speed and resolution. Fortunately, the recent developmentof improved gel filtration media makes this choice less difficult and the vastmajority of separations can be completed in a few hours at the most.
Resolution (Rs) =
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High performance gel filtrationIn high performance gel filtration, specialized media and equipment areemployed to get the best possible combination of resolution and speed.Since zone broadening is a principle cause of loss of resolution, every effortis made to reduce dispersion. By using small bead sizes and columns packedto the highest possible efficiency, combined with small sample volumes, anddetectors and other equipment designed for the purpose, excellent resultscan be obtained in the fractionation of complex samples in less than anhour.
CapacityThe quantity of sample which can be fractionated successfully depends onits volume and concentration. These variables affect the resolutiondifferently.
Sample concentrationThe concentration of protein in the sample has little direct result on resolu-tion (Fig. 28). However, even moderately concentrated solutions of nucleic
Fig. 28. Influence ofsample concentration onthe resolution oftransferrin and IgG onSuperdex 200 prep grade.
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acids and polysaccharides may be so viscous relative to the eluent thatviscous fingering in the column leads to a catastrophic loss of resolution(5). Within the limits set by viscosity, see page 57, any sample concent-ration may be used. Using a concentrated sample is thus a simple andeffective way of increasing the preparative capacity of a procedure.
Sample volumeResolution depends on the ratio of sample volume to column volume, largesample to column volume ratios giving lower resolution than smaller ones(Fig. 29). Recommended sample volumes for obtaining good resolution
with different media are given in Table 8. The relationship between samplevolume, bead size and resolution has been described by Hagel (41). Theactual sample volume which can be applied for a given separation problemcan only be found by experiment.
Fig. 29. Influence of sample volume on the resolution oftransferrin and IgG on Superdex 200 prep grade.
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Table 8. Recommended sample volumes as a per cent of the total bed volume forgood resolution. Many separations will show adequate resolution for larger samplevolumes.
Medium Recommended sample volume
%Vt
Sephadex 2 - 5%Sepharose 2 - 5%Sepharose CL 2 - 5%Sephacryl HR 1 - 2%Superdex prep grade 1 - 2%Superose prep grade 1 - 2%Superdex 0.5%Superose 0.5%
Process considerationsIn large scale applications of gel filtration it is not only important to obtainthe desired separation result; the method used must be shown to perform tostringent demands for economy.
ProductivityAn important concept in discussing the performance of gel filtration inproduction processes is the productivity, or mass of material which can beprocessed per bed volume per hour (g . ml bed-1 . hour-1). For a given size ofcolumn, productivity is thus increased by increasing the sample concent-ration, the sample volume and the flow rate whilst maintaining adequateresolution (42). The exact conditions which lead to the highest productivitycan only be ascertained by systematic experimentation.
Scale upGel filtration is simple to scale up from the laboratory to process scale (43).Columns with volumes of 2 500 litres can be operated in favourable cases.The bed height, linear flow rate and sample concentration should be keptconstant and the sample volume and volumetric flow rate increased inproportion to the increase in bed volume.
Choice of gelChoice of an appropriate gel depends on two main considerations, thepurpose of the experiment and the sizes of the molecules to be separated. Insome cases it may be important to consider other characteristics of thesample or the molecules to be separated.
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The purpose of the experiment
AnalysisAnalytical applications place special demands on the run-to-run reproduci-bility of the gel filtration column. It is thus specially important to choose aseparation medium which gives a perfectly stable bed under the conditionsof the analysis. Columns pre-packed with Sephacryl HR, Superdex orSuperose are suitable, both for reproducibility and efficiency. Pre-packedcolumns are recommended to ensure the maximum column efficiencywhich is essential for the highest resolution. When the column is to bepacked in the laboratory, special care should be taken to obtain a stable,well-packed bed and the packing recommendations should be followed indetail (see p 63–71).
Purity determinationWhen gel filtration is used for purity determination, it is usually necessaryto obtain the maximum resolution between the target protein and knowncontaminants. Small differences between the selectivities of different mediacan have a significant effect on the resolution and it may be necessary torun preliminary analyses with several different media since the optimum gelcan only be ascertained by experiment.
Molecular weight determination and molecular weightdistribution analysis
For molecular weight determinations a gel should be chosen so that thesample’s expected molecular weight falls on the linear part of the selectivitycurve and in the middle of a suitable range of calibration standards.Selectivity curves for the different media are shown on pages 25, 30, 33,34, 37, 40. If the molecular weight is unknown, a gel with a widefractionation range, e.g. Sephacryl HR, will be most suitable. A widefractionation range is also recommended for molecular weight distributionanalysis. If necessary, gels with different fractionation ranges can be usedtogether in the same column or separately in two or more columns in series(42).
Binding equilibriaStudies of binding equilibria require that the reacting species be separated.In many applications, one of the species is much larger than the other andchoice of gel is simple. Sephadex G-25 is suitable for studies of bindingequilibria between proteins and small molecules (20).
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Preparative
Group separations and desaltingThe choice of gel is most easy for group separations such as desalting andbuffer exchange, since there is a large difference in molecular weightbetween the two groups of components and complete resolution is notdifficult to achieve.
The gel is chosen so that the high molecular weight substances are eluted atthe void volume (Kav = 0). This will give the minimum zone broadeningand dilution and reduce the time the components of interest are on thecolumn. The low molecular weight substances should be eluted near Vt(Kav = 1).
Sephadex G-25 Fine is the recommended gel for the majority of desaltingapplications. It is easy to work with and has excellent separation capacityfor desalting molecules down to about 5000 MW.
Table 9. Pre-packed disposable columns for group separations
Column designation Medium Max. sample volume
HiTrap Desalting Sephadex G-25 Superfine 1.5 mlFast Desalting Sephadex G-25 Superfine 0.5 mlPD-10 Columns Sephadex G-25 Medium 2.5 mlNAP 5 Columns Sephadex G-25 DNA grade 500 µlNAP 10 Columns Sephadex G-25 DNA grade 1.0 mlNAP 25 Columns Sephadex G-25 DNA grade 2.5 mlNICK Columns Sephadex G-50 DNA grade 100 µlNICK Spin Columns Sephadex G-50 DNA grade 150 µlcDNA Spun Columns Sephacryl S-300 HR 100 µlMiniprep Spun Columns Sephacryl S-400 HR 50 µlSizeSelect-400 Spun Columns Sephacryl S-400 HR 100 µl
Small disposable columns (Table 9) should be used if there is a risk ofbiological or radio-active contamination of the gel to reduce hazards inhandling used materials. Disposable columns are also recommended whenmany small samples must be treated or when no risk of carry-over betweenone sample and another can be tolerated.
The Fast Desalting Desalting Column HR 10/10 is recommended for rapidbuffer exchange and other group separations of samples up to 2 ml in highperformance separation schemes.
Group separations of DNA and oligonucleotidesSephadex DNA grade should be used for group separations of DNA or anoligonucleotide (greater than 10-mer) from salts, unincorporated
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nucleotides etc. NAP-columns (Sephadex G-25 DNA grade) arerecommended for desalting and buffer exchange and NICK-columns(Sephadex G-50 DNA grade) for removal of unincorporated nucleotides.cDNA or Size Select-400 spun columns should be used for group separationof cDNA from small molecules like linkers and adapters. Group separationof plasmids from proteins, RNA and NaOH in DNA sequencing may becarried out on Sephacryl S-400 packed in Miniprep spun columns thuseliminating the need for phenol extraction (30).
Larger quantities of plasmids are quickly and conveniently prepared by gelfiltration on Superose 6 prep grade (44).
Preparative fractionationFractionation by size using gel filtration is a natural part of manyprocedures for purifying proteins and other biomacromolecules. The exactchoice of gel will depend on the circumstances. The following guide-linesshould be useful in most situations.
Gel filtration is used in the early stages of a purification scheme to removehigh molecular weight contaminants which may cause problems later onand to adjust the ionic composition of the sample to the pH and ionicstrength required for a subsequent chromatographic step. The sample willbe relatively crude and the ability to clean the gel from lipids and/or proteincontamination is important. The sample will also be relatively large involume and a large bed volume may be needed. The likely presence ofproteases or other enzymes which might degrade the target molecules alsomakes it desirable to work with high flow rates. These considerationssuggest that a gel from the Sephacryl HR series will generally be mostsuitable.
Later on in the purification scheme, the sample will be simpler incomposition and remaining impurities may require high performance gelfiltration media to remove them. Pre-packed columns containing Superdexor Superose are available which ensure maximum resolution forparticularly difficult fractionations.
Removal of dimers and aggregatesElimination of dimers and aggregates requires a gel with a selectivity suchthat the monomeric form of the molecule of interest is eluted in the latterpart of the chromatogram. High resolution media, Superdex, Superose orSephacryl HR, should be used for separation of dimers from monomers ifbaseline resolution is required.
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Removal of degradation products and incomplete moleculesWhen the impurities to be removed are known to be smaller than the targetmolecules, the gel should be chosen so that the target elutes early in thechromatogram. Sephacryl S-100 HR and Superdex 75 may berecommended when the protein of interest has a molecular weight in therange 50 000 – 100 000.
Solute characteristics
Molecular weights of the components The molecular sizes of the molecules to be separated are the mostimportant of the factors which govern the choice of separation medium.The medium must have a separation range which covers the molecular sizesof the target molecules. The selectivity curves of the different media are thebest guide to the separation to be expected. Where there is a choice ofmatrix for a given molecular size range, the media with the steeperselectivity curves and the smaller bead sizes, e.g. Superdex, are to bepreferred if high resolution is needed. Media with relatively shallowselectivity curves, e.g. Sephacryl HR or Sepharose CL, are preferable iffractionation over a wide range of molecular size is required.
Type of molecules to be separatedThe class of molecule, DNA, protein, peptide, polysaccharide etc, to beseparated has an important bearing on the choice of gel since differentclasses of molecule may have very different shapes and thus very differentexclusion properties for a given molecular weight (45–47). The selectivitycurves on pages 25, 30, 33, 34, 37, 40 show this clearly and may be used asa guide to the selection of the correct medium for the main classes ofbiomacromolecules. Note that Sephacryl HR types are available withexclusion limits which make them useful for fractionation of even nativenucleic acids and polysaccharides as well as membrane vesicles and multi-component complexes.
Sample characteristics
Sample sizeSample size has an indirect influence on the choice of gel in that largesample volumes require large bed volumes; the medium chosen musttherefore be capable of being run economically in a sufficiently large bed(see ‘Choice of column dimensions’ below).
EluentSome samples contain proteins or other components which have a limitedrange of solubility or stability. There is thus always a risk that changing the
52
pH or other conditions of the sample will cause inactivation or evenprecipitation. If the sample precipitates in a gel filtration column, thecolumn will be blocked, possibly irreversibly, and the sample may be lost.Care should always be taken to work within the solubility and stabilityrange of the sample. The free choice of eluent in gel filtration enables thisrequirement to be met in almost every case.
The composition of the eluent does not affect the choice of gel except whenit can be expected to alter the conformation of the molecules to beseparated or when it places special demands on the stability of the gel. Gelfiltration in dissociating eluents, e.g. 6 M guanidine hydrochloride or urea,is extremely useful for molecular weight determination (14). However,because of the more extended configuration of proteins and polypeptides inthese eluents, a more porous gel is usually required than predicted fromselectivity curves prepared for native globular proteins. Thus, in manycases, Sephacryl HR or Sepharose CL will be found the most appropriategels. Their high chemical and physical stability also makes themparticularly suitable for use in these eluents.
Detergents are particularly useful as solubilizing agents for proteins withlow aqueous solubility, for example, membrane components (48–50). Onceagain the effect of these agents on protein conformation usually requires amore open gel to be used for gel filtration. Detergents do not appear toinfluence the pore structure of the gels (51).
The more recently developed media, Sephacryl HR, Superdex and Superose,are in general more suitable than the traditional media for work indissociating media, eluents containing organic solvents and eluents ofextreme pH.
Choice of running conditionsChoice of column
To obtain the best results from gel filtration experiments care in the choiceof column equipment is necessary.
Ideal featuresGel filtration on a laboratory, pilot plant or industrial scale should becarried out on suitably designed columns.
53
The dead volumes at the outlet and inlet should always be as small aspossible. Many simple columns have large dead volumes and should,therefore, be avoided. Bed supports made from coarse sintered glass orglass wool cannot be recommended for long term use, because they soonbecome clogged, are difficult to clean and can give artifactual results (52).
Pharmacia has developed a series of standard columns suitable for gelfiltration. Their design is based on many years experience in the field of gelfiltration. They are manufactured from materials which do not causedestruction of labile biological substances. All are easy to dismantle andreassemble to allow thorough cleaning, which is particularly importantwhen handling biological samples.
Other important characteristics include
• Dead space at the outlet of less than 0.1% of the column volume.Minimizes dilution and prevents remixing of separated zones.
• Advanced design bed supports which give uniform flow.• All of the columns, except for K9 types, can be fitted with flow adaptors
for easy sample application (p 73–77).
Pressure and solvent resistancesPharmacia columns, packing equipment and accessories are designed inseveral resistance classes. XK columns are recommended for use in allcommon buffer systems at pressures up to 5 bars. Columns in theC-column series are for laboratory applications at pressures of up to 1 bar.SR-columns are for use with organic solvents.
Column dimensionsThe resolution of two separated zones in gel filtration increases as thesquare root of column length. Long columns should, therefore, be used toobtain the best resolution in analytical fractionation. Bed heights of greaterthan 1 m are seldom required. If a very long bed is judged to be necessary,the effective bed height can often be increased simply by recycling (Fig. 30)or by using columns coupled in series. Both techniques require the use ofadaptors or end pieces (p 70).
For analytical purposes a column with an internal diameter ofapproximately 1.0 cm often proves satisfactory. In general the length ofcolumn is decided by the resolution required and the diameter by thesample volume (see “Sample size” page 55).
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Fig. 30. Increasing the effective column height by recycling. Eluent and sample areconnected to the 3-way valve which can be closed during recycling. The 4-way valveconnects the column outlet to the inlet, or the sample/eluent to the column and the columnoutlet to the fraction collector.
For group separations short columns often suffice and gel beds less than50 cm can be used with the Coarse grade of Sephadex. With Fine andSuperfine grades still shorter beds give satisfactory resolution forcomparable separations.
Flow rateResolution, under conditions which are usually encountered in gel filt-ration, decreases with increase in flow rate. The optimum flow rate formaximum resolution of proteins is of the order of 5 ml.cm-2.h-1 inlaboratory columns, although flows up to 5 times faster can often be usedwithout much deterioration. Maximum resolution is obtained with a longcolumn and a low flow rate and the fastest run is obtained with a shortcolumn and a high flow rate. Good resolution and short running times maythus appear to be basically incompatible.
Rec102
55
Using media with small particles will give increased efficiency and higherresolution. This may allow higher flow rates to be used, but usually at theexpense of sample capacity.
If peaks are well separated at a low flow rate using a long column, theexcess resolution may be traded off for speed. The flow rate can beincreased and a shorter column can be used, or alternatively more samplecan be applied.
For preparative purposes the advantage of a higher flow rate (andconsequently a faster separation) often outweighs the loss of resolution inthe chromatographic run. Full details of the maximum permissible flowrates of the various gel types, governed by their mechanical properties, aregiven on page 66.
Sample characteristics
Sample sizeFor analytical purposes and difficult fractionation experiments wheremaximum resolution is required the starting zone must be narrow relativeto the length of the column. A sample volume of 0.5–5% of the bed volumeis recommended, see Table 8. Smaller volumes do not normally improveresolution.
In group separations and some fractionation experiments, where peaks arewell resolved, it is often appropriate to improve the experimental design byincreasing the sample size. In order to minimize sample dilution, which isan inevitable consequence of gel filtration, a maximum sample volumeshould be used within the limits set by the separation distance. Figure 31shows how, if no zone-broadening were to occur during passage down acolumn, the maximum sample volume could be as great as the separationvolume (VSep).
VSep = VeB - VeA
However, due to eddy diffusion, non-equilibrium between the stationaryphase and the mobile phase, and longitudinal diffusion in the bed, thezones will always be broadened (40). The sample size must, therefore,always be smaller than the separation volume. In desalting and bufferexchange volumes up to approximately 30% of the total bed volume (Vt)
56
Fig. 31. Elution curves fordifferent sample sizes. Thetop diagram corresponds tothe application of a smallsample. The centre diagramcorresponds to the maximumsample size giving completeseparation if no zonebroadening were to takeplace. The bottom diagramcorresponds to the maximumsample volume to be appliedto obtain completeseparation in the conditionsof the experiment. Theshaded areas correspond tothe elution profiles thatwould be obtained if no zonebroadening were to occur.
can be used to minimize dilution and still retain good separation. Wherecomplete recovery of desalted sample is the major requirement, samplevolumes of between 15 and 20% of Vt are recommended. For routine smallscale desalting with the PD-10 columns (p 49) use of the maximumrecommended sample volume of 2.5 ml results in the effectively completerecovery of desalted material in a volume of 3.5 ml, a dilution factor ofonly 1.4 (Fig. 32).
Fig. 32. Desalting of albuminsolution. A column PD-10was equilibrated withdistilled water. The samplecontained human serumalbumin (25 mg) dissolved inNaCl (0.5 M, 2.5 ml). Yieldof albumin in 3.5 ml aftersample application (betweenarrows) 95.3%; salt content,2.0% of total salt originallypresent.
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When designing an optimized desalting system, the volume of gel requiredshould be packed in a short, wide column rather than a long, narrow one.This allows more rapid recovery of desalted materials since higher volumeflow rates can be achieved with the shorter column.
Sample compositionDue to the linear partition isotherms, gel filtration is to a large extentindependent of mass of solute in the sample. However, in addition to thesolubility of the solutes, the viscosity of the sample often limits the concent-ration that can be used. A high sample viscosity causes instability of thezone and an irregular flow pattern. This leads to very broad and skewzones. The critical variable is the viscosity of the sample relative to theeluent. This is illustrated in Figure 33 which shows the elution profiles ofhaemoglobin and NaCl at different sample viscosities.
Fig. 33. Elution diagramsobtained when haemoglobinand NaCl were separated.Experimental conditionswere identical except that theviscosities were altered bythe addition of increasingamounts of dextran. Anincreasing deterioration ofthe separation becomesstrikingly apparent. (A lowerflow rate will not improvethe separation.)
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In practice the relative viscosities of sample and eluent should not differ bymore than a factor of about 2, corresponding to a protein concentration inthe sample of about 70 mg/ml when a dilute aqueous buffer is used as theeluent. Approximate relative viscosities can be quickly estimated bycomparing emptying times from a pipette.
EluentEluent composition does not directly influence the resolution which can beobtained in gel filtration. Completely uncharged substances may be elutedwith distilled water. For chromatography of substances carrying chargedgroups an eluent containing a buffer, e.g. Tris-HCl, sodium phosphate etc.,is often used to control pH, and an ionic strength of at least 0.02 forSephadex and Sepharose or 0.15 for Sephacryl HR is recommended tosafeguard against possible ionic interactions with the gel matrix. Sodiumchloride can be used for this purpose. It should also be noted that someproteins may precipitate in solutions of low ionic strength.
If the product is to be lyophilized, volatile buffers such as ammoniumacetate, ammonium bicarbonate or ethylenediamine acetate can be used. Indesalting, the separation volume is so large that, in general, chargedsubstances can also be treated with distilled water as eluent. Completeremoval of salt is not possible, but the amount of ions excluded and,therefore, eluted with the HMW fraction is so small it can be neglected inmost cases.
The most important consideration in the choice of eluent in gel filtration isits effect on the sample molecules. The pH and ionic composition of thebuffer, and the presence of dissociating media or detergents can causeconformation changes, dissociation of proteins into subunits, dissociationof enzymes and cofactors, dissociation of hormones and carrier proteinsetc. which must be taken into account when choosing the gel and wheninterpreting the results of an experiment.
OptimizationOptimization of a separation can only be carried out by careful experiment,since each separation problem is different. The behaviour of a threecomponent model system of proteins A, B and C (Fig. 34) is used here toillustrate some of the factors which might need to be considered.Components A and B are separated at the lowest flow rate. However, thisseparation can not be speeded up significantly without serious loss of
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Fig. 34. A model gel filtrationexperiment to demonstrateoptimization of theseparation of model proteinsA, B and C.
L2 =
resolution. Protein C is well separated from protein B and this particularseparation can be speeded up by a factor of 50. (An alternative view ofoptimization could be to retain the flow rate and increase sample size toutilise more completely the separation volume between proteins C and B).
In the first stage of the optimization, increasing the flow rate, the time forseparating protein C from B is reduced from about 30 hours to about 2.5hours. The resolution (Rs) is still 2.25. The bed height (L2) required to giveRs = 1 can be calculated from
L1Rs1
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where Rs1 is the resolution obtained with a column length L1. In thisexample L2=17 cm. Reducing the bed height to about 20 cm should stillgive adequate resolution of protein C and protein B. The result obtainedwith a bed height of 19.5 cm at 24.5 ml.cm-2.h-1 is shown in Figure 34. Theseparation time for protein C and protein B has been reduced from 30hours to 35 minutes. For a full discussion of the optimization of gel filt-ration see (40).
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Table 10. Bed volume and swellings times for Sephadex G-types.
Gel type Approx. bed Swelling time (h)volume (ml/g) 20 °C 90 °C
Sephadex G-10 2 – 3 3 1Sephadex G-15 2.5 – 3.5 3 1Sephadex G-25 (all grades) 4 – 6 3 1Sephadex G-50 (all grades) 9 – 11 3 1Sephadex G-75 (all grades) 12 – 15 24 3Sephadex G-100 (all grades) 15 – 20 72 5Sephadex G-150 20 – 30 72 5Sephadex G-150 Superfine 18 – 22 72 5Sephadex G-200 30 – 40 72 5Sephadex G-200 Superfine 20 – 25 72 5
Performing a gel filtrationexperiment
Gel filtration is essentially simple to perform once a well packed columnhas been obtained. Providing that a column is used and maintainedcarefully it can be expected to give many months of reliable service.
Preparing the gelPre-swollen media (Sephacryl HR, Superdex prep grade,
Superose prep grade, Sepharose CL and Sepharose)These gels are supplied swollen and ready to use as a suspension containing20% ethanol as a preservative. The suspension is too thick to be poureddirectly 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 inexcess solvent before use. During swelling excessive stirring should beavoided as it may break the beads. Do not use magnetic stirrers. For allSephadex G-types, the process of swelling can be accelerated by using aboiling water bath which also serves to deaereate the buffer (Table 10).
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Preparation of Sephacryl HR, Superdex prep grade,Superose prep grade or Sepharose CL
for use in organic solventsThe aqueous medium in which a gel is swollen can be exchanged for avariety of organic solvents. (Note: Because the gel structure of Sepharose isonly stabilized by hydrogen bonding, it is not suitable for gel filtration inmany organic solvents. However, Sepharose CL can be used quite safely ina wide range of organic solvents.) To ensure efficient replacement of thewater by the required solvent, the transfer must be made through a gradedseries of solvent mixtures. Thus to transfer from one pure solvent (A) toanother pure solvent (B), the gel is transferred first to 70% A/30% B thento 30% A/70% B and finally to pure B. If A and B are not mutually
Fig. 35. Suggested routes for changing to organic solvents.
miscible, the transfer is made via an intermediate solvent e.g. from water tochloroform via acetone (Fig. 35).
Transfer the required amount of gel to a sintered glass Buchner funnel andremove the excess aqueous medium by gentle suction. Add the next solvent(see Fig. 35) and resuspend the gel by gentle stirring. Suck off the excesssolvent and resuspend in the same solvent. Repeat the process with the nextsolvent of the series, allowing two resuspensions and proceed until therequired solvent composition is reached.
Note that the gel volume of Sephacryl HR may be reduced by up to 15%on transfer to organic solvents (Table 2).
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Gel filtration in organic solventsAfter transferring the gel to the solvent of choice it may be packed in theusual way (see below). Sepharose CL floats in dense solvents, e.g.chloroform, and in these cases packing should be carried out as describedin the booklet “Sephadex LH-20. Chromatography in organic solvents”.Pharmacia columns SR 10/50, SR 25/45 or SR 25/100 should be used.Flow rates will depend on the viscosity of the eluent. Certain solventswhich disrupt hydrogen bonds, e.g. DMSO, may cause softening ofSepharose CL so that flow rates become impracticably low.
Packing a column (Fig. 36-43)This is a very critical stage in any gel filtration experiment. A poorlypacked column will give rise to uneven flow, zone broadening and loss ofresolution. The flow rates obtainable are also affected. It is important tonote that Sephacryl HR, Superdex prep grade and Superose prep grade,which have a relatively rigid bead form, are packed in a rather differentmanner than the Sephadex G-types and Sepharose CL or B types.
Packing Sephadex G types, Sepharose and Sepharose CL
Filling the column1. Prepare the gel as described above. The suspension of gel should be
adjusted so that it is a fairly thick slurry. It should not be so thick itretains air bubbles. Usually about 75% settled gel is suitable. Fineparticles can be removed at this stage by decantation, if desired.
Fig. 36.
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The suspension should be de-gassed under vacuum (Fig. 36) This is notnecessary for Sephadex which has been swollen on a boiling waterbath. The gel suspension should reach the temperature of columnoperation before packing is begun.
2. Mount the column on a stable laboratory stand. It is important toavoid locations which are exposed to draughts or direct sunlight andwhich can cause temperature changes and the formation of bubbles ina packed column. Use of degassed buffers and a filled water-jacket willalso help safeguard against the effects of rapid temperature changes.
3. Ensure that there are no air bubbles trapped in the dead space underthe net by drawing water (or 20% ethanol) through it. This is bestdone by submerging the plunger in a beaker of water (or 20% ethanol)and attaching the tubing to a pump or a syringe. Alternatively, injecteluent into the outlet tubing until it passes through the bed support net(Fig. 37). When the dead space is properly filled, close the outlettubing.
4. Tilt the column and pour the well-mixed gel suspension down theinside wall of the column. Immediately readjust the column to avertical position. Alternatively the gel can be poured directly into thevertically mounted column using a glass rod. If the slurry volume isgreater than the volume of the column, a gel reservoir (XK, C, K seriescolumns) or a column extension (SR-10, HR 10 and HR 16 columns)should be attached (Fig. 38 and Fig. 39). All the gel required should bepoured in a single operation. Preparing the gel from too thin a suspen-sion or, for other reasons, packing the column in stages, often results ina badly packed bed.
Fig. 37.
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Fig. 39.Fig. 38.
5. The next step involves closing the column without trapping air inside.If an adaptor is used, follow the instructions given below (p 70–71).With C or K columns, a top piece can be used in the following way.Close the column outlet and fill the space above the gel with eluent toabout 1 cm from the top of the glass tube. Fit the top piece, removingair via the air vent valve in the top piece This step is unnecessary if acolumn packing reservoir or extension is being used.
6. The flow should be started as soon as possible after filling the columnto obtain even sedimentation. The top piece air vent should be closedand the column outlet opened to allow the packing to continue. Checkfor air bubbles and repeat steps if necessary. Do not exceed the maxi-mum operating pressures given in Table 11.
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Equilibrating the bed1. Before opening the column outlet tubing, check the operating pressure.
This depends on the difference between the free surface of eluent in thereservoir and the outlet as illustrated in Figure 40. With the softer gelsthe operating pressure must not exceed the limits given in Table 11.With rigid gels such as Sephadex G-10 or G-50 such precautions areunnecessary and packing can be rapidly achieved with a pump orgravity feed.
Table 11. Approx maximum flow rates and pressures. Data has been obtained fromcolumns 2.5 cm diameter with a bed height of 30 cm.
Gel type Max. operating Approx.pressure cm H2O max. flow rate
ml/min ml.cm.–2 h–1
Sephacryl S-100 HR 500 2.5 30*Sephacryl S-200 HR 500 2.5 30*Sephacryl S-300 HR 500 2.5 30*Sephacryl S-400 HR 500 2.5 30*Sephacryl S-500 HR 500 2.5 30*
Sepharose CL-6B >200 2.5 30Sepharose CL-4B 120 2.17 26Sepharose CL-2B 50 1.25 15
Sepharose 6B 200 1.16 14Sepharose 4B 80 0.96 11.5Sepharose 2B 40 0.83 10
Sephadex G-10 - G-50 These gels obeyDarcy’s Law(see p 78)
Sephadex G-75 160 6.4 77Sephadex G-75 Superfine 160 1.5 18Sephadex G-100 96 4.2 50Sephadex G-100 Superfine 96 1.0 12Sephadex G-100 36 1.9 23Sephadex G-150 Superfine 36 0.5 6Sephadex G-200 16 1.0 12Sephadex G-200 Superfine 16 0.25 3
* For flow rates used during packing please see Table 12.
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Fig. 40. Definition of operating pressure (A-D) and sample application from a samplereservoir (D).A and B. Pressure (cm water) is measured as the distance between the free surface in thecolumn or reservoir and the end of the outlet tubing.C and D. Pressure (cm water) is measured from the bottom of the air inlet tube in theMariotte flask to the end of the outlet tubing, no matter whether the flow through thecolumn is downward (C) or upward (D).
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2. Open the column outlet and start the flow. Two or three columnvolumes of eluent should be passed through the column in order tostabilize the bed and equilibrate with eluent buffer. A slightly higherflow rate than is to be used in the experiments should be used forpacking.
Packing Sephacryl HR, Superdex prep gradeand Superose prep grade
Sephacryl HR, Superdex prep grade and Superose prep grade should bepacked and equilibrated at a high flow rate in a column in the XK-seriesusing a suitable pump (e.g. P-l, P-50, P-500). The packing methoddescribed below ensures that the gel is optimally packed at the point ofentry of the sample, the most critical area, and it is highly recommendedthat this method be followed.
Pack the column at the temperature at which it will be used.
1. Make sure the column is not damaged and that all parts are reallyclean. It is of special importance that the nets, net fasteners and glasstube are not damaged.
2. Attach the packing reservoir tightly and mount the column verticallyon a stand.
3. Wet the adaptor by drawing water (or 20% ethanol) through it, ma-king sure no air bubbles are trapped under the net. This is best done bysubmerging the plunger in a beaker of water (or 20% ethanol) andattaching the tubing to a pump or a syringe. Close the tubing with astopper when all air bubbles have been removed.
4. Insert the adaptor at the bottom of the column far enough to give thedesired bed height.
5. Wet the column glass tube with eluent leaving a few centimetres offluid in the bottom making sure that the net is completely free from airbubbles.
6. Prepare the gel as described above. The suspension of gel should beadjusted so that it is a fairly thick slurry. It should not be so thick itretains air bubbles. Usually about 75% settled gel is suitable.Resuspend the gel and pour the well-mixed gel suspension carefullydown the wall of the column using a glass rod. Pour all the gel in asingle operation and fill the reservoir to the top with eluent.
7. Screw on the reservoir cap tightly, connect it to the pump and open theoutlet.
8. Pack the column in two steps using the flow rates given in Table 12,
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Table 12. Recommended flow rates for packing Sephacryl HR
Column Flow rates (ml/h)Step 1 Step 2
XK 26/40 240 490XK 26/70 240 360XK 26/100 180 320
XK 50/60 600 1 400XK 50/100 600 950
pack the gel in STEP 1 for 2 hours or until the gel bed has reached aconstant height, then increase the flow rate to the value listed for STEP2 and pack for 60 minutes.
9. Stop the pump, close the column outlet and remove the packingreservoir. This is most easily done by first removing the column fromthe stand and then unscrewing the reservoir over a sink. It may beeasier to use a siphon when working with a large column.Using one adaptor and a bottom piece:
10. Remove excess gel carefully with a small spoon or a plastic spatulauntil the bed surface is about 2 mm below the end of the glass tube.When the bottom piece is inserted in point 14 it will be pressed about5 mm into the gel. If there is not enough gel in the column, it will benecessary to use a second adaptor or to repack the column with moregel.
11. Mount the column vertically on the stand and fill the column to the topwith buffer.
12. Wet the bottom piece with eluent or 20% ethanol.13. Insert the bottom piece carefully so that no air bubbles are trapped
under the net.14. Open the bottom piece outlet (NOT the outlet from the adaptor at the
bottom of the column). Press down and tighten the bottom piece. Thiswill cause eluent to flow out of the tubing attached to the bottompiece. Close the bottom piece outlet again.Using two adaptors:
10. Remove excess gel by gently stirring the top of bed with a glass rod andremoving the suspended gel with a Pasteur pipette. Remove enough gelso that the plunger will be visible below the end piece.
11. Mount the column vertically on the stand and fill the column to the topwith buffer.
12. Wet the second adaptor with eluent or 20% ethanol.13. Remove the stopper from the second adaptor tubing (NOT from the
adaptor at the bottom of the column) and insert the adaptor carefullyso that no air bubbles are trapped under the net.
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14. Bring the adaptor to the gel surface and then a further 5 mm into thegel bed. Liquid will flow out of the tubing attached to the adaptorduring this process.
AdaptorsAdaptors are adjustable column end pieces which support the bed matrix.They allow automatic methods of sample application which eliminatedisturbance of the bed surface and protect the bed from insoluble particlesin the sample.
After an extended period of use, flow rates through a column packed withSephadex at a given hydrostatic pressure will be reduced. An upward flowarrangement using adaptors will discourage this process (Fig. 40 D).
Adaptors are available for most Pharmacia columns. They should be fittedas follows.
1. The gel should be packed completely as described above, oralternatively should be allowed to settle sufficiently so that there are2–3 cm of clear eluent at the top of the column.
2. Ensure the column outlet is closed otherwise the gel may becompressed when the adaptor is fitted. Carefully add more eluent to fillthe column and form an upward meniscus (Fig. 41)
3. Slacken the adaptor tightening mechanism and insert it at an angle intothe column so that no air is trapped under the net (Fig. 42).
Fig. 41. Fig. 42.
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Fig. 43.
4. Adjust the tightening mechanism to give a sliding seal between thecolumn wall and O-ring. Screw the adaptor top piece onto the columnend piece.
5. Make all tubing connections at this stage. Slide the plunger slowlydown the column so that air in the adaptor above the net and in thecapillary tubings is displaced by eluent. Valves on the inlet side of thecolumn should be turned in all directions during this procedure toremove air from all connections.
Lock the adaptor in position with the tightening mechanism andlocking screw (Fig. 43), open the outlet and start the eluent flow.Readjust the position of the adaptor at the bed surface after packinghas been completed (if the column has already been packed when theadaptor is fitted, only about 10 minutes further packing is requiredbefore re-adjustment).
Checking the packed bedBefore starting any experiment, it is advisable to check the homogeneity ofthe bed by running a freshly prepared and filtered solution of a colouredsubstance. Blue Dextran 2000 at a concentration of 2 mg/ml can be usedfor this purpose and to determine the void volume of the bed. The qualityof the packing can be checked by watching the progress of a zone of thissubstance through the bed. Visual inspection of the bed in transmitted lightmay also reveal heterogeneities and air-bubbles.
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Using pre-packed columnsThe initial setting up of a gel filtration experiment is considerablysimplified if a pre-packed column is used.1. Connect the column to the sample application device (see below for
alternatives) and to the monitor.2. Follow the instructions supplied with the column to equilibrate it with
the chosen eluent.
Sample applicationSample application without an adaptor
Note that these methods are not suitable for use with pre-packed columnssince removing the upper adaptor to apply the sample would disturb thecolumn packing. One of the methods described under ‘Sample applicationwith an adaptor’ should be used.
Sample application on a drained bed surfaceAlthough this is the method which requires least equipment, it is not thesimplest and considerable care must be taken to avoid disturbing the bedsurface. An uneven bed surface leads to uneven separated bands and poorresolution. A sample applicator cup (available for columns series XK, Cand K columns with diameters 16 and 26 mm) helps prevent disturbance ofthe bed surface.
1. Close the outlet and remove most of the eluent above the gel surface bysuction.
2. Open the outlet and allow the remaining eluent to drain away. Underno circumstances should the bed be allowed to run dry.
3. Layer the sample on top of the bed.4. Open the column outlet and allow the sample to drain into the bed. Do
not allow the bed to run dry.5. Wash the sample which remains on the bed surface and on the column
wall into the bed with a small amount of eluent.6. Refill the column with eluent and reconnect to a Mariotte flask or
pump.
Sample application under the eluent (Fig. 44)The sample must be denser than the eluent. If not, it can be made so by theaddition of a small amount of glucose, sodium chloride, buffer salt oranother suitable inert material. Take care not to increase the sampleviscosity too much.
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Fig. 44.
1. Use a syringe fitted with a piece of fine capillary tubing or a pipettewith a bent tip. Draw the sample into the syringe, then draw a little airinto the capillary tubing to prevent mixture of sample with eluent.
2. Close the column outlet and place the tubing in the eluent so that its tipis held a few mm above the bed surface and dispense the sample slowlyand evenly as a layer under the eluent.
3. After application draw a small amount of eluent into the tubing toprevent sample mixing with eluent when the tubing is withdrawn. Norinsing is required and eluent flow can be restarted directly.
Sample application with an adaptorThese methods are always used with pre-packed columns and are also themost convenient for other columns if the column is to be used frequently.Samples must be applied by one of these methods if upward flow elution isused.
Syringe method. (Fig. 45).The valves LV-3 and LV-4 can be used as syringe holders to give a verysimple method for the application of small samples .
Sample reservoir (Fig. 46).In a similar way, a sample reservoir (e.g. RK or R) can be connected via a3-way valve to apply larger samples. Figure 46 also demonstrates sampleapplication and elution with upward flow.
74
Fig. 45.
Fig. 46.
75
Sample applicators SA-5, SA-50 (Fig. 47).These are reservoirs which allow the sample to be introduced as a layerbelow the eluent using a syringe and needle without disturbing thechromatographic bed. They can also be used in a sample loop system (Fig.47) where their large capacity (up to 5 ml for the SA-5 and 45 ml for theSA-50) and lack of tailing due to wall effects offer distinct advantages.
Fig. 47. A Sample Applicator SA-5 used as a sample loop.
Sample loops with valves LV-4 or SRV-4 (Fig. 48).This method is convenient for application of small samples. By using thesame sample loop very reproducible sample volumes can be applied,although exact knowledge of the applied volume requires calibration of thecapillary tubing loop.
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Fig. 48. Sample application with a sample loop and two SRV-4 valves.
Fig. 49. Seven-port valves, V-7 and MV-7, have three operating positions which makesample application and changing eluents particularly convenient.
columns in FPLC System. Superloop (Fig. 50) is a unique sampleapplication device from which a sample of any volume up to the capacity ofthe Superloop (10 ml or 50 ml) can be applied to a column without tailing.A movable seal separates the sample from the eluent. As eluent is pumped
Sample loops or Superloop with valves V-7 or MV-7 (Fig. 49).This method is used for sample application when using high performancecolumns (e.g. Superdex HR 10/30 and Superose HR 10/30) and other
77
Fig. 50. Principle of operation of Superloop. Sample is drawn into the space in front of themoving seal and applied to the column when eluent is pumped into the space behind theseal. When the seal reaches the end of its travel, eluent by-passes the seal and washes the lastof the sample quantitatively onto the column.
into the Superloop, the sample moves ahead of the seal and onto thecolumn. When nearly all the sample has been applied, eluent flows roundthe seal to wash the remainder of the sample quantitatively onto thecolumn. A Superloop should be used for applying sample volumes greaterthan approximately 1 ml. Very reproducible sample volumes can beapplied, particularly when the system is operated automatically usingLCC-501 Plus or FPLCdirector.
ElutionThe flow of liquid through the column can be controlled by difference inhydrostatic pressure or by a pump. Accurate and reproducible control offlow rate is particularly important when repeating experiments orperforming routine preparative work. It is most easily achieved using agood peristaltic pump such as the P-l pump. Note that a pump shouldalways be connected into the system so that it pumps the eluent onto thecolumn rather than connecting it after the column. This reduces the risk ofbubble formation in the column which can result from suction. When theflow is to be maintained by gravity feed a constant pressure flask, such as a
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Gel and Eluent Reservoir used as a Mariotte flask, is recommended.Mention has already been made (see page 70) of the advantage of upwardflow for long term use of a column.
A problem which can occur when an experiment is continued withoutsupervision, for example, overnight, is that of stopping the experimentbefore the eluent runs out. When using a pump this can be achieved byattaching it to the power supply via a timing/cut-off device or via a fractioncollector which can control the pump. If gravity feed is used, a safety loop,of the type illustrated in Figure 51 can easily be arranged to prevent airfrom entering the column.
Flow ratesRecommendations concerning flow rates for use in experiments are givenon page 66. It is not possible to apply one set of rules for calculating flowrates over the whole range of gel types. The arguments, equations and datagiven here apply only to laboratory columns with diameters up to 5 cm.
Sephadex G-10, G-15, G-25 and G-50These gels may be assumed to behave as rigid spheres in gel filtration andtherefore obey Darcy’s Law, i.e.
U = K ∆P L-1 (1)
Fig. 51. Safety looparrangements:A. The safety loop is placedafter the column and the endof the outlet tubing is placedabove the column. The flowstops when the eluent in theinlet tubing reaches the levelof the outlet.B. The safety loop is placedbefore the column with thecolumn outlet tubing in anyposition above the lowerloop on the inlet side. Theflow stops when the eluentin the inlet tubing reachedthe level of the outlet.
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where U is the linear flow rate expressed in cm/h (ml. cm-2.h-1), ∆p is thepressure drop over gel bed expressed in cm H2O, L is the bed heightexpressed in cm and K is a constant of proportionality depending on theproperties of the bed material and the eluent. Assuming an eluent withviscosity of 1 cP one can write
U = Ko ∆P L-1 (2)
where Ko is the “specific permeability” depending on the particle size of thegel beads and their water regain. Observe that the flow rate is proportionalto the pressure drop over the bed and, assuming a constant pressure head,inversely proportional to the bed height. Notice that (to a good approxima-tion) the flow rates are independent of the column diameter.
Flow rates at viscosities greater than 1 cP can be obtained by using therelation: flow rate inversely proportional to viscosity. At first sight, itwould appear that high eluent viscosities lead to poor flow rates but theoperating pressure can be increased to compensate for the viscosity effect.Temperature influences the viscosity of the eluent. Lower flow rates areobtainable, for a given pressure head, in a cold room than at roomtemperature.
Theoretical flow rates (not maximum) can be calculated from equation (2)by inserting values for ∆p and L. Specific permeabilities are given inTable 13.
Sephadex type Permeability K
Sephadex G-10 19Sephadex G-15 18Sephadex G-25 Superfine 9Sephadex G-25 Fine 30Sephadex G-25 Medium 80Sephadex G-25 Coarse 290Sephadex G-50 Superfine 13.5Sephadex G-50 Fine 36Sephadex G-50 Medium 145Sephadex G-50 Coarse 400
Table 13. Specific permeabilities of Sephadex G-types.
Flow rates in columns packed with other mediaCalculation of flow rates in other less rigid gels is somewhat morecomplicated since Darcy’s Law is not applicable. Not only is the flow ratedependent upon the factors already mentioned but also on the columndiameter. Wider columns do not allow as high a pressure and linear flow
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rate (ml.cm-2.h-1) as narrower ones. These gels do not have a linearrelationship between pressure and flow, exceeding the maximumrecommended values can lead to gel compression, reduction in flow rateand loss of resolution. Recommended maximum operating pressures andcorresponding approximate flow rates at room temperatures are given inTable 11.
A bed diameter of 2.6 cm (column XK 26) and bed height of 30 cm havebeen assumed. Maximum operating pressures are independent of bedheight but flow rates decrease with bed height. To calculate the flow ratefor a different bed height it may be assumed to be inversely proportional tothe bed height. Slightly reduced maximum operating pressures must be usedwith columns wider than 2.6 cm.
To utilize fully the high rigidity and the excellent flow properties ofSephacryl HR a liquid delivery system including a pump should be used. Aperistaltic pump can still be used with the other softer gels provided care istaken. It is recommended to determine the maximum flow rate for thecolumn in question with gravity feed, taking care not to exceed thepressures given in Table 11, and then use flow rates not exceeding 75 % ofthis value with the pump. The maximum flow rates which are given inTable 11 serve as a guide only and may vary depending upon columnpacking or eluent viscosity (Note especially the effect of temperature onviscosity).
Cleaning gels and packed columnsGeneral cleaning procedures
When a column has been in use for some time, it may be necessary toremove precipitated proteins or other contaminants which have built up onthe gel bed. The need for cleaning may show itself as the appearance of acoloured band at top of the column, as a space between the upper adaptorand the bed surface, as a loss in resolution or as a significant increase inback-pressure. General procedures are given below for different mediafollowed by special procedures for removing specific contaminants. In allcases, prevention is better than cure. The use of filtered eluents and samplesis essential for high performance columns and will greatly reduce thenumber and severity of the problems encountered with any medium.Similarly, only fresh buffer solutions should be used. Many buffersubstances are excellent supporters of microbial growth. See also thesection on ‘Prevention of microbial growth’ (p 84).
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If an increase in back-pressure is observed, for example by the level of thegel falling, make sure that the high back-pressure is in fact caused by thecolumn before starting the cleaning procedure. Disconnect one piece ofequipment at a time (starting at the fraction collector) with the pumpworking and check the pressure as each piece is disconnected. A dirty filteris often the cause of increased back-pressure. The pressure should bechecked at the same stage during each run, since the back-pressure can varywithin a run during sample injection or when changing to a differenteluent.
Note that cleaning solutions should also be filtered before use. Aftercleaning, the column must be carefully re-equilibrated with 2–3 columnvolumes of eluent buffer before it is used again.
Cleaning Sephadex G-typesPacked columns may be cleaned with 2 column volumes of a non-ionicdetergent solution. Sephadex may also be washed with NaOH (0.2 M) on aBuchner funnel.
Cleaning SepharoseWash with a non-ionic detergent.
Cleaning Sepharose CLTreat the gel with one bed volume of 0.5 M NaOH or a non-ionic deter-gent solution (1%) either in the column or on a Buchner funnel.
Cleaning Sephacryl HRSephacryl HR may be cleaned and sanitized in the column with 1–2 columnvolumes of NaOH (0.2–0.5 M) at a flow rate which gives a contact timebetween the gel and the cleaning solution of at least 1 hour. This contacttime is sufficient to solubilize most protein precipitates. Sephacryl HR mayalso be washed with a solution of a non-ionic detergent.
Use the same method if the gel is severely contaminated, but reverse thedirection of flow in the column during treatment with NaOH.
Cleaning Superose prep gradea. Cleaning a packed column:
Do NOT reverse the flow during cleaning since this may cause a loss ofefficiency.
1. Change the filter on the top of the gel bed and check the bed support,changing it if necessary.
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2. Wash with at least 1/3 of the column volume NaOH (0.1 M-0.2 M).3. Rinse with high purity water or buffer. Small aliquots (3 x 100 or
200 µl) of acetic acid (50%) may be added during this rinse.4. Equilibrate with buffer until the baseline is stable.
b. Cleaning the gel before repacking:1. Put a large enough beaker under the column to collect the gel, remove
the bottom piece and empty the column by pumping high purity wateror buffer through it. Clean all column parts with soapy water orlaboratory detergents. Inspect the top and bottom filters and changethem.
2. Wash the gel on a glass filter with NaOH (0.1-0.2 M), then water andlastly ethanol (20%).
3. Re-suspend the gel in at least 5 times the gel volume of high puritywater in a beaker.
4. Allow the gel to sediment and pour off the supernatant.5. Repeat the washing procedure once more before re-packing the
column.
Cleaning Superose HR 10/30 pre-packed columnsRegular cleaning
The following procedure will help prevent contaminants from building upon the column.
1. Wash with 5 ml 0.1 M NaOH2. Wash with 5 ml 50% acetic acid3. Re-equilibrate with buffer until the base line is stable
Rigourous cleaning to remove contaminationDo NOT reverse the flow during cleaning since this may cause a loss ofefficiency and NEVER exceed the maximum pressure for the column.
1. Change the filter at the column inlet. Since the contaminants areintroduced with the liquid flow, many of them are caught by the filter.
2. Set the pressure limit to the maximum for the column.3. Wash in sequence with 25 ml acetic acid (50%), 25 ml water, 25 ml
ethanol (20%, run at a low flow rate), 25 ml NaOH (0.1 M), 25 mlwater and three aliquots of 100 or 200 ml of acetic acid (50%). Thisprocedure is most conveniently carried under automatic control from aLiquid Chromatography Controller LCC-501 Plus.
4. Equilibrate with buffer until the baseline and the pH of the eluentleaving the column are stable.
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The suggested cleaning volume of 25 ml is only a guide-line; thepractical requirements are best determined by monitoring the baseline,which should be stable after each step in the sequence.
In special cases only, it may be necessary to change the bottom filter orto remove and discard the top 2-3 mm of the gel. These operationsmust be carried out extremely carefully to avoid serious loss of resolu-tion.
Cleaning Superdex pre-packed columnsSuperdex columns may be cleaned with one bed volume of NaOH (0.5 M).This treatment will remove most proteins from the gel. The frequency ofcleaning depends on the degree of contamination, but a cleaning cycle atleast every 10–20 separation cycles is recommended.Re-equilibrate the column with two bed volumes of buffer immediatelyafter cleaning. Wait until the UV baseline is steady before applying the nextsample.
If the column still is contaminated, it may be washed with 0.5 bed volumeof 1 M NaOH solution and 0.5 bed volume of 30% isopropanol or 0.1 MHCl. Replace the filters if necessary.
Procedures to remove specific contaminantsIf the general cleaning methods fail to give the desired result, the followingmethods may be used to remove specific contaminants. Various alternativesare given for each type of contaminant – choose the most convenientaccording to the reagents you have available; if this does not work, try oneof the alternatives. Since some of the cleaning solutions are more viscousthan normal buffer solutions, care must be taken not to exceed the maxi-mum operating pressure which the gel can sustain.
Hydrophilic proteins and peptidesWash the column with the solution which previously dissolved the materialduring sample preparation e.g. an extraction solution, detergent, etc. (over-night, at low flow rate).
Wash overnight in 30–50% acetic acid at 0.1 ml/min.
Fill the column with 1 mg/ml pepsin in 0.1 M acetic acid and 0.5 NaCl andleave overnight at room temp. or 1 hour at 37 °C. Note: after enzymaticdigestion, careful rinsing is required to remove trace amounts of enzymeremaining in the system.
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Hydrophobic proteins and peptidesThese are usually soluble in polar organic solvents e.g. ethanol (24%),acetonitrile (30%). If the organic solvent which best dissolves thecontaminant is known, run this overnight at a slow flow rate.
Nucleic acidsRNA and DNA are very soluble in solutions of low ionic strength and maybe re-dissolved by running low ionic strength buffer (e.g. 10 mMTris-HCl, 1 mM EDTA, pH 8.0) through the column at low flow rate for24 hours.
RNAHydrolyse the RNA with NaOH (0.1–2 M, 1 hour) and rinse with water orwith ribonuclease I solution (2 ml, 1 mg/ml in 0.1 M NaCl, 50 mMTris-HCl, pH 7.5, 37 °C, 2 hours) and rinse with 10 mM Tris-HCl, 1 mMEDTA, pH 8.0.
DNAHydrolyse the DNA with deoxyribonuclease I solution (2 ml, 1 mg/ml in0.1 M NaCl,10 mM MgCl2, 50 mM Tris-HCl, pH 7.5, 37 C, 2 hours) andrinse with 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.Note: after enzymatic digestion, careful rinsing is required to remove traceamounts of enzyme remaining in the system; special caution isrecommended if subsequent separations of RNA or DNA are planned.
LipidsWash the gel overnight at 0.1 ml/min with a non-ionic detergent (e.g.0.2–1% NP-40 or Lubrol) in a basic or acidic solution and remove thedetergent by washing with methanol or ethanol.
Storage of gels and columnsPrevention of microbial growth
Microbial growth rarely occurs in columns during use but steps shouldalways be taken to prevent infection of packed columns, buffers and gelsuspension. Antimicrobial agents may be eluted from columns beforechromatographic runs or they may be present in the eluent duringchromatography. Antimicrobial agents which interact with samplesubstances must be avoided if they are to be used in eluents, otherwise anyagent which does not interact with the gel may be used. Some of the morecommonly used antimicrobial agents are described below.
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Antimicrobial agents
ChlorhexidineChlorhexidine (e.g. Hibitane®), 0.002%, is a very effective antimicrobialagent. It is incompatible with only a very few substances but is notrecommended for use with Sepharose. Precipitation may occur on storageof Hibitane with appreciable concentrations of chloride or sulphate ions.
Chloroform, butanol and tolueneThese organic solvents are not recommended as antimicrobial agents forSephadex G-100, G-150 or G-200, as they cause the gel particles to shrinkslightly. In addition they are effective only in very concentrated solutions.They penetrate plastic parts of chromatographic equipment, softening theplastic and leaving the liquid without antimicrobial activity. Oxidizingsubstances should not be used as antimicrobial agents. For sterilization ofgels by autoclaving of the gel see pages 24, 28, 31, 39, 41.
Ethanol 20%Superose, Superdex, Sephacryl HR, Sepharose CL and Sepharose aresupplied in 20% ethanol. This solution can also be used as a bacteriostaticagent for storage of used gel.
Phenyl mercuric saltsPhenyl mercuric salts (acetate, borate, nitrate), 0.001–0.01%, are effectiveonly in weakly alkaline solutions.
Sodium azideNote. The use of sodium azide is discouraged in many countries. It can leadto explosions when disposed of via lead pipe waste disposal systems and isbelieved to be a mutagen.
Sodium azide, NaN3, 0.02–0.05%, is very widely used. It does not interactnotably with proteins or carbohydrates or change their chromatographicbehaviour. Sodium azide interferes with fluorescent marking of proteins,the anthrone reaction and inhibits certain enzymes.
Sodium hydroxideSodium hydroxide, 0.01 M, is an effective bacteriostatic agent. At higherconcentrations (0.1–0.5 M) it is an effective disinfectant even for resistantbacteria such as Pseudomonas. Treatment with sodium hydroxideinactivates endotoxins and will, in many cases, solubilize substances
86
precipitated on the column. Its low toxicity is an advantage that reduces therisk of sample contamination.
Sodium hydroxide is not recommended for use with Sepharose and forstorage of Sephacryl HR.
ThimerosalThimerosal (ethylmercurithiosalicylate e.g. Merthiolate®), 0.005%, is mosteffective in weakly acidic solutions. It is bound to and inactivated bysubstances containing thiol groups. It is not recommended for use withSephacryl HR.
TrichlorbutanolTrichlorbutanol (e.g. Chloretone®), 0.05%, is effective only in weaklyacidic solutions.
Storage of unused mediaUnused media should be kept at +4–25 °C. Note that it is important thatswollen media are not allowed to freeze as the beads may be disrupted byice crystals leading to the generation of fines.
Storage of used mediaUsed media should be stored at +4–8 °C, pH 6-8 in the presence of asuitable bacteriostatic, e.g. sodium azide (0.05%) or ethanol (20%). Notethat it is important that swollen media are not allowed to freeze as thebeads may be disrupted by ice crystals leading to the generation of fines.Thimerosal should not be used with Sephacryl.
Sephadex can also be stored partially shrunk in, for example, 60–70%alcohol. For special purposes Sephadex can be dried and restored to itsoriginal state by the following procedure. The swollen gel is thoroughlywashed with water to remove salts and contaminants. After removal ofexcess water, the gel can be shrunk by successive addition of alcoholsolutions of increasing percentage alcohol. The gel should be allowed toequilibrate in between each addition. Final shrinking should be with 96%alcohol. The gel is then sucked dry on a Büchner funnel and finally dried at60–80 °C. A last wash with diethyl ether reduces the drying time. Clumpsmay appear during the shrinking process but they disperse on re-swelling.Risk for clump formation is reduced by slow shrinking and ether washing.
87
Storage of packed columnsPacked columns should be stored at +4–8 °C in the presence of a suitablebacteriostat. Sodium azide (0.05%) or ethanol (20%) may be used forSuperdex, Superose, Sephacryl, Sepharose and Sepharose CL, but onlysodium azide (0.05%) is recommended for columns packed with SephadexG-types.
For short-term storage, e.g. overnight, the column should be left connectedto the system; a low flow rate through the column will prevent bacterialgrowth.
For long-term storage, the gel bed should be thoroughly cleaned beforere-equilibration with the storage buffer. If ethanol (20%) is used in thestorage buffer, de-gas the ethanol/water mixture well, start theequilibration at a low flow rate and check the back-pressure whileequilibrating the column (water/ethanol mixtures are more viscous thannormal aqueous buffer solutions which will increase the back-pressure).
Disconnect the column from the system, close the bottom tubing of thecolumn and insert the tubing from the top of the column in a Parafilm®
covered vessel (e.g. test tube) containing ethanol (20%). Pre-packed HRcolumns are supplied with a rubber tubing between the inlet and the outletof the column; this tubing may be filled with buffer and re-connected toprevent the column drying out.
Note that columns may need to be re-packed if they are exposed to tempe-ratures widely different from the temperature at which they were packed.
88
89
Problem Cause Remedy
Fault finding chart
Column is clogged Presence of lipoproteins Prior to chromatography,or protein aggregates. precipitate with 10%
Dextran Sulphate or 3%polyvinylpyrrolidone.See cleaning proceduresp 80–84.
Precipitation of proteins Modify the eluent toin the column caused by maintain stability.removal of stabilizingagents during fractionation.
Filter is clogged. Replace the filter. Alwaysfilter samples and bufferbefore use.
Microbial growth has Microbial growth rarelyoccurred in the column. occurs in columns
during use, but stepsshould always be taken toprevent infection of packedcolumns, buffers and gelsuspensions. Store gel in thepresence of 20% ethanolor 0.05% sodium azide.See p 86–87.
The sample Microbial growth has See abovesubstance is occurred in the column.poorly resolvedfrom othermajor peaks
90
Problem Cause Remedy
The sample Sample volume is too Decrease sample volumesubstance is large or the sample has and apply the samplepoorly resolved been improperly applied. carefully. For maximumfrom other resolution do not exceedmajor peaks the sample volumes given
in Table 10. Contaminatedgel surface or top net willspoil sample application.
Sample is too viscous. Dilute the sample with theelution buffer. Keep proteinconcentration below 30 mg/ml.
Improper filtration of the Regenerate the column,sample before application filter the sample andto the column. repeat the chromatography
step.
Column is not mounted Try again with the columnvertically. mounted in a vertical
position. You may need torepack the column.
Column is poorly packed. Check the packing byrunning a colouredcompound, e.g. BlueDextran and observing theband. Repack the column ifnecessary.
Too much sample mass Decrease the sample load.has been loaded onto thecolumn.
The column is dirty. Clean and regenerate thecolumn.
91
Problem Cause Remedy
Detector cell volume is Change the flow cell.too big.
Wrong gel type. Substances which arerelatively close in molecularweight require Superose orSuperdex HR. Checkselectivity curve. Check forpossibility of adsorptioneffects. Consider the effectsof dissociating agents ordetergents if present.
Large mixing spaces in See p 53.or after column.
Column too short. See p 53.
Flow rate too high. See p 54.
Proteins or lipids Choose elution conditionsprecipitated on column. which stabilize the sample.
See p 80–84 for cleaningprocedures.
Uneven temperature in Use a column withthe bed. a water-jacket.
Leading or very Overloaded column. Decrease the sample loadrounded peaks and repeat the run.observed in thechromatogram
Column is poorly packed. Check the packing byrunning a colouredcompound, e.g. BlueDextran and observing theband. Repack the column ifnecessary.
92
Problem Cause Remedy
Earlier elution The sample has altered Prepare fresh sample.profile can not during storage.be reproduced
Larger sample mass load Keep volume and mass ofapplied compared to sample constant whenearlier run. High protein repeating runs.concentration can causeprotein protein interactionresulting in a change inelution profile.
The sample volume is Resolution is dependent ondifferent from earlier the sample volume. Keepruns. sample volume constant
when repeating runs.
Proteins or lipids have Clean and regenerate theprecipitated on the column.column.
Sample has not been Regenerate the column,filtered properly. filter the sample carefully
and repeat this step.
Sample substance The molecular weight –elutes at an or shape is other thanunexpected elution expected.position
Ionic interactions Keep the ionic strengthbetween the protein and above 0.05 M to minimizethe matrix. ionic interactions.
Hydrophobic interactions Reduce the saltbetween the protein and concentration to minimizethe matrix. hydrophobic interactions.
Add a suitable detergent.
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Problem Cause Remedy
Sample substance Retardation is probably If possible decrease theelutes later than due to hydrophobic ionic strength, increase pHexpected or even interactions between or introduce organicafter the total protein and the matrix. solvent in the elutioncolumn volume buffer, e.g. 5%
isopropanol, in order tominimize hydrophobicinteractions.
Late elution and The column has become Clean and regenerate thebroad peaks dirty. column.observed in thechromatogram
Elution times are Leakage before the gel Eliminate the leak.too long, but bed.elution volumes arecorrect
Pump wrongly calibrated. Re-calibrate the pump.
More sample Sample substance Try to optimize yoursubstance is co-elutes with other separation in order torecovered than substances. resolve the peak.expected Alternatively, combine
several techniques toremove contaminantscompletely.
Low recovery of Sample substance may Change the eluent.activity while not be stable in thenormal recovery chosen eluent and isof protein therefore inactivated.
Enzyme separated from Test by pooling fractionsco-factor or similar. and repeating the assay.
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Problem Cause Remedy
Microbial growth. See p 84.
Protein amount in The protein may have Add protease inhibitors tothe eluted fractions been degraded by the buffers to preventis much less than proteases. proteolytic digestion.expected
Microbial growth has See p 84.occurred in the column.
Non-specific adsorption. Try adding ethylene glycol(e.g. 10%) to the buffers toprevent any hydrophobicinteractions.
Elution conditions too Choose elution conditionsharsh. which stabilize the sample.
Specific adsorption. Lectins may bind to sugarresidues in the matrix. Tryspecific elution with ananalogous sugar.
Sample precipitates. May be caused by removalof salts or sample dilution.
More activity is Different assay conditions Use the same assayrecovered than have been used before conditions for all the assayswas applied to the and after the in your purification scheme.column chromatographic step.
Peaks too small Wrong sensitivity range Adjust.on detector.
Sample absorbs poorly at Use a different wavelength.the chosen wavelength.
Recorder Adjust.range incorrectly set.
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Problem Cause Remedy
Sample size is smaller –than intended.
Excessive zone Check the column packingbroadening and re-pack if necessary.
No peaks Monitor switched off. Switch it on.
Monitor not connected Check the electricalto recorder. connections and cables.
UV-lamp not working. Replace the lamp.
Sample not applied. Check the operation of thesample application device.
Range button depressed Check the recorder settings.or no sensitivity set onrecorder.
No flow through Outlet closed. –the column
No flow from pump. With peristaltic pumpscheck the condition of thetubings. Check for leaks atall connections.
Air-lock in outlet tubing See page 69.or bottom-piece.
Clogged end-piece or Bed supports of porousadaptor or tubing. glass or polythene are
prone to clogging by gelparticles.
Reduced or poor See above. –flow through thecolumn
96
Problem Cause Remedy
Bed surface blocked by Remove contaminated gelprecipitated proteins. from the bed surface. Stir
the top 1–2 cm and allowto re-settle as an even layer.
Bed compressed. Special care is needed whenpacking Sephadex G-75 –G-200 (see Table 13). Canalso occur after prolongeduse. Upward elution retardsthe process. Repacking thecolumn may be necessary.
Microbial growth. For prevention see page 84.
Gel not fully swollen See page 61.(Sephadex G-types).
Fines (Sephadex G-types). Do not use a magneticstirrer; it can break thebeads. Fine particles can beremoved by decanting froma settling suspension.
Bubbles in the bed Column packed or stored Small bubbles can often beat cool temperature and removed by passing wellthen warmed up. de-gassed buffer upwards
through the column.Column may need to berepacked. Take special careif buffers are used afterstorage in a fridge orcold-room.Do not allow column towarm up due to sunshine orheating system. A water-jacket is a good safeguard.Use de-gassed buffers.
97
Problem Cause Remedy
Eluent not properly De-gas the eluentde-gassed. thoroughly.
Cracks in the bed Large air leak in column. Check all connections forleaks. Repack the column.
Column violently –mishandled.
Distorted bands as Air bubble at the top of Re-install the adaptorsample runs into the column or in the taking care to avoid airthe bed adaptor. bubbles. See p 70–71.
Particles in eluent or Filter or centrifuge thesample. sample. Protect eluents
from dust.
Particles on the bed Remove small amount ofsurface or uneven bed gel and stir up the topsurface. 1–2 cm, allowing it to
re-settle as an even layer.
Clogged or damaged net Dismantle the adaptor,in upper adaptor. clean or replace the net.
Keep particles out ofsamples and eluents.
Distorted bands as Column poorly packed. Gel suspension too thicksample passes down or too thin. Bed packedbed at a temperature different
from run. Bed insufficientlypacked (too low packingpressure, too shortequilibration). Columnpacked at too highpressure.
98
Problem Cause Remedy
Gel beads in eluent Bed support loose or Replace or re-fasten.broken.
Column operated at too Do not exceed thehigh pressure. recommended operating
pressure for the gelmedium.
99
References1. Gel filtration: A method for desalting and group separation. Nature 183 (1959) 1657–1659, Porath, J., Flodin, P.
2. On the history of the development of Sephadex. Chromatographia 23 (1987) 361–369, Janson, J.-C.
3. Gel filtration. In Protein Purification. Principles, high resolution methods, and applications, pp 63–106. (ed. J.-C.Janson, L.Rydén), VCH Publishers Inc., New York, Weinheim, Cambridge 1989 Hagel, L.
4. The gel filtration behaviour of proteins related to their molecular weights over a wide range. Biochem. J. 96(1965) 595–606, Andrews, P.
5. Dextran gels and their applications in gel filtration. Dissertation 85 pp., AB Pharmacia, Uppsala, Sweden, 1962Flodin, P.
6. Some recently developed fractionation procedures and their application to peptide and protein hormones J. Appl.Chem. 6 (1963) 233–244, Porath, J.
7. A relationship between the molecular weights of macromolecules and their elution volumes based on a model forSephadex gel filtration. Arch. Biochem. Biophys. 107 (1964) 471–478, Squire, P.G.
8. A theory of gel filtration and its experimental verification. J. Chromatogr. 14 (1964) 317–330, Laurent, T.C.,Killander, J.
9. A new calibration procedure for gel filtration columns. J. Biol. Chem. 242 (1967) 3237–3238, Ackers, G.K.
10. Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration anddensity gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductase.Biochim. Biophys. Acta 112 (1966) 346–362, Siegel, L.M., Monty, K.J.
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13. Estimation of molecular size and molecular weights of biological compounds by gel filtration. In Methods ofBiochemical Analysis vol. 18 pp. 1–53 (ed. Glick, D.) Interscience Publishers, New York, London 1970 Andrews,P.
14. Molecular-weight estimation of proteins using Sepharose CL-6B in guanidine hydrochloride. J. Chromatogr. 140(1977) 98–102, Ansari, A.A., Mage, R.G.
15. Fractionation of dextran by the gel filtration method. Makromolekulare Chem. 48 (1961) 160–71, Granath, K.A.,Flodin, P.
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34. Column lifetime of Superose 6 at 37 Celsius and basic pH. J. Chromatogr. 351 (1986) 136–139, Johansson, B-L.,AAhsberg, L.
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36. A new general method for separation of nucleic acids. Prep. Biochem. 4 (1974) 509–522, Petrovic, S.L., Petrovic,J.S., Markovic, R.A. et al.
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Superdex Peptide HR 10/30 17-1453-01Superdex Peptide PE 7.5/300 17-5003-01Superdex Peptide PC 3.2/30 17-1458-01Superdex 75 PC 3.2/30 17-0771-01Superdex 200 PC 3.2/30 17-1089-01
Superose 6 PC 3.2/30 17-0673-01Superose 12 PC 3.2/30 17-0674-01Superose 6 HR 10/30 17-0537-01Superose 12 HR 10/30 17-0538-01
Superdex 75 HR 10/30 17-1047-01Superdex 200 HR 10/30 17-1088-01
HiLoad 16/60 Superdex 30 pg 17-1139-01HiLoad 26/60 Superdex 30 pg 17-1140-01HiLoad 16/60 Superdex 75 pg 17-1068-01HiLoad 26/60 Superdex 75 pg 17-1070-01HiLoad 16/60 Superdex 200 pg 17-1069-01HiLoad 26/60 Superdex 200 pg 17-1071-01
HiPrep 16/60 Sephacryl S-100 HR 17-1165-01HiPrep 26/60 Sephacryl S-100 HR 17-1194-01HiPrep 16/60 Sephacryl S-200 HR 17-1166-01HiPrep 26/60 Sephacryl S-200 HR 17-1195-01HiPrep 16/60 Sephacryl S-300 HR 17-1167-01HiPrep 26/60 Sephacryl S-300 HR 17-1196-01
HiTrap Desalting (5 columns) 17-1408-01Fast Desalting Column HR 10/10 17-0591-01
PD-10 Prepacked Disposable 30 17-0851-01NAP-5 20 17-0853-01NAP 5 50 17-0853-02NAP-10 20 17-0854-01NAP 10 50 17-0854-02NAP-25 20 17-0852-01NAP 25 50 17-0852-02NICK Column 20 17-0855-01NICK Column 50 17-0855-02NICK Spin Columns 20 17-0862-01NICK Spin Columns 50 17-0862-02
Superose 6 prep grade 125 ml 17-0489-01Superose 12 prep grade 125 ml 17-0536-01
Superdex 30 prep grade 25 ml 17-0905-10Superdex 30 prep grade 150 ml 17-0905-01Superdex 30 prep grade 1 litre 17-0905-03Superdex 30 prep grade 5 litres 17-0905-04
Superdex 75 prep grade 25 ml 17-1044-10Superdex 75 prep grade 150 ml 17-1044-01Superdex 75 prep grade 1 litre 17-1044-02Superdex 75 prep grade 5 litres 17-1044-04
Superdex 200 prep grade 25 ml 17-1043-10Superdex 200 prep grade 150 ml 17-1043-01Superdex 200 prep grade 1 litre 17-1043-02Superdex 200 prep grade 5 litres 17-1043-04
Sephacryl S-100 HR 150 ml 17-0612-10Sephacryl S-100 HR 750 ml 17-0612-01Sephacryl S-100 HR 10 litres 17-0612-05Sephacryl S-200 HR 150 ml 17-0584-10Sephacryl S-200 HR 750 ml 17-0584-01Sephacryl S-200 HR 10 litres 17-0584-05Sephacryl S-300 HR 150 ml 17-0599-10Sephacryl S-300 HR 750 ml 17-0599-01Sephacryl S-300 HR 10 litres 17-0599-05Sephacryl S-400 HR 150 ml 17-0609-10Sephacryl S-400 HR 750 ml 17-0609-01Sephacryl S-400 HR 10 litres 17-0609-05Sephacryl S-500 HR 150 ml 17-0613-10Sephacryl S-500 HR 750 ml 17-0613-01Sephacryl S-500 HR 10 litres 17-0613-05Sephacryl S-1000 SF 750 ml 17-0476-01
Sepharose 6B 1 litre 17-0110-01Sepharose 6B 10 litres 17-0110-05Sepharose 4B 1 litre 17-0120-01Sepharose 4B 10 litres 17-0120-05Sepharose 2B 1 litre 17-0130-01Sepharose 2B 10 litres 17-0130-05
Sepharose CL-6B 1 litre 17-0160-01Sepharose CL-6B 10 litres 17-0160-05Sepharose CL-4B 1 litre 17-0150-01Sepharose CL-4B 10 litres 17-0150-05Sepharose CL-2B 1 litre 17-0140-01Sepharose CL-2B 10 litres 17-0140-05
Sephadex G-10 100 g 17-0010-01Sephadex G-10 500 g 17-0010-02Sephadex G-10 5 kg 17-0010-03
Sephadex G-15 500 g 17-0020-01Sephadex G-15 5 kg 17-0020-02Sephadex G-15 500 g 17-0020-02
Column Code No. Media Pack Code No.size
Ordering information
Column Qty. Code No.
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Sephadex G-25 25 g 17-0572-01DNA Grade SF 100 g 17-0572-02
Sephadex G-50 25 g 17-0573-01DNA Grade F 100 g 17-0573-02
Sephadex G-100 25 g 17-0045-01DNA Grade M 100 g 17-0045-02
Sephadex G-100 25 g 17-0574-01DNA Grade SF 100 g 17-0574-02
Handbook. 18-1022-18Gel filtration, Principlesand Methods
Media Pack Code No.size
Media Pack Code No.size
Sephadex G-25 Fine 100 g 17-0032-01Sephadex G-25 Fine 500 g 17-0032-02Sephadex G-25 Fine 5 kg 17-0032-03Sephadex G-25 Medium 100 g 17-0033-01Sephadex G-25 Medium 500 g 17-0033-02Sephadex G-25 Medium 5 kg 17-0033-03Sephadex G-25 Coarse 100 g 17-0034-01Sephadex G-25 Coarse 500 g 17-0034-02Sephadex G-25 Coarse 5 kg 17-0034-03Sephadex G-25 Superfine 100 g 17-0031-01Sephadex G-25 Superfine 5 kg 17-0031-03
Sephadex G-50 Fine 100 g 17-0042-01Sephadex G-50 Fine 500 g 17-0042-02Sephadex G-50 Fine 5 kg 17-0042-03Sephadex G-50 Medium 100 g 17-0043-01Sephadex G-50 Medium 500 g 17-0043-02Sephadex G-50 Medium 5 kg 17-0043-03Sephadex G-50 Coarse 100 g 17-0044-01Sephadex G-50 Coarse 500 g 17-0044-02Sephadex G-50 Coarse 5 kg 17-0044-03Sephadex G-50 Superfine 100 g 17-0041-01Sephadex G-50 Superfine 5 kg 17-0041-03
Sephadex G-75 100 g 17-0050-01Sephadex G-75 500 g 17-0050-02Sephadex G-75 5 kg 17-0050-03Sephadex G-75 Superfine 100 g 17-0051-01Sephadex G-75 Superfine 5 kg 17-0051-03
Sephadex G-100 100 g 17-0060-01Sephadex G-100 500 g 17-0060-02Sephadex G-100 5 kg 17-0060-03Sephadex G-100 Superfine 100 g 17-0061-01Sephadex G-100 Superfine 5 kg 17-0061-03
Standards Pack Code No.size
Gel Filtration LMWCalibration kit 1 kit 17-0442-01Gel Filtration HMWCalibration kit 1 kit 17-0441-01Blue Dextran 2000 10 g 17-0360-01
Contents of the gel filtration calibration kits.
Low Molecular Weight Gel Filtration Calibration Kit
Protein M Weight Stokes’ Radius Å Source
ribonuclease A 13 700 16.4 bovine pancreaschymotrypsinogen A 25 000 20.9 bovine pancreasovalbumin 43 000 30.5 hen eggalbumin 67 000 35.5 bovine serumBlue Dextran 2000
High Molecular Weight Gel Filtration Calibration Kit
Aldolase* 158 000 48.1 rabbit musclecatalase 232 000 52.2 bovine liverferritin* 440 000 61.0 horse spleenthyroglobulin 669 000 85.0 bovine thyroidBlue Dextran 2000
Each Kit contains 50 mg of each protein and 50 mg of Blue Dextran 2000.
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