Gel filtration

Principles and Methods

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8 th edition
18-1022-18
 Gel filtration
Principles and Methods

ISBN 91-97-0490-2-6
 Contents
Introduction ............................................................................................. 4
Principles ................................................................................................. 6
Gel filtration ...................................................................................... 6
Gel media .......................................................................................... 6
Separation by size ............................................................................... 6
Experimental parameters ..................................................................... 7
Sample concepts ........................................................................... 7
Column parameters ...................................................................... 7
Eluent parameters ......................................................................... 8
Running conditions ...................................................................... 8
Characterization of solute behaviour ................................................ 8
Theoretical considerations ........................................................... 12
Deviations from ideal behaviour in gel filtration ............................. 13
Application examples ..................................................................... 14
Fractionation by size ................................................................... 14
Separation of monomers from dimers and higher aggregates ............ 16
MW estimation, native and other forms ........................................ 17
Determination of molecular weight distribution of polymers ............ 18
Determination of equilibrium constants ........................................ 19
Desalting ................................................................................... 19
Industrial applications ................................................................... 21
Properties of gel filtration media ............................................................ 22
Sephacryl HR ................................................................................. 22
Chemical and physical properties ................................................. 22
Chromatographic properties ........................................................ 24
Availability ................................................................................ 27
Further information .................................................................... 27
Superdex ......................................................................................... 27
Chemical and physical properties ................................................. 27
Chromatographic properties ........................................................ 29
Availability ................................................................................ 30
Further information .................................................................... 30
Superose .......................................................................................... 31
Chemical and physical properties ................................................. 31
Chromatographic properties ........................................................ 32
Availability ................................................................................ 34
Further information .................................................................... 34
 Sephadex ......................................................................................... 35
Chemical and physical properties ................................................. 35
Chromatographic properties ........................................................ 36
Availability ................................................................................ 37
Further information .................................................................... 38
Sepharose ....................................................................................... 38
Chemical and physical properties ................................................. 38
Chromatographic properties ........................................................ 40
Availability ................................................................................ 41
Further information .................................................................... 41
Sepharose CL ................................................................................. 41
Chemical and physical properties ................................................. 41
Chromatographic properties ........................................................ 42
Availability ................................................................................ 43
Further information .................................................................... 43
Experimental design ............................................................................... 44
Performance ................................................................................... 44
Resolution ................................................................................. 44
Separation time ........................................................................ 44
High performance gel filtration .................................................... 45
Capacity .................................................................................... 45
Process considerations ................................................................. 47
Choice of gel .................................................................................. 47
The purpose of the experiment ..................................................... 48
Solute characteristics ................................................................... 51
Sample characteristics ................................................................. 51
Choice of running conditions ......................................................... 52
Choice of column ....................................................................... 52
Flow rate ................................................................................... 54
Sample characteristics ................................................................. 55
Eluent ....................................................................................... 58
Optimization ............................................................................. 58
Performing a gel filtration experiment ................................................... 61
Preparing the gel ............................................................................. 61
Pre-swollen media (Sephacryl HR, Superose prep grade,
Sepharose CL and Sepharose) ...................................................... 61
Media which require swelling (Sephadex G-types) .......................... 61
Preparation of Sephacryl HR, Superose prep grade or
Sepharose CL for use in organic solvents ....................................... 62
Gel filtration in organic solvetns ................................................... 63
Packing a column ........................................................................... 63

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

3
 Introduction
For more than thirty years since the introduction of Sephadex (l,2), gel
filtration has occupied a key position in the purification of thousands of
enzymes, polysaccharides, nucleic acids, proteins and other biological
macromolecules. Its continuing value depends partly on the special nature
of the macromolecules studied by the biochemist and partly on the
reliability and simplicity of gel filtration as a separation technique.

Biological macromolecules form a class of substances with special functions

which are controlled in vivo by small changes in the environment. Changes
in the pH, concentrations of metal ions, cofactors etc may have a profound
effect on the molecules being studied and it is clearly necessary to have
available mild separation techniques which operate independently of these
factors. Gel filtration is one of these techniques. A gel filtration separation
can be performed in the presence of essential ions or cofactors, detergents,
urea, at high or low ionic strength, at 37 °C or in the cold room according
to the requirements of the experiment. The stability of gel filtration media
from Amersham Pharmacia Biotech and their inertness towards
biopolymers under a wide range of conditions have made them the stan-
dard in practically every biochemistry laboratory. No less valuable than its
widespread usefulness, is the reliability and simplicity of gel filtration as an
experimental procedure. Little equipment is required, the procedure is
straightforward and good separations and yields are usually obtained even
in the first experiment. The reliability of gel filtration stems from the
reliability of Sephadex and other gel filtration media from Amersham
Pharmacia Biotech, backed by over 30 years of experience in developing
biochemical separation techniques and by thousands of publications
describing their use. Among the new developments described in this hand-
book are Sephacryl HR for standard chromatography and Superdex and
Superose for high speed gel filtration. These new media are the result of
continuing efforts of Pharmacia to improve the separation techniques
which you need for your work.

This handbook is designed as a laboratory aid in the selection and practical

use of gel filtration media from Amersham Pharmacia Biotech. For specific
separation problems where the substances in question may have special
properties, or for theoretical aspects where ideas are undergoing continual
evolution, it is essential to refer to the original literature.

4
Our aim remains to provide better tools for the life sciences and we believe
that to achieve this it is important to maintain close cooperation between
user and manufacturer. We hope that this handbook will aid our
cooperation and that you will find the information helpful. Should we at
any time be able to give you further information, please contact us.

5
 Principles
Gel filtration
In gel filtration molecules in solution are separated according to differences
in their sizes as they pass through a column packed with a chromatographic
medium which is a gel.

Gel media
A gel is a heterogeneous phase system in which a continuous liquid phase,
usually aqueous, is contained within the pores of a continuous solid phase,
the gel matrix. In gels made for gel filtration, the pores have a carefully
controlled range of sizes, and the matrix is chosen for its chemical and
physical stability, and inertness (lack of adsorptive properties).

Gels may be formed from polymers by cross-linking to form a

three-dimensional network; for example Sephadex which is formed by
cross-linking dextran. Some polymers, like agarose, form gels 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 are
covalently bonded to a highly cross-linked agarose gel matrix. Composite
gels are of interest since they can combine valuable properties from more
than one gel-forming system.

Separation by size
The pores in the gel matrix which are filled by the liquid phase are
comparable in size to the molecules we may wish to separate. Relatively
small molecules can diffuse into the gel from a surrounding solution,
whereas relatively large molecules will be prevented by their size from
diffusing into the gel to the same degree. Sufficiently large molecules are
completely unable to diffuse into the gel and are thus confined to the
solution outside.

In a gel filtration column, gel particles in bead form are packed to form a
separation bed through which a buffer solution, the eluent, is passed.

6
Sample molecules which are to be separated are added in solution as a zone
to the top of the bed. The sample zone moves down the bed as eluent is
added to the top. The small molecules which diffuse into the gel beads are
delayed in their passage down the column compared with the large
molecules which cannot diffuse into the gel and move continuously down
the column in the flowing eluent. The large molecules thus leave the
column first followed by the smaller molecules in the order of their sizes.

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

Column parameters
The most important characteristic of a gel filtration column is the way in
which the gel filtration medium has been packed. If the column is evenly
packed so the sample zone is not unnecessarily broadened as it passes down
the column then good results can be obtained. If the column is packed
unevenly then good results will never be obtained from it.

The length of the column, cm, is significant since it affects both the 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 under
otherwise comparable conditions and is chosen depending on the sample
volume.

7
 Eluent parameters
Since the separation depends only on the sizes of the molecules being
separated, the composition of the eluent is unimportant for the separation
mechanism. The eluent can thus be whatever is convenient with regard to
the overall requirements of the experiment. Usually it is a buffer solution
with a well defined pH and ionic composition chosen to preserve the
structure and biological activity of the substances of interest. An ionic
strength of 0.15 or greater is generally used to avoid any unwanted ionic
interactions between the solute molecules and the gel matrix.

Running conditions
The experimental variable of significance which remains to be considered is
the rate at which the eluent flows through the column. This affects not only
the speed at which the separation is obtained but also the resolution which
can be achieved. Generally speaking, the lower the flow rate the better the
resolution, at least for large molecules. Flow rates are measured in simple
volume terms, e.g. ml/min, but when comparing results between columns of
different sizes it is useful to use the linear superficial flow rate, e.g. cm/
hour.

Linear superficial flow rate (cm/h) =

Volume flow rate (ml/min) x 60

Cross-sectional area of the column (cm2)

The flow rate defined in this was is usually simply referred to as the linear
flow rate. Results obtained at the same linear flow rate will be comparable
as far as the effects of flow rate are concerned.

Characterization of solute behaviour

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

8
Fig. 1. Gel filtration of an
oligosaccharide mixture from
the acetolysis and hydrolysis
of cellulose on Sephadex
G-25. The numbers above
the peaks indicate the degree
of polymerisation. Column,
4.5x150 cm; eluent, water;
flow rate, 1.9 ml.cm-2.h-1
(Flodin, P., Aspberg, K.,
Biological Structure and
Function 1 (1961) 345-349.
Reproduced by kind
permission of the Authors
and the Publisher).

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

9
• When very small samples are applied (small enough to be neglected
compared with the elution volume), the position of the peak maximum
in the elution diagram should be taken as Ve (Fig. 2A).
• If the sample volume cannot be neglected compared with the elution
volume, the elution volume is measured from half the sample volume to
the position of the peak maximum (Fig. 2B).
• When very large sample volumes are used (giving a plateau region in the
elution curve), the volume eluted from the start of sample application to
the inflexion point (or half height) of the rising part of the elution peak
should be taken as Ve. This criterion is often incorrectly applied to
samples not giving plateau regions. (Fig. 2C).
In gel filtration solutes normally give symmetrical peaks. Elution volumes
are, therefore, easily determined by these methods. More sophisticated
criteria are seldom useful in practice. Ve is not in itself sufficient to define
the behaviour of the sample substance, since this parameter varies with the
total volume of the packed bed (Vt) and with the way the column has been
packed. By analogy with other types of partition chromatography the
elution of a solute is best characterized by a distribution coefficient (Kd)
The volume of the mobile phase is equal to the void volume, Vo, the elution
volume of molecules which are confined to the mobile phase because they
are larger than the largest pores in the gel. The volume of the stationary
phase, Vs, in gel filtration is equal to Vi, the volume of solvent inside the gel
which is available to very small molecules, i.e. the elution volume of a
solute which will distribute freely between the mobile and stationary sol-
vent phases minus the void volume. Thus Kd represents the fraction of the
stationary phase which is available for diffusion of a given solute species. In
practice the volume of the stationary phase defined in this way is rather
difficult to determine. Methods which can be used often involve
measurement of the elution volumes of radioactive ions such as 23Na. It is
much more convenient to substitute the term (Vt-Vo) for Vs, when we
obtain
Kav = (Ve - Vo)/(Vt-Vo)
Since (Vt-Vo) includes the volume of the gel forming substance, which is
inaccessible to all solute molecules, Kav is not a true partition coefficient
(Fig. 3). However, for a given gel there is a constant ratio of Kav:Kd which
is independent of the nature of the solute or its concentration. Kav is easily
determined and, like Kd, defines solute behaviour independently of the bed
dimensions and packing. Other methods of normalizing data give values
which vary depending upon how well the column is packed. The
approximate relationships between some of these terms are shown in
Figure 4.

10
Fig. 3. Diagrammatic
representation of Vt and Vo.
Note that Vt-Vo will include
the volume of the solid
material which forms the
matrix. (Fischer, L.
Laboratory Techniques in
Biochemistry and Molecular
Biology. Vol. 1 part II. An
introduction to Gel
Chromatography. North
Holland Publishing
Company, Amsterdam.
Reproduced by kind
permission of the Authors
and the Publisher).

Fig. 4. Relationship between

several expressions used for
normalizing elution
behaviour.

11
 Theoretical considerations
The use of gel filtration for the determination of molecular weight or size,
particularly of proteins, is well documented (3). In practice it is found that
for a series of compounds of similar molecular shape and density a
sigmoidal relationship exists between their Kav values and the logarithms
of their molecular weights (MW). Calibration curves constructed in this
way for a particular gel type are often termed selectivity curves (see p 25,
30, 33, 34, 37, 40), and over a considerable range there is a conveniently
linear relationship between Kav and log MW. In ideal gel filtration
behaviour no molecules can be eluted with a Kav greater than 1 or less than
0. If the Kav is greater than 1, some kind of adsorption is indicated. If the
Kav is less than 0 after calibration then channelling in the chromatographic
bed is indicated and the column must be repacked.

Several models have been proposed to describe the behaviour of solutes

during gel filtration (3, 4). Most have regarded the partition of solute
molecules between the gel particles and surrounding fluid as an entirely
steric effect. Thus Flodin (5) divided the gel into permitted and forbidden
regions. The larger the molecular dimensions of the solute the greater is the
proportion of the gel which is forbidden. In the permitted region the con-
centration of solute is assumed to be identical to that in the surrounding
liquid.

The steric approach has been extended in various ways. Porath (6) derived
a theoretical relationship between Kd and Stokes radius assuming that the
pores in Sephadex are conical. In another treatment Squire (7) considered
pores and crevices as well as cones. An interesting approach by Laurent and
Killander (8) assumes that the gel network is composed of rigid rods
randomly arranged. Good correlation was found between Kav and
molecular radius with this model.

All of these models have been successfully applied to predict the elution
behaviour of solutes. However, as has been pointed out by Ackers (9), none
of them may be accurate in a structural sense. Results are equally in
accordance with a formulation where the fractions of the stationary phase
available to molecules of different radii are defined by a Gaussian
probability curve and no assumption is made about the geometric shape of
the pores. From a practical stand-point, for molecular weight determina-
tion, it is still most common to construct a calibration curve and perform
the estimations graphically.

12
 Deviations from ideal behaviour in gel filtration
In ideal gel filtration the only effects which contribute to the behaviour of
solute molecules are steric effects. Insensitivity of gel filtration to the
composition of the eluent is a major advantage and is observed in the vast
majority of cases in aqueous systems at neutral pH in the presence of
electrolyte. However, deviation from a Kav:log MW calibration curve may
still occur if a compound being studied does not have the same molecular
shape as the standards. Under certain conditions, factors other than the size
and shape of the molecules being studied can influence the separation.
These effects can usually be avoided and are generally only significant when
chromatographing highly acidic or basic substances at low ionic strength or
aromatic materials on those gels which have a high matrix content. Often
they have proved highly advantageous. Thus an important application of
Sephadex types G-10, G-15 and G-25 is in the separation of aromatic
peptides and other substances which may differ only slightly in molecular
weight. Further details of these effects can be found under the
chromatographic properties of the different gels (see pages 22–43).

13
 Application examples
Fractionation by size
The special ability of gel filtration to separate macromolecules on the basis
of small differences in size between them makes it an essential fractionation
tool. Here the correct choice of gel and operating conditions are critical if
good results are to be obtained. The peptide separation shown in Figure 5

Fig. 5. Gel filtration of a tryptic digest of citraconylated

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

illustrates this type of experiment. With the proper experimental design, a

protein can be separated from other species differing in molecular weight
by a factor of two or slightly less (Fig. 6). Practical simplicity, excellent
recovery, free choice of elution conditions and straight forward interpreta-

14
tion of results make fractionation by gel filtration an invaluable part of any
purification scheme. Details about experimental design are given on p 44–
60. Practical details are given on p 61–87.

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

15
 Separation of monomers from dimers and
higher aggregates
It is frequently found that proteins with a high degree of homogeneity with
respect to the protein species present contain dimers and higher aggregates.
High resolution gel filtration provides an excellent and gentle means for
separating the monomer from the aggregates, either as part of an analysis
or for their final purification. Gel filtration is thus especially useful as a
polishing step in a more complex purification scheme and finds widespread
use for this purpose in the purification of recombinant and other proteins
in industrial processes (Fig. 7) where protein aggregates must be reduced to
below a specified level in the final product.

Fig. 7. Final purification of a mouse monoclonal antibody, IgG2a, on Superdex 200

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

16
 MW estimation, native and other forms
A calibrated column of Sephacryl HR, Superdex, Superose, Sephadex or
Sepharose provides a simple and well-documented way of determining the
molecular weights of proteins during a natural stage in their purification
(10–14).

Unlike electrophoretic techniques, gel filtration provides a means of

determining the molecular weight or size (Stokes radius) of native or
denatured globular proteins under a wide variety of conditions of pH, ionic
strength, temperature etc., freeing the researcher from the constraints
imposed by the charge state of the molecules. Gel filtration in the presence
of urea or guanidine hydrochloride, dissociating agents which transform
polypeptides and proteins to a random coil configuration reducing
structural differences, has proved particularly useful for molecular weight
determinations. Figure 8 illustrates this kind of application.

Fig. 8. Gel filtration of proteins on Sepharose CL-6B under

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

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

17
molecular weight determination in 6M guanidine hydrochloride over a
period of 10 months, without any change in the elution pattern.
The only special requirements for molecular weight determination are a
column packed with the appropriate gel and a series of protein standards to
calibrate it. Gel Filtration Calibration Kits HMW and LMW from
Pharmacia provide a series of well characterized globular protein standards
carefully chosen to give reliable calibration points in the molecular weight
range 13 700 to 669 000 (see p 87–88). An elution profile obtained with
Sephadex G-200 Superfine is shown in Figure 9.

Fig. 9. Calibration proteins being used to calibrate Sephadex

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

Determination of molecular weight distribu-

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

18
The ability of gel filtration to fractionate molecules according to molecular
size offers an improved method for distribution analysis of many polymers
(15). The elution curve can be recorded continuously or determined by
investigating individual fractions. It is unnecessary to determine molecular
weights of the polymers in the effluent fractions of individual experiments,
as the chromatographic behaviour of the columns is very reproducible. A
calibration curve once determined for the column can be applied to a large
number of runs. The precision of this method of distribution analysis is
very high (16). The method is used for the determination of molecular
weight distributions of e.g. dextrans, polyvinylpyrrolidones, gelatin
preparations and other water-soluble polymers (3).

Determination of equilibrium constants

Gel filtration has proved to be a valuable technique for the study of
chemical equilibria. In the case of slow reactions, where the reactants and
the products can be separated on a gel filtration column, these substances
can be quantitatively determined in the effluent, thereby establishing the
position of the equilibrium (17–19).

Gel filtration can also be used to determine the position of equilibrium of

complex formation where the reactions are rapid. In this case, one of the
reactants is chromatographed in an eluent containing the other reactant.
From the elution curve of the reactants the complex formation can be
studied. This method is of great value in the study of protein binding of low
molecular weight substances such as drugs (20–26) and can be extended to
the study of, for instance, competition of two molecules for the same site
(22). Gel filtration has also been applied to the estimation of reaction rates
(27, 28).

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

19
 chromatography and other LC techniques which use an aqueous mobile
phase.
• phenol removal from preparations of nucleic acids.
• removal of unincorporated nucleotides during DNA sequencing (29, 30).
• removal of free low molecular weight labels, e.g. 125I, FITC, from
solutions of labelled proteins (Fig. 10).

Fig. 10. Removal of free 125l

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

• termination of reactions between macromolecules and low molecular

weight reactants.
• removal of products, cofactors, inhibitors etc. from enzymes.
• phenol red removal from culture fluids prior to anion exchange
chromatography.

20
 Industrial applications
The advantages of gel filtration for the separation of biological molecules
can be equally well realized in large-scale applications as they are in the
research laboratory. In particular, scale-up from pilot-plant to production
capacity has proved a straightforward and trouble-free operation.
Pharmacia has built up considerable experience in this area and you are
invited to contact us directly should you require further information.

21
 Properties of gel filtration
media

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

Fig. 11. Hypothetical partial structure of Sephacryl HR.

22
The porosity of the gel is controlled by the dextran component to give five
types with different fractionation ranges. The wet bead diameter is between
25–75 µm, with an average bead diameter of approximately 50 µm.
Sephacryl HR types are supplied pre-swollen and ready to use. Table 1
summarizes some of the properties of Sephacryl HR.

Table 1. Properties of Sephacryl HR

Gel type Bead size Fractionation range Fractionation range Exclusion

µm Globular proteins Dextrans limit
DNA
Sephacryl S-100 HR 25 – 75 1 000 – 100 000 ND ND
Sephacryl S-200 HR 25 – 75 5 000 – 250 000 1 000 – 80 000 118
Sephacryl S-300 HR 25 – 75 10 000 – 1 500 000 2 000 – 400 000 118
Sephacryl S-400 HR 25 – 75 20 000 – 8 000 000 10 000 – 2 000 000 271
Sephacryl S-500 HR 25 – 75 ND 40 000 – 20 000 000 1078

Chemical stability
Sephacryl HR is stable in all aqueous buffers commonly used in
biochemistry within the pH range 3–11. Short term exposure, e.g. for
cleaning, to pH´s in the range 2-13 has no adverse effect on subsequent
chromatographic properties. The separation properties of the gel are not
affected by detergents e.g. 1% SDS, chaotropic salts or dissociating agents
e.g. 8 M urea and 6 M guanidine HCI.

Strong solutions of NaOH (0.5M) can be used for cleaning if the gel is
washed immediately afterwards with buffer or water. Sephacryl HR can
also be used with organic solvents. A method for transferring Sephacryl HR
from water to organic solvents is described on page 62. Table 2 lists the gel
volumes in various organic solvents for an original 100 ml of gel
sedimented in water.

Table 2. Bed volumes of Sephacryl HR-types in common organic solvents starting from
100 ml sedimented gel.

Gel type Formamide DMSO Methanol Ethanol Acetone

Sephacryl S-100 HR 110 100 100 100 85
Sephacryl S-200 HR 115 110 100 100 85
Sephacryl S-300 HR 100 90 100 95 85
Sephacryl S-400 HR 100 90 100 95 85
Sephacryl S-500 HR 100 95 100 100 90

23
 Physical stability
Sephacryl 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/flow
diagram in Fig. 12.

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

Columns of all dimensions, including wide diameter production columns

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

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

24
including monoclonal antibodies and serum proteins, use Sephacryl S-200
HR or S-300 HR respectively. Sephacryl S-400 HR and S-500 HR are the
choice for separation of larger proteins, polysaccharides, nucleic acids,
DNA fragments and small particles e.g. plasmids.

Table 1 gives the fractionation ranges for Sephacryl HR. Figure 13 shows
the selectivity curves for globular proteins and dextrans respectively.

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

The narrow particle size distribution of Sephacryl HR allows columns to be

packed easily and with high efficiency giving > 9000 plates per metre if the
packing instruction is followed.

A typical example of results obtainable with Sephacryl HR is shown in

Figure 14.

25
Fig. 14. Separation of integral membrane proteins from
human erythrocytes on Sephacryl S-300 HR.
Human erythrocyte membrane were solubilized in phosphate
buffer (0.1 M, pH 7.4) containing SDS (0.1 M), EDTA (1
mM), DTE (1 mM).
Peaks, 1. dimeric anion transporter protein; 2. monomer of
the anion transporter protein; 3. dimeric glycophorin A; 4.
glucose transporter protein; 5. possibly lipids and SDS;
column, K 26/70; bed height, 61 cm; sample concentration, 2
mg/ml; sample volume, 2 ml; eluent, phosphate buffer (0.1 M,
pH 7.4) containing SDS (0.05 M), EDTA (1 mM), DTE (1
mM); flow rate, 58 ml/hour. (Work by E. Greijer and P.
Lundahl, Institute of Biochemistry, University of Uppsala,
Uppsala, Sweden.)

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

FPLC and industrial scale

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

26
 Availability
Sephacryl HR types are supplied pre-swollen as a suspension containing
20% ethanol in packs of 750 ml. Sephacryl S-100, S-200 and S-300 are
also supplied pre-packed in XK-columns with the designation HiLoad
columns.
Further information
Further information on Sephacryl HR is given in the data sheet: “Sephacryl
High Resolution gel filtration media” with details of applications. For
information on scale-up and the operation of large scale chromatography
systems, please contact Pharmacia.

Superdex
Chemical and physical properties
Superdex is based on highly cross-linked porous agarose beads to which
dextran has been covalently bonded. Superdex is thus a composite gel (Fig.
15) in which the high physical and chemical stabilities are chiefly due to the
agarose matrix and the gel filtration properties are principally determined
by the dextran chains.

Different types with different fractionation ranges are available. Superdex

30 prep grade, Superdex 75 prep grade and Superdex 200 prep grade have

Fig. 15. Structure of

Superdex. Dextran chains are
covalently linked to a highly
cross-linked agarose matrix.

27
an average wet bead diameter of 34 µm with a range of 24–44 µm.
Superdex peptide, Superdex 75 and Superdex 200 for high performance gel
filtration have a bead size of 13 µm (mean). Table 3 summarizes some of
the properties of Superdex and Superdex prep grade; HiLoad columns can
be used both with standard chromatography systems and with FPLC sys-
tem. Superdex packed in HR columns is for use with FPLC system.

Table 3. Properties of Superdex

Gel type Bead size µm Fractionation range Fractionation range

Globular 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 – 30 000
Superdex 75 11 – 15 3 000 – 70 000 500 – 30 000
Superdex 200 prep grade 24 – 44 10 000 – 600 000 1 000 – 100 000
Superdex 200 11 – 15 10 000 – 600 000 1 000 – 100 000

Chemical stability
Superdex can be used with all aqueous buffers commonly used in
biochemistry within the pH range 3–12, and withstands strong bases, e.g.
0.2 M NaOH, and acids, e.g. 0.01 M HCI or 0.1 M acetic acid. Short term
exposure to extremes of pH (1-14) e.g. for cleaning, has no adverse effect
on subsequent chromatographic properties. The separation properties of
the gel are not affected by detergents, e.g. 1% SDS, chaotropic salts or
dissociating agents, e.g. 8 M urea or 6 M guanidine HCI.

Strong solutions of NaOH (0.5 M) or HCl (0.5 M) can be used for cleaning
if the gel is immediately washed with buffer or water. The high stability of
Superdex prep grade makes it very suitable for use in industrial processes
where high flow rates and effective cleaning-in-place are required.

Superdex can also be used with organic solvents. A method for transferring
Superdex from water to organic solvents is described on page 62.

Physical stability
Columns pre-packed with Superdex may be used at temperatures in the
range +4–40 °C. Exposure to temperatures outside this range will destroy
the efficiency of the packed bed and the column will need to be re-packed.

Typical flow rates for a bed 60 cm long packed with Superdex prep grade

28
Fig. 16. Pressure drop as a function of flow rate for HiLoad
columns packed with Superdex prep grade. Bed height
approximately 60 cm in distilled water at 25 °C. To calculate
volumetric flow rate, multiply linear flow rate by cross-sectional
area of column (2 cm2 for XK16, 5.3 cm2 for XK26).

can be seen in the pressure/flow diagrams in Figure 16.

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

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

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

29
Fig. 17. Selectivity curves for Superdex.

the separation of a wide range of recombinant DNA products. Superdex

200 prep grade is a suitable choice when the molecular weight of the pro-
tein of interest is unknown. It is especially suitable for the separation of
monoclonal antibodies from dimers and from contaminants of lower
molecular weight, for example albumin and transferrin.

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

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

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

30
 Superose
Chemical and physical properties
Superose (32) is composed of highly cross-linked porous agarose beads in
two different particle sizes and two different fractionation ranges.
The average wet bead diameter is 10 ±2 µm or 13 ±2 µm for Superose 6
and Superose 12 respectively, and 20–40 µm for the corresponding prep
grades. Table 4 summarizes some of the properties of Superose.

Table 4. Properties of Superose

Gel type Bead size µm Fractionation range Fractionation range

Globular proteins Dextrans

Superose 12 prep grade 20 – 40 1 000 – 300 000 ND

Superose 12 8 – 12 1 000 – 300 000 ND
Superose 6 prep grade 20 – 40 5 000 – 5 000 000 ND
Superose 6 11 – 15 5 000 – 5 000 000 ND

Chemical stability
Superose can be used with all aqueous buffers commonly used in
biochemistry within the pH range 3–12. Short term exposure to extremes
of pH (1–14) e.g. for cleaning, has no adverse effect on subsequent
chromatographic properties and the media withstand strong bases (33, 34)
e.g. 0.2 M NaOH, and acids e.g. 0.01 M HCI and 1 M acetic acid.

The separation properties of the gels are not affected by detergents e.g. 1%
SDS, chaotropic salts or dissociating agents e.g. 8 M urea and 6 M
guanidine HCl.

Strong solutions of NaOH (0.5 M) or HCl (0.5 M) can be used for cleaning
if the gel is immediately washed with buffer or water. Superose can also be
used with organic solvents. Superose prep grade may be transferred from
water to organic solvents by the method described on page 62.

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

31
Recommended flow rates for Superose prep grade are in the range 0.3–0.5
ml/min for a column 16 mm i.d., 50 cm long. Pressure/flow diagrams for
Superose 6 HR 10/30 and Superose 12 HR 10/30 columns are shown in
figure 18.

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

The mechanical rigidity of Superose prep grade allows even relatively

viscous eluents, such as 8 M urea, to be run at practicable flow rates.

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

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

32
Fig. 19. Selectivity curves of Superose 6 and 12, globular
proteins.

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

33
 Fig. 21. Selectivity curves
of Superose 6 and 12,
DNA-fragments.

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

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

34
 Sephadex
Chemical and physical properties
Sephadex is a bead-formed gel prepared by cross-linking dextran with
epichlorohydrin (Fig. 22). Table 5 lists the different G-types of Sephadex
and their physical properties.

Fig. 22. Partial structure of Sephadex.

Sephadex swells in aqueous solutions, and in dimethylsulphoxide and

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

35
Table 5. Properties of Sephadex.

Fractionation Fractionation range

Gel type Dry bead size range Dextrans Swelling
µm Globular factor ml/g
proteins
Sephadex G-10 40 – 120 – 700 – 700 2– 3
Sephadex G-15 40 – 120 – 1 500 – 1 500 2.5 – 3.5
Sephadex G-25 Coarse 100 – 300 1 000 – 5 000 100 – 5 000 4– 6
Sephadex G-25 Medium 50 – 150 1 000 – 5 000 100 – 5 000 4– 6
Sephadex G-25 Fine 20 – 80 1 000 – 5 000 100 – 5 000 4– 6
Sephadex G-25 Superfine 10 – 40 1 000 – 5 000 100 – 5 000 4– 6
Sephadex G-50 Coarse 100 – 300 1 500 – 30 000 500 – 10 000 9 – 11
Sephadex G-50 Medium 50 – 150 1 500 – 30 000 500 – 10 000 9 – 11
Sephadex G-50 Fine 20 – 80 1 500 – 30 000 500 – 10 000 9 – 11
Sephadex G-50 Superfine 10 – 40 1 500 – 30 000 500 – 10 000 9 – 11
Sephadex G-75 40 – 120 3 000 – 80 000 1 000 – 50 000 12 – 15
Sephadex G-75 Superfine 10 – 40 3 000 – 70 000 1 000 – 50 000 12 – 15
Sephadex G-100 40 – 120 4 000 – 150 000 1 000 – 100 000 15 – 20
Sephadex G-100 Superfine 10 – 40 4 000 – 100 000 1 000 – 100 000 15 – 20
Sephadex G-150 40 – 120 5 000 – 300 000 1 000 – 150 000 20 – 30
Sephadex G-150 Superfine 10 – 40 5 000 – 150 000 1 000 – 150 000 18 – 22
Sephadex G-200 40 – 120 5 000 – 600 000 1 000 – 200 000 30 – 40
Sephadex G-200 Superfine 10 – 40 5 000 – 250 000 1 000 – 150 000 20 – 25

Chromatographic properties
The G-types of Sephadex differ in their degree of cross-linking and hence in
their degree of swelling and fractionation range (Fig. 23 and 24).

Sephadex is available in different particle size grades. The different grades

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

36
Fig. 23. Selectivity curves of Sephadex G-types Superfine,
globular proteins.

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

proteins.

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

37
 Further information
Further information on Sephadex with details of applications is given in the
data sheet: “Sephadex gel filtration media”. For information on scale-up
and the operation of large scale chromatography systems, please contact
Pharmacia.

Sepharose
Chemical and physical properties
Sepharose is a bead-formed gel prepared from agarose. In its natural state
agarose occurs as part of the complex mixture of charged and neutral
polysaccharides referred to as agar. The agarose used to make Sepharose is
obtained by a purification process which removes the charged
polysaccharides to give a gel with only a very small number of residual
charged groups.

A gel forms spontaneously as a hot solution of agarose is cooled. The

individual polysaccharide chains form double helices which subsequently
aggregate to form bundles during the formation of a stable gel (Fig. 25).

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

The individual polysaccharide chains may be represented as polymers of the

repeating unit shown in Figure 26.

38
Fig. 26. Structure of the repeating unit of agarose. Note the presence of the unusual sugar
3,6-anhydro-L-galactose.

Sepharose is available in three types with different agarose concentrations

and different fractionation ranges (Table 6).

Table 6. Properties of Sepharose.

Gel type Approx. % Bead size Fractionation range Fractionation range

agarose µm Globular proteins. Dextrans
Sepharose 6B 6 45 – 165 10 000 – 4 000 000 10 000 – 1 000 000
Sepharose 4B 4 45 – 165 60 000 – 20 000 000 30 000 – 5 000 000
Sepharose 2B 2 60 – 200 70 000 – 40 000 000 100 000 – 20 000 000

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

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

39
The mechanical strength of Sepharose depends on the agarose concent-
ration in the beads. Thus Sepharose 6B, (approx. 6% agarose) is considera-
bly stronger than Sepharose 2B (approx. 2% agarose). Practical details on
flow rates obtainable with Sepharose are given on page 66.

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

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

globular proteins.

makes it difficult to estimate the exclusion limits for proteins with

confidence and these figures should be taken as a guide only.

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

40
 Availability
Sepharose is supplied as a ready-to-use suspension in 1 litre packs
containing 20% ethanol as a preservative.

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

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

Sepharose CL is available in three types corresponding to the parent gels

Sepharose 2B, 4B and 6B (Table 6).

Chemical stability
Stability in aqueous media
Sepharose CL can be used in aqueous media in the range pH 3–13. Its
stability in alkaline media is particularly high (37). Short term exposure to
extremes of pH (2–14) e.g. for clening, has no adverse effect on subsequent

Table 7. Stability of Sepharose CL in the presence of 3 M KSCN.

Sample Carbohydrate concentration

µg/ml
First effluent 6.5
After 24 h exposure <0.5
After 48 h exposure <0.5
After 72 h exposure <0.5

41
chromatographic properties. Under oxidizing conditions, limited hydrolysis
of the polysaccharide chains may occur.
The data in Table 7 demonstrate the stability of Sepharose CL-4B in
solutions of chaotropic ions. Sepharose CL-4B was packed in a Pharmacia
Column K 16/40 (bed volume 57.4 ml) and equilibrated with sodium
phosphate buffer (0.02 M) containing NaCl (0.15 M). One bed volume of
the same buffer containing the chaotropic salt KSCN (3 M) was passed
through the column and the carbohydrate content of the effluent was
estimated by the anthrone reaction. The column was allowed to stand
overnight in the KSCN solution and the determination was repeated. After
the initial elution the concentration of carbohydrate in the effluent was
below the lower limit of sensitivity of the detection method.

Stability in organic solvents

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

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

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

42
 Adsorption
Sepharose CL shows even lower non-specific adsorption than the parent
non cross-linked gel, in part due to its extremely low content of charged
groups. Sepharose CL, for example, has been shown to separate nucleic
acids in order of molecular size (39).

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

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

43
 Experimental design
Performance
Resolution
The principle objective of any fractionation experiment is to achieve
adequate separation of the components of interest. In chromatography, the
degree of separation, or resolution, is defined by
distance between separated zones
Resolution (Rs) =
average zone width
Values of Rs greater than about 1.5 indicate baseline separation when the
two components are present in approximately equal proportions. Greater
resolution is needed if one of the components is present in considerable
excess. Resolution can be increased in a number of ways (40) e.g. using a
longer column, a gel medium with smaller particles and/or greater
selectivity, a small sample volume, a low flow rate etc. However, the
conditions which lead to the highest resolution are usually in conflict with
other experimental objectives and a careful evaluation of the overall
requirements is necessary. In particular, there is no need to design an
experiment to give the ultimate in resolution unless the circumstances
actually demand it.

In many experiments adequate resolution is not difficult to achieve. More

consideration can then be given to other objectives of a well designed
experiment. In the case of preparative separations, these may be to achieve
maximum recovery with least sample dilution in the shortest possible time.
For analytical work we may be more interested in reducing sample
consumption and maximizing run-to-run reproducibility.

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

44
 High performance gel filtration
In high performance gel filtration, specialized media and equipment are
employed to get the best possible combination of resolution and speed.
Since zone broadening is a principle cause of loss of resolution, every effort
is made to reduce dispersion. By using small bead sizes and columns packed
to the highest possible efficiency, combined with small sample volumes, and
detectors and other equipment designed for the purpose, excellent results
can be obtained in the fractionation of complex samples in less than an
hour.

Capacity
The quantity of sample which can be fractionated successfully depends on
its volume and concentration. These variables affect the resolution
differently.
Sample concentration
The concentration of protein in the sample has little direct result on resolu-
tion (Fig. 28). However, even moderately concentrated solutions of nucleic

Fig. 28. Influence of

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

45
acids and polysaccharides may be so viscous relative to the eluent that
viscous fingering in the column leads to a catastrophic loss of resolution
(5). Within the limits set by viscosity, see page 57, any sample concent-
ration may be used. Using a concentrated sample is thus a simple and
effective way of increasing the preparative capacity of a procedure.

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

Fig. 29. Influence of sample volume on the resolution of

transferrin and IgG on Superdex 200 prep grade.

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

46
Table 8. Recommended sample volumes as a per cent of the total bed volume for
good resolution. Many separations will show adequate resolution for larger sample
volumes.

Medium Recommended sample volume

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

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

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

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

47
 The purpose of the experiment
Analysis
Analytical applications place special demands on the run-to-run reproduci-
bility of the gel filtration column. It is thus specially important to choose a
separation medium which gives a perfectly stable bed under the conditions
of the analysis. Columns pre-packed with Sephacryl HR, Superdex or
Superose are suitable, both for reproducibility and efficiency. Pre-packed
columns are recommended to ensure the maximum column efficiency
which is essential for the highest resolution. When the column is to be
packed in the laboratory, special care should be taken to obtain a stable,
well-packed bed and the packing recommendations should be followed in
detail (see p 63–71).

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

Molecular weight determination and molecular weight

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

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

48
 Preparative
Group separations and desalting
The choice of gel is most easy for group separations such as desalting and
buffer exchange, since there is a large difference in molecular weight
between the two groups of components and complete resolution is not
difficult to achieve.

The gel is chosen so that the high molecular weight substances are eluted at
the void volume (Kav = 0). This will give the minimum zone broadening
and dilution and reduce the time the components of interest are on the
column. The low molecular weight substances should be eluted near Vt
(Kav = 1).

Sephadex G-25 Fine is the recommended gel for the majority of desalting
applications. It is easy to work with and has excellent separation capacity
for desalting molecules down to about 5000 MW.

Table 9. Pre-packed disposable columns for group separations

Column designation Medium Max. sample volume

HiTrap Desalting Sephadex G-25 Superfine 1.5 ml
Fast Desalting Sephadex G-25 Superfine 0.5 ml
PD-10 Columns Sephadex G-25 Medium 2.5 ml
NAP 5 Columns Sephadex G-25 DNA grade 500 µl
NAP 10 Columns Sephadex G-25 DNA grade 1.0 ml
NAP 25 Columns Sephadex G-25 DNA grade 2.5 ml
NICK Columns Sephadex G-50 DNA grade 100 µl
NICK Spin Columns Sephadex G-50 DNA grade 150 µl
cDNA Spun Columns Sephacryl S-300 HR 100 µl
Miniprep Spun Columns Sephacryl S-400 HR 50 µl
SizeSelect-400 Spun Columns Sephacryl S-400 HR 100 µl

Small disposable columns (Table 9) should be used if there is a risk of

biological or radio-active contamination of the gel to reduce hazards in
handling used materials. Disposable columns are also recommended when
many small samples must be treated or when no risk of carry-over between
one sample and another can be tolerated.

The Fast Desalting Desalting Column HR 10/10 is recommended for rapid

buffer exchange and other group separations of samples up to 2 ml in high
performance separation schemes.

Group separations of DNA and oligonucleotides

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

49
nucleotides etc. NAP-columns (Sephadex G-25 DNA grade) are
recommended for desalting and buffer exchange and NICK-columns
(Sephadex G-50 DNA grade) for removal of unincorporated nucleotides.
cDNA or Size Select-400 spun columns should be used for group separation
of cDNA from small molecules like linkers and adapters. Group separation
of plasmids from proteins, RNA and NaOH in DNA sequencing may be
carried out on Sephacryl S-400 packed in Miniprep spun columns thus
eliminating the need for phenol extraction (30).

Larger quantities of plasmids are quickly and conveniently prepared by gel

filtration on Superose 6 prep grade (44).

Preparative fractionation
Fractionation by size using gel filtration is a natural part of many
procedures for purifying proteins and other biomacromolecules. The exact
choice of gel will depend on the circumstances. The following guide-lines
should be useful in most situations.

Gel filtration is used in the early stages of a purification scheme to remove

high molecular weight contaminants which may cause problems later on
and to adjust the ionic composition of the sample to the pH and ionic
strength required for a subsequent chromatographic step. The sample will
be relatively crude and the ability to clean the gel from lipids and/or protein
contamination is important. The sample will also be relatively large in
volume and a large bed volume may be needed. The likely presence of
proteases or other enzymes which might degrade the target molecules also
makes it desirable to work with high flow rates. These considerations
suggest that a gel from the Sephacryl HR series will generally be most
suitable.

Later on in the purification scheme, the sample will be simpler in

composition and remaining impurities may require high performance gel
filtration media to remove them. Pre-packed columns containing Superdex
or Superose are available which ensure maximum resolution for
particularly difficult fractionations.

Removal of dimers and aggregates

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

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

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

Type of molecules to be separated

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

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

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

51
pH or other conditions of the sample will cause inactivation or even
precipitation. If the sample precipitates in a gel filtration column, the
column will be blocked, possibly irreversibly, and the sample may be lost.
Care should always be taken to work within the solubility and stability
range of the sample. The free choice of eluent in gel filtration enables this
requirement to be met in almost every case.

The composition of the eluent does not affect the choice of gel except when
it can be expected to alter the conformation of the molecules to be
separated or when it places special demands on the stability of the gel. Gel
filtration in dissociating eluents, e.g. 6 M guanidine hydrochloride or urea,
is extremely useful for molecular weight determination (14). However,
because of the more extended configuration of proteins and polypeptides in
these eluents, a more porous gel is usually required than predicted from
selectivity curves prepared for native globular proteins. Thus, in many
cases, Sephacryl HR or Sepharose CL will be found the most appropriate
gels. Their high chemical and physical stability also makes them
particularly suitable for use in these eluents.

Detergents are particularly useful as solubilizing agents for proteins with

low aqueous solubility, for example, membrane components (48–50). Once
again the effect of these agents on protein conformation usually requires a
more open gel to be used for gel filtration. Detergents do not appear to
influence the pore structure of the gels (51).

The more recently developed media, Sephacryl HR, Superdex and Superose,
are in general more suitable than the traditional media for work in
dissociating media, eluents containing organic solvents and eluents of
extreme pH.

Choice of running conditions

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

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

52
The dead volumes at the outlet and inlet should always be as small as
possible. Many simple columns have large dead volumes and should,
therefore, be avoided. Bed supports made from coarse sintered glass or
glass wool cannot be recommended for long term use, because they soon
become clogged, are difficult to clean and can give artifactual results (52).

Pharmacia has developed a series of standard columns suitable for gel

filtration. Their design is based on many years experience in the field of gel
filtration. They are manufactured from materials which do not cause
destruction of labile biological substances. All are easy to dismantle and
reassemble to allow thorough cleaning, which is particularly important
when handling biological samples.

Other important characteristics include

• Dead space at the outlet of less than 0.1% of the column volume.
Minimizes dilution and prevents remixing of separated zones.
• Advanced design bed 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 resistances

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

Column dimensions
The resolution of two separated zones in gel filtration increases as the
square root of column length. Long columns should, therefore, be used to
obtain the best resolution in analytical fractionation. Bed heights of greater
than 1 m are seldom required. If a very long bed is judged to be necessary,
the effective bed height can often be increased simply by recycling (Fig. 30)
or by using columns coupled in series. Both techniques require the use of
adaptors or end pieces (p 70).

For analytical purposes a column with an internal diameter of

approximately 1.0 cm often proves satisfactory. In general the length of
column is decided by the resolution required and the diameter by the
sample volume (see “Sample size” page 55).

53
 Rec102

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

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

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

54
Using media with small particles will give increased efficiency and higher
resolution. This may allow higher flow rates to be used, but usually at the
expense of sample capacity.

If peaks are well separated at a low flow rate using a long column, the
excess resolution may be traded off for speed. The flow rate can be
increased and a shorter column can be used, or alternatively more sample
can be applied.

For preparative purposes the advantage of a higher flow rate (and

consequently a faster separation) often outweighs the loss of resolution in
the chromatographic run. Full details of the maximum permissible flow
rates of the various gel types, governed by their mechanical properties, are
given on page 66.

Sample characteristics
Sample size
For analytical purposes and difficult fractionation experiments where
maximum resolution is required the starting zone must be narrow relative
to the length of the column. A sample volume of 0.5–5% of the bed volume
is recommended, see Table 8. Smaller volumes do not normally improve
resolution.

In group separations and some fractionation experiments, where peaks are

well resolved, it is often appropriate to improve the experimental design by
increasing the sample size. In order to minimize sample dilution, which is
an inevitable consequence of gel filtration, a maximum sample volume
should be used within the limits set by the separation distance. Figure 31
shows how, if no zone-broadening were to occur during passage down a
column, the maximum sample volume could be as great as the separation
volume (VSep).

VSep = VeB - VeA

However, due to eddy diffusion, non-equilibrium between the stationary

phase and the mobile phase, and longitudinal diffusion in the bed, the
zones will always be broadened (40). The sample size must, therefore,
always be smaller than the separation volume. In desalting and buffer
exchange volumes up to approximately 30% of the total bed volume (Vt)

55
 Fig. 31. Elution curves for
different sample sizes. The
top diagram corresponds to
the application of a small
sample. The centre diagram
corresponds to the maximum
sample size giving complete
separation if no zone
broadening were to take
place. The bottom diagram
corresponds to the maximum
sample volume to be applied
to obtain complete
separation in the conditions
of the experiment. The
shaded areas correspond to
the elution profiles that
would be obtained if no zone
broadening were to occur.

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

Fig. 32. Desalting of albumin

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

56
When designing an optimized desalting system, the volume of gel required
should be packed in a short, wide column rather than a long, narrow one.
This allows more rapid recovery of desalted materials since higher volume
flow rates can be achieved with the shorter column.

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

Fig. 33. Elution diagrams

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

57
In practice the relative viscosities of sample and eluent should not differ by
more than a factor of about 2, corresponding to a protein concentration in
the sample of about 70 mg/ml when a dilute aqueous buffer is used as the
eluent. Approximate relative viscosities can be quickly estimated by
comparing emptying times from a pipette.

Eluent
Eluent composition does not directly influence the resolution which can be
obtained in gel filtration. Completely uncharged substances may be eluted
with distilled water. For chromatography of substances carrying charged
groups an eluent containing a buffer, e.g. Tris-HCl, sodium phosphate etc.,
is often used to control pH, and an ionic strength of at least 0.02 for
Sephadex and Sepharose or 0.15 for Sephacryl HR is recommended to
safeguard against possible ionic interactions with the gel matrix. Sodium
chloride can be used for this purpose. It should also be noted that some
proteins may precipitate in solutions of low ionic strength.

If the product is to be lyophilized, volatile buffers such as ammonium

acetate, ammonium bicarbonate or ethylenediamine acetate can be used. In
desalting, the separation volume is so large that, in general, charged
substances can also be treated with distilled water as eluent. Complete
removal of salt is not possible, but the amount of ions excluded and,
therefore, eluted with the HMW fraction is so small it can be neglected in
most cases.

The most important consideration in the choice of eluent in gel filtration is

its effect on the sample molecules. The pH and ionic composition of the
buffer, and the presence of dissociating media or detergents can cause
conformation changes, dissociation of proteins into subunits, dissociation
of enzymes and cofactors, dissociation of hormones and carrier proteins
etc. which must be taken into account when choosing the gel and when
interpreting the results of an experiment.

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

58
 Fig. 34. A model gel filtration
experiment to demonstrate
optimization of the
separation of model proteins
A, B and C.

resolution. Protein C is well separated from protein B and this particular

separation can be speeded up by a factor of 50. (An alternative view of
optimization could be to retain the flow rate and increase sample size to
utilise more completely the separation volume between proteins C and B).

In the first stage of the optimization, increasing the flow rate, the time for
separating protein C from B is reduced from about 30 hours to about 2.5
hours. The resolution (Rs) is still 2.25. The bed height (L2) required to give
Rs = 1 can be calculated from

L1
L2 =
Rs1

59
where Rs1 is the resolution obtained with a column length L1. In this
example L2=17 cm. Reducing the bed height to about 20 cm should still
give adequate resolution of protein C and protein B. The result obtained
with a bed height of 19.5 cm at 24.5 ml.cm-2.h-1 is shown in Figure 34. The
separation time for protein C and protein B has been reduced from 30
hours to 35 minutes. For a full discussion of the optimization of gel filt-
ration see (40).

60
 Performing a gel filtration
experiment
Gel filtration is essentially simple to perform once a well packed column
has been obtained. Providing that a column is used and maintained
carefully it can be expected to give many months of reliable service.

Preparing the gel

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

Media which require swelling (Sephadex G-types)

Sephadex is supplied as a dry powder and must be allowed to swell in
excess solvent before use. During swelling excessive stirring should be
avoided as it may break the beads. Do not use magnetic stirrers. For all
Sephadex G-types, the process of swelling can be accelerated by using a
boiling water bath which also serves to deaereate the buffer (Table 10).

Table 10. Bed volume and swellings times for Sephadex G-types.

Gel type Approx. bed Swelling time (h)

volume (ml/g) 20 °C 90 °C
Sephadex G-10 2 – 3 3 1
Sephadex G-15 2.5 – 3.5 3 1
Sephadex G-25 (all grades) 4 – 6 3 1
Sephadex G-50 (all grades) 9 – 11 3 1
Sephadex G-75 (all grades) 12 – 15 24 3
Sephadex G-100 (all grades) 15 – 20 72 5
Sephadex G-150 20 – 30 72 5
Sephadex G-150 Superfine 18 – 22 72 5
Sephadex G-200 30 – 40 72 5
Sephadex G-200 Superfine 20 – 25 72 5

61
 Preparation of Sephacryl HR, Superdex prep grade,
Superose prep grade or Sepharose CL
for use in organic solvents
The aqueous medium in which a gel is swollen can be exchanged for a
variety of organic solvents. (Note: Because the gel structure of Sepharose is
only stabilized by hydrogen bonding, it is not suitable for gel filtration in
many organic solvents. However, Sepharose CL can be used quite safely in
a wide range of organic solvents.) To ensure efficient replacement of the
water by the required solvent, the transfer must be made through a graded
series of solvent mixtures. Thus to transfer from one pure solvent (A) to
another pure solvent (B), the gel is transferred first to 70% A/30% B then
to 30% A/70% B and finally to pure B. If A and B are not mutually

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

miscible, the transfer is made via an intermediate solvent e.g. from water to
chloroform via acetone (Fig. 35).

Transfer the required amount of gel to a sintered glass Buchner funnel and
remove the excess aqueous medium by gentle suction. Add the next solvent
(see Fig. 35) and resuspend the gel by gentle stirring. Suck off the excess
solvent and resuspend in the same solvent. Repeat the process with the next
solvent of the series, allowing two resuspensions and proceed until the
required solvent composition is reached.

Note that the gel volume of Sephacryl HR may be reduced by up to 15%

on transfer to organic solvents (Table 2).

62
 Gel filtration in organic solvents
After transferring the gel to the solvent of choice it may be packed in the
usual way (see below). Sepharose CL floats in dense solvents, e.g.
chloroform, and in these cases packing should be carried out as described
in the booklet “Sephadex LH-20. Chromatography in organic solvents”.
Pharmacia columns SR 10/50, SR 25/45 or SR 25/100 should be used.
Flow rates will depend on the viscosity of the eluent. Certain solvents
which disrupt hydrogen bonds, e.g. DMSO, may cause softening of
Sepharose CL so that flow rates become impracticably low.

Packing a column (Fig. 36-43)

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

Packing Sephadex G types, Sepharose and Sepharose CL

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

Fig. 36.

63
 The suspension should be de-gassed under vacuum (Fig. 36) This is not
necessary for Sephadex which has been swollen on a boiling water
bath. The gel suspension should reach the temperature of column
operation before packing is begun.
2. Mount the column on a stable laboratory stand. It is important to
avoid locations which are exposed to draughts or direct sunlight and
which can cause temperature changes and the formation of bubbles in
a packed column. Use of degassed buffers and a filled water-jacket will
also help safeguard against the effects of rapid temperature changes.
3. Ensure that there are no air bubbles trapped in the dead space under
the net by drawing water (or 20% ethanol) through it. This is best
done by submerging the plunger in a beaker of water (or 20% ethanol)
and attaching the tubing to a pump or a syringe. Alternatively, inject
eluent into the outlet tubing until it passes through the bed support net
(Fig. 37). When the dead space is properly filled, close the outlet
tubing.

Fig. 37.

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

64
Fig. 38. Fig. 39.

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

65
Table 11. Approx maximum flow rates and pressures. Data has been obtained from
columns 2.5 cm diameter with a bed height of 30 cm.

Gel type 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 30
Sepharose CL-4B 120 2.17 26
Sepharose CL-2B 50 1.25 15
Sepharose 6B 200 1.16 14
Sepharose 4B 80 0.96 11.5
Sepharose 2B 40 0.83 10
Sephadex G-10 - G-50 These gels obey
Darcy’s Law
(see p 78)
Sephadex G-75 160 6.4 77
Sephadex G-75 Superfine 160 1.5 18
Sephadex G-100 96 4.2 50
Sephadex G-100 Superfine 96 1.0 12
Sephadex G-100 36 1.9 23
Sephadex G-150 Superfine 36 0.5 6
Sephadex G-200 16 1.0 12
Sephadex G-200 Superfine 16 0.25 3

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

Equilibrating the bed

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

66
Fig. 40. Definition of operating pressure (A-D) and sample application from a sample
reservoir (D).
A and B. Pressure (cm water) is measured as the distance between the free surface in the
column or reservoir and the end of the outlet tubing.
C and D. Pressure (cm water) is measured from the bottom of the air inlet tube in the
Mariotte flask to the end of the outlet tubing, no matter whether the flow through the
column is downward (C) or upward (D).

67
2. Open the column outlet and start the flow. Two or three column
volumes of eluent should be passed through the column in order to
stabilize the bed and equilibrate with eluent buffer. A slightly higher
flow rate than is to be used in the experiments should be used for
packing.

Packing Sephacryl HR, Superdex prep grade

and Superose prep grade
Sephacryl HR, Superdex prep grade and Superose prep grade should be
packed and equilibrated at a high flow rate in a column in the XK-series
using a suitable pump (e.g. P-l, P-50, P-500). The packing method
described below ensures that the gel is optimally packed at the point of
entry of the sample, the most critical area, and it is highly recommended
that this method be followed.

Pack the column at the temperature at which it will be used.

1. Make sure the column is not damaged and that all parts are really
clean. It is of special importance that the nets, net fasteners and glass
tube are not damaged.
2. Attach the packing reservoir tightly and mount the column vertically
on a stand.
3. Wet the adaptor by drawing water (or 20% ethanol) through it, ma-
king sure no air bubbles are trapped under the net. This is best done by
submerging the plunger in a beaker of water (or 20% ethanol) and
attaching the tubing to a pump or a syringe. Close the tubing with a
stopper when all air bubbles have been removed.
4. Insert the adaptor at the bottom of the column far enough to give the
desired bed height.
5. Wet the column glass tube with eluent leaving a few centimetres of
fluid in the bottom making sure that the net is completely free from air
bubbles.
6. Prepare the gel as described above. The suspension of gel should be
adjusted so that it is a fairly thick slurry. It should not be so thick it
retains air bubbles. Usually about 75% settled gel is suitable.
Resuspend the gel and pour the well-mixed gel suspension carefully
down the wall of the column using a glass rod. Pour all the gel in a
single operation and fill the reservoir to the top with eluent.
7. Screw on the reservoir cap tightly, connect it to the pump and open the
outlet.
8. Pack the column in two steps using the flow rates given in Table 12,

68
Table 12. Recommended flow rates for packing Sephacryl HR

Column Flow rates (ml/h)

Step 1 Step 2
XK 26/40 240 490
XK 26/70 240 360
XK 26/100 180 320

XK 50/60 600 1 400

XK 50/100 600 950

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

69
14. Bring the adaptor to the gel surface and then a further 5 mm into the
gel bed. Liquid will flow out of the tubing attached to the adaptor
during this process.

Adaptors
Adaptors are adjustable column end pieces which support the bed matrix.
They allow automatic methods of sample application which eliminate
disturbance of the bed surface and protect the bed from insoluble particles
in the sample.

After an extended period of use, flow rates through a column packed with
Sephadex at a given hydrostatic pressure will be reduced. An upward flow
arrangement using adaptors will discourage this process (Fig. 40 D).

Adaptors are available for most Pharmacia columns. They should be fitted
as follows.
1. The gel should be packed completely as described above, or
alternatively should be allowed to settle sufficiently so that there are
2–3 cm of clear eluent at the top of the column.
2. Ensure the column outlet is closed otherwise the gel may be
compressed when the adaptor is fitted. Carefully add more eluent to fill
the column and form an upward meniscus (Fig. 41)
3. Slacken the adaptor tightening mechanism and insert it at an angle into
the column so that no air is trapped under the net (Fig. 42).

Fig. 41. Fig. 42.

70
Fig. 43.

4. Adjust the tightening mechanism to give a sliding seal between the

column wall and O-ring. Screw the adaptor top piece onto the column
end piece.
5. Make all tubing connections at this stage. Slide the plunger slowly
down the column so that air in the adaptor above the net and in the
capillary tubings is displaced by eluent. Valves on the inlet side of the
column should be turned in all directions during this procedure to
remove air from all connections.

Lock the adaptor in position with the tightening mechanism and

locking screw (Fig. 43), open the outlet and start the eluent flow.
Readjust the position of the adaptor at the bed surface after packing
has been completed (if the column has already been packed when the
adaptor is fitted, only about 10 minutes further packing is required
before re-adjustment).

Checking the packed bed

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

71
 Using pre-packed columns
The initial setting up of a gel filtration experiment is considerably
simplified if a pre-packed column is used.
1. Connect the column to the sample application device (see below for
alternatives) and to the monitor.
2. Follow the instructions supplied with the column to equilibrate it with
the chosen eluent.

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

Sample application on a drained bed surface

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

Sample application under the eluent (Fig. 44)

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

72
Fig. 44.

1. Use a syringe fitted with a piece of fine capillary tubing or a pipette

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

Sample application with an adaptor

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

Syringe method. (Fig. 45).

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

Sample reservoir (Fig. 46).

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

73
Fig. 45.

Fig. 46.

74
 Sample applicators SA-5, SA-50 (Fig. 47).
These are reservoirs which allow the sample to be introduced as a layer
below the eluent using a syringe and needle without disturbing the
chromatographic bed. They can also be used in a sample loop system (Fig.
47) where their large capacity (up to 5 ml for the SA-5 and 45 ml for the
SA-50) and lack of tailing due to wall effects offer distinct advantages.

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

Sample loops with valves LV-4 or SRV-4 (Fig. 48).

This method is convenient for application of small samples. By using the
same sample loop very reproducible sample volumes can be applied,
although exact knowledge of the applied volume requires calibration of the
capillary tubing loop.

75
Fig. 48. Sample application with a sample loop and two SRV-4 valves.

Sample loops or Superloop with valves V-7 or MV-7 (Fig. 49).

This method is used for sample application when using high performance
columns (e.g. Superdex HR 10/30 and Superose HR 10/30) and other

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

columns in FPLC System. Superloop (Fig. 50) is a unique sample

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

76
Fig. 50. Principle of operation of Superloop. Sample is drawn into the space in front of the
moving seal and applied to the column when eluent is pumped into the space behind the
seal. When the seal reaches the end of its travel, eluent by-passes the seal and washes the last
of the sample quantitatively onto the column.

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

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

77
Gel and Eluent Reservoir used as a Mariotte flask, is recommended.
Mention has already been made (see page 70) of the advantage of upward
flow for long term use of a column.

A problem which can occur when an experiment is continued without

supervision, for example, overnight, is that of stopping the experiment
before the eluent runs out. When using a pump this can be achieved by
attaching it to the power supply via a timing/cut-off device or via a fraction
collector which can control the pump. If gravity feed is used, a safety loop,
of the type illustrated in Figure 51 can easily be arranged to prevent air
from entering the column.

Fig. 51. Safety loop

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

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

Sephadex G-10, G-15, G-25 and G-50

These gels may be assumed to behave as rigid spheres in gel filtration and
therefore obey Darcy’s Law, i.e.
U = K ∆P L-1 (1)

78
where U is the linear flow rate expressed in cm/h (ml. cm-2.h-1), ∆p is the
pressure drop over gel bed expressed in cm H2O, L is the bed height
expressed in cm and K is a constant of proportionality depending on the
properties of the bed material and the eluent. Assuming an eluent with
viscosity of 1 cP one can write
U = Ko ∆P L-1 (2)
where Ko is the “specific permeability” depending on the particle size of the
gel beads and their water regain. Observe that the flow rate is proportional
to the pressure drop over the bed and, assuming a constant pressure head,
inversely proportional to the bed height. Notice that (to a good approxima-
tion) the flow rates are independent of the column diameter.

Flow rates at viscosities greater than 1 cP can be obtained by using the

relation: flow rate inversely proportional to viscosity. At first sight, it
would appear that high eluent viscosities lead to poor flow rates but the
operating pressure can be increased to compensate for the viscosity effect.
Temperature influences the viscosity of the eluent. Lower flow rates are
obtainable, for a given pressure head, in a cold room than at room
temperature.

Theoretical flow rates (not maximum) can be calculated from equation (2)
by inserting values for ∆p and L. Specific permeabilities are given in
Table 13.

Table 13. Specific permeabilities of Sephadex G-types.

Sephadex type Permeability K
Sephadex G-10 19
Sephadex G-15 18
Sephadex G-25 Superfine 9
Sephadex G-25 Fine 30
Sephadex G-25 Medium 80
Sephadex G-25 Coarse 290
Sephadex G-50 Superfine 13.5
Sephadex G-50 Fine 36
Sephadex G-50 Medium 145
Sephadex G-50 Coarse 400

Flow rates in columns packed with other media

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

79
rate (ml.cm-2.h-1) as narrower ones. These gels do not have a linear
relationship between pressure and flow, exceeding the maximum
recommended values can lead to gel compression, reduction in flow rate
and loss of resolution. Recommended maximum operating pressures and
corresponding approximate flow rates at room temperatures are given in
Table 11.

A bed diameter of 2.6 cm (column XK 26) and bed height of 30 cm have

been assumed. Maximum operating pressures are independent of bed
height but flow rates decrease with bed height. To calculate the flow rate
for a different bed height it may be assumed to be inversely proportional to
the bed height. Slightly reduced maximum operating pressures must be used
with columns wider than 2.6 cm.

To utilize fully the high rigidity and the excellent flow properties of
Sephacryl HR a liquid delivery system including a pump should be used. A
peristaltic pump can still be used with the other softer gels provided care is
taken. It is recommended to determine the maximum flow rate for the
column in question with gravity feed, taking care not to exceed the
pressures given in Table 11, and then use flow rates not exceeding 75 % of
this value with the pump. The maximum flow rates which are given in
Table 11 serve as a guide only and may vary depending upon column
packing or eluent viscosity (Note especially the effect of temperature on
viscosity).

Cleaning gels and packed columns

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

80
If an increase in back-pressure is observed, for example by the level of the
gel falling, make sure that the high back-pressure is in fact caused by the
column before starting the cleaning procedure. Disconnect one piece of
equipment at a time (starting at the fraction collector) with the pump
working and check the pressure as each piece is disconnected. A dirty filter
is often the cause of increased back-pressure. The pressure should be
checked at the same stage during each run, since the back-pressure can vary
within a run during sample injection or when changing to a different
eluent.

Note that cleaning solutions should also be filtered before use. After
cleaning, the column must be carefully re-equilibrated with 2–3 column
volumes of eluent buffer before it is used again.

Cleaning Sephadex G-types

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

Cleaning Sepharose
Wash with a non-ionic detergent.

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

Cleaning Sephacryl HR
Sephacryl HR may be cleaned and sanitized in the column with 1–2 column
volumes of NaOH (0.2–0.5 M) at a flow rate which gives a contact time
between the gel and the cleaning solution of at least 1 hour. This contact
time is sufficient to solubilize most protein precipitates. Sephacryl HR may
also be washed with a solution of a non-ionic detergent.

Use the same method if the gel is severely contaminated, but reverse the
direction of flow in the column during treatment with NaOH.

Cleaning Superose prep grade

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

81
2. Wash with at least 1/3 of the column volume NaOH (0.1 M-0.2 M).
3. Rinse with high purity water or buffer. Small aliquots (3 x 100 or
200 µl) of acetic acid (50%) may be added during this rinse.
4. Equilibrate with buffer until the baseline is stable.

b. Cleaning the gel before repacking:

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

Cleaning Superose HR 10/30 pre-packed columns

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

Rigourous cleaning to remove contamination

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

82
 The suggested cleaning volume of 25 ml is only a guide-line; the
practical requirements are best determined by monitoring the baseline,
which should be stable after each step in the sequence.
In special cases only, it may be necessary to change the bottom filter or
to remove and discard the top 2-3 mm of the gel. These operations
must be carried out extremely carefully to avoid serious loss of resolu-
tion.

Cleaning Superdex pre-packed columns

Superdex columns may be cleaned with one bed volume of NaOH (0.5 M).
This treatment will remove most proteins from the gel. The frequency of
cleaning depends on the degree of contamination, but a cleaning cycle at
least every 10–20 separation cycles is recommended.
Re-equilibrate the column with two bed volumes of buffer immediately
after cleaning. Wait until the UV baseline is steady before applying the next
sample.

If the column still is contaminated, it may be washed with 0.5 bed volume
of 1 M NaOH solution and 0.5 bed volume of 30% isopropanol or 0.1 M
HCl. Replace the filters if necessary.

Procedures to remove specific contaminants

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

Hydrophilic proteins and peptides

Wash the column with the solution which previously dissolved the material
during sample preparation e.g. an extraction solution, detergent, etc. (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 and
leave overnight at room temp. or 1 hour at 37 °C. Note: after enzymatic
digestion, careful rinsing is required to remove trace amounts of enzyme
remaining in the system.

83
 Hydrophobic proteins and peptides
These are usually soluble in polar organic solvents e.g. ethanol (24%),
acetonitrile (30%). If the organic solvent which best dissolves the
contaminant is known, run this overnight at a slow flow rate.

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

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

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

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

Storage of gels and columns

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

84
 Antimicrobial agents
Chlorhexidine
Chlorhexidine (e.g. Hibitane®), 0.002%, is a very effective antimicrobial
agent. It is incompatible with only a very few substances but is not
recommended for use with Sepharose. Precipitation may occur on storage
of Hibitane with appreciable concentrations of chloride or sulphate ions.

Chloroform, butanol and toluene

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

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

Phenyl mercuric salts

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

Sodium azide
Note. The use of sodium azide is discouraged in many countries. It can lead
to explosions when disposed of via lead pipe waste disposal systems and is
believed to be a mutagen.

Sodium azide, NaN3, 0.02–0.05%, is very widely used. It does not interact
notably with proteins or carbohydrates or change their chromatographic
behaviour. Sodium azide interferes with fluorescent marking of proteins,
the anthrone reaction and inhibits certain enzymes.

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

85
precipitated on the column. Its low toxicity is an advantage that reduces the
risk of sample contamination.

Sodium hydroxide is not recommended for use with Sepharose and for
storage of Sephacryl HR.

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

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

Storage of unused media

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

Storage of used media

Used media should be stored at +4–8 °C, pH 6-8 in the presence of a
suitable bacteriostatic, e.g. sodium azide (0.05%) or ethanol (20%). Note
that it is important that swollen media are not allowed to freeze as the
beads may be disrupted by ice crystals leading to the generation of fines.
Thimerosal should not be used with Sephacryl.

Sephadex can also be stored partially shrunk in, for example, 60–70%
alcohol. For special purposes Sephadex can be dried and restored to its
original state by the following procedure. The swollen gel is thoroughly
washed with water to remove salts and contaminants. After removal of
excess water, the gel can be shrunk by successive addition of alcohol
solutions of increasing percentage alcohol. The gel should be allowed to
equilibrate in between each addition. Final shrinking should be with 96%
alcohol. The gel is then sucked dry on a Büchner funnel and finally dried at
60–80 °C. A last wash with diethyl ether reduces the drying time. Clumps
may appear during the shrinking process but they disperse on re-swelling.
Risk for clump formation is reduced by slow shrinking and ether washing.

86
 Storage of packed columns
Packed columns should be stored at +4–8 °C in the presence of a suitable
bacteriostat. Sodium azide (0.05%) or ethanol (20%) may be used for
Superdex, Superose, Sephacryl, Sepharose and Sepharose CL, but only
sodium azide (0.05%) is recommended for columns packed with Sephadex
G-types.

For short-term storage, e.g. overnight, the column should be left connected
to the system; a low flow rate through the column will prevent bacterial
growth.

For long-term storage, the gel bed should be thoroughly cleaned before
re-equilibration with the storage buffer. If ethanol (20%) is used in the
storage buffer, de-gas the ethanol/water mixture well, start the
equilibration at a low flow rate and check the back-pressure while
equilibrating the column (water/ethanol mixtures are more viscous than
normal aqueous buffer solutions which will increase the back-pressure).

Disconnect the column from the system, close the bottom tubing of the
column and insert the tubing from the top of the column in a Parafilm®
covered vessel (e.g. test tube) containing ethanol (20%). Pre-packed HR
columns are supplied with a rubber tubing between the inlet and the outlet
of the column; this tubing may be filled with buffer and re-connected to
prevent the column drying out.

Note that columns may need to be re-packed if they are exposed to tempe-
ratures widely different from the temperature at which they were packed.

87
88
 Fault finding chart
Problem Cause Remedy
Column is clogged Presence of lipoproteins Prior to chromatography,
or protein aggregates. precipitate with 10%
Dextran Sulphate or 3%
polyvinylpyrrolidone.
See cleaning procedures
p 80–84.

Precipitation of proteins Modify the eluent to

in the column caused by maintain stability.
removal of stabilizing
agents during fractionation.

Filter is clogged. Replace the filter. Always

filter samples and buffer
before use.

Microbial growth has Microbial growth rarely

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

The sample Microbial growth has See above

substance is occurred in the column.
poorly resolved
from other
major peaks

89
Problem Cause Remedy
The sample Sample volume is too Decrease sample volume
substance is large or the sample has and apply the sample
poorly resolved been improperly applied. carefully. For maximum
from other resolution do not exceed
major peaks the sample volumes given
in Table 10. Contaminated
gel surface or top net will
spoil sample application.

Sample is too viscous. Dilute the sample with the

elution buffer. Keep protein
concentration below 30 mg/
ml.

Improper filtration of the Regenerate the column,

sample before application filter the sample and
to the column. repeat the chromatography
step.

Column is not mounted Try again with the column

vertically. mounted in a vertical
position. You may need to
repack the column.

Column is poorly packed. Check the packing by

running a coloured
compound, e.g. Blue
Dextran and observing the
band. Repack the column if
necessary.

Too much sample mass Decrease the sample load.

has been loaded onto the
column.

The column is dirty. Clean and regenerate the

column.

90
Problem Cause Remedy
Detector cell volume is Change the flow cell.
too big.

Wrong gel type. Substances which are

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

Large mixing spaces in See p 53.

or after column.

Column too short. See p 53.

Flow rate too high. See p 54.

Proteins or lipids Choose elution conditions

precipitated on column. which stabilize the sample.
See p 80–84 for cleaning
procedures.

Uneven temperature in Use a column with

the bed. a water-jacket.

Leading or very Overloaded column. Decrease the sample load

rounded peaks and repeat the run.
observed in the
chromatogram

Column is poorly packed. Check the packing by

running a coloured
compound, e.g. Blue
Dextran and observing the
band. Repack the column if
necessary.

91
Problem Cause Remedy
Earlier elution The sample has altered Prepare fresh sample.
profile can not during storage.
be reproduced

Larger sample mass load Keep volume and mass of

applied compared to sample constant when
earlier run. High protein repeating runs.
concentration can cause
protein protein interaction
resulting in a change in
elution profile.

The sample volume is Resolution is dependent on

different from earlier the sample volume. Keep
runs. sample volume constant
when repeating runs.

Proteins or lipids have Clean and regenerate the

precipitated 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 than
unexpected elution expected.
position

Ionic interactions Keep the ionic strength

between the protein and above 0.05 M to minimize
the matrix. ionic interactions.

Hydrophobic interactions Reduce the salt

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

92
Problem Cause Remedy
Sample substance Retardation is probably If possible decrease the
elutes later than due to hydrophobic ionic strength, increase pH
expected or even interactions between or introduce organic
after the total protein and the matrix. solvent in the elution
column volume buffer, e.g. 5%
isopropanol, in order to
minimize hydrophobic
interactions.

Late elution and The column has become Clean and regenerate the
broad peaks dirty. column.
observed in the
chromatogram

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

Pump wrongly calibrated. Re-calibrate the pump.

More sample Sample substance Try to optimize your

substance is co-elutes with other separation in order to
recovered than substances. resolve the peak.
expected Alternatively, combine
several techniques to
remove contaminants
completely.

Low recovery of Sample substance may Change the eluent.

activity while not be stable in the
normal recovery chosen eluent and is
of protein therefore inactivated.

Enzyme separated from Test by pooling fractions

co-factor or similar. and repeating the assay.

93
Problem Cause Remedy

Microbial growth. See p 84.

Protein amount in The protein may have Add protease inhibitors to

the eluted fractions been degraded by the buffers to prevent
is 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 to
prevent any hydrophobic
interactions.

Elution conditions too Choose elution conditions

harsh. which stabilize the sample.

Specific adsorption. Lectins may bind to sugar

residues in the matrix. Try
specific elution with an
analogous sugar.

Sample precipitates. May be caused by removal

of salts or sample dilution.

More activity is Different assay conditions Use the same assay

recovered than have been used before conditions for all the assays
was 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.

94
Problem Cause Remedy

Sample size is smaller –

than intended.

Excessive zone Check the column packing

broadening and re-pack if necessary.

No peaks Monitor switched off. Switch it on.

Monitor not connected Check the electrical

to recorder. connections and cables.

UV-lamp not working. Replace the lamp.

Sample not applied. Check the operation of the

sample application device.

Range button depressed Check the recorder settings.

or no sensitivity set on
recorder.

No flow through Outlet closed. –

the column

No flow from pump. With peristaltic pumps

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

Air-lock in outlet tubing See page 69.

or bottom-piece.

Clogged end-piece or Bed supports of porous

adaptor or tubing. glass or polythene are
prone to clogging by gel
particles.

Reduced or poor See above. –

flow through the
column

95
Problem Cause Remedy
Bed surface blocked by Remove contaminated gel
precipitated proteins. from the bed surface. Stir
the top 1–2 cm and allow
to re-settle as an even layer.

Bed compressed. Special care is needed when

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

Microbial growth. For prevention see page 84.

Gel not fully swollen See page 61.

(Sephadex G-types).

Fines (Sephadex G-types). Do not use a magnetic

stirrer; it can break the
beads. Fine particles can be
removed by decanting from
a settling suspension.

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

96
Problem Cause Remedy
Eluent not properly De-gas the eluent
de-gassed. thoroughly.

Cracks in the bed Large air leak in column. Check all connections for
leaks. Repack the column.

Column violently –
mishandled.

Distorted bands as Air bubble at the top of Re-install the adaptor

sample runs into the column or in the taking care to avoid air
the bed adaptor. bubbles. See p 70–71.

Particles in eluent or Filter or centrifuge the

sample. sample. Protect eluents
from dust.

Particles on the bed Remove small amount of

surface or uneven bed gel and stir up the top
surface. 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 of
samples and eluents.
Distorted bands as Column poorly packed. Gel suspension too thick
sample passes down or too thin. Bed packed
bed at a temperature different
from run. Bed insufficiently
packed (too low packing
pressure, too short
equilibration). Column
packed at too high
pressure.

97
Problem Cause Remedy

Gel beads in eluent Bed support loose or Replace or re-fasten.

broken.
Column operated at too Do not exceed the
high pressure. recommended operating
pressure for the gel
medium.

98
 References
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2. On the history of the development of Sephadex. Chromatographia 23 (1987) 361–369, Janson, J.-C.

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

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

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

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

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

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

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

10. Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and
density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductase.
Biochim. Biophys. Acta 112 (1966) 346–362, Siegel, L.M., Monty, K.J.

11. The correlation between molecular weight and elution behaviour in the gel chromatography of proteins. J.
Chromatogr. 25 (1966) 303–313, Determann, H., Michel, W.

12. Estimation of molecular weights of proteins by agarose gel filtration. J. Chromatogr. 40 (1969) 453–457,
Locascio, G.A., Tigier, H.A., Batlle, A.M. del C.

13. Estimation of molecular size and molecular weights of biological compounds by gel filtration. In Methods of
Biochemical Analysis vol. 18 pp. 1–53 (ed. Glick, D.) Interscience Publishers, New York, London 1970 Andrews,
P.

14. Molecular-weight estimation of proteins using Sepharose CL-6B in guanidine hydrochloride. J. Chromatogr. 140
(1977) 98–102, Ansari, A.A., Mage, R.G.

15. Fractionation of dextran by the gel filtration method. Makromolekulare Chem. 48 (1961) 160–71, Granath, K.A.,
Flodin, P.

16. Molecular-weight distribution determination of clinical dextran by gel permeation chromatography. J.

Chromatogr. 101 (1974) 137–153, Nilsson, G., Nilsson, K.

17. Drug-plasma binding measured by Sephadex. J. Pharm. Pharmacol. 9 (1962) 550–555, Barlow, C.F., Firemark,
H., Roth, L.J.

18. Separation of human heme- and hemoglobin-binding plasma proteins, ceruloplasmin and albumin by gel
filtration. Biochim. Biophys. Acta 93 (1964) 1–14, Killander, J.

19. Estimation of serum haemoglobin-binding capacity (haptoglobin) on Sephadex G-100. J. Clin. Pathol. 17 (1964)
676–679, Ratcliff, A.P., Hardwicke, J.

20. Measurement of protein-binding phenomena by gel filtration. Biochim. Biophys. Acta 63 (1962) 530–532,
Hummel, J.P., Dreyer, W.J.

21. Studies of chemically reacting systems on Sephadex. 1. Chromatographic demonstration of the Gilbert Theory.
Biochemistry 2 (1963) 1263–1267, Winzor, D.J., Scheraga, H.A.

99
22. Peptide-protein interaction as studied by gel filtration. Biochemistry 5 (1966) 673–683, Fairclough, Jr., G.F.,
Fruton, J.S.

23. Binding of sulfonamides to serum proteins: physico-chemical and immuno-chemical studies. J. Pharmacol. Exp.
Therap. 153 (1966) 167–175, Clausen, J.

24. Protein-binding of small molecules. New gel filtration method. J. Pharm. Pharmacol. 20 (Suppl.) (1968) 1505–
1565, Cooper, P.F., Wood, G.C.

25. The application of gel filtration to the study of protein-binding of small molecules. Chromatogr. Rev. 12 (1970)
88–107, Wood, G.C., Cooper, P.F.

26. Determination by gel filtration of association constants for metal-nucleotide interaction. Anal. Biochem. 46
(1972) 358-363, Colman, R.F.

27. A hydrogen exchange method using tritium and Sephadex: its application to ribonuclease. Biochemistry 2 (1963)
798–807, Englander, S.W.

28. Determination of stoichiometry and equilibrium constants for reversibly associating systems by molecular sieve
chromatography. Proc. Nat. Acad. Sci. USA 53 (1965) 342–349, Ackers, G.K., Thompson, T.E.

29. Labeling deoxyribonucleic acid to high specific activty in vitro by nick translation with DNA polymerase I. J. Mol.
Biol. 113 (1977) 237–251, Rigby, P.W.J., Dieckmann, M., Rhodes, C. et al.

30. “Molecular Cloning. A laboratory manual”, T. Maniatis, E.F. Fritsch, J. Sambrook (Cold Spring Harbor 1982)

31. Properties, in theory and practice, of novel gel filtration media for standard liquid chromatography. J.
Chromatogr. 476 (1989) 329–344, Hagel, L., Lundström, H., Andersson, T. et al.

32. Agarose-based media for high-resolution gel filtration of biopolymers. J. Chromatogr. 326 (1985) 33–44,
Andersson, T., Carlsson, M., Hagel, L. et al.

33. Column lifetime of a new agarose medium for high-performance gel filtration chromatography at basic pH. J.
Chromatogr. 330 (1985) 360–364, Johansson, B-L., Ellstrom, C.

34. Column lifetime of Superose 6 at 37 Celsius and basic pH. J. Chromatogr. 351 (1986) 136–139, Johansson, B-L.,
AAhsberg, L.

35. Separation of transfer ribonucleic acid by Sepharose chromatography using reverse salt gradients. Proc. Nat.
Acad. Sci. USA 72 (1975) 1068–1071, Holmes, W.M., Hurd, R.E., Reid, B.R. et al.

36. A new general method for separation of nucleic acids. Prep. Biochem. 4 (1974) 509–522, Petrovic, S.L., Petrovic,
J.S., Markovic, R.A. et al.

37. Agar derivatives for chromatography, electrophoresis and gel-bound enzymes. 1. Desulphated and reduced
crosslinked agar and agarose in spherical bead form. J. Chromatogr. 60 (1971) 167–177, Porath, J., Janson, J.-C.,
Låås, T.

38. The agarose double helix and its function in agarose gel structure. J. Mol. Biol. 90 (1974) 269-284, Arnott, S.,
Fulmer, A., Scott, W.E. et al.

39. Resolution of ribonucleic acids by Sepharose 4B column chromatography. Biochemistry 16 (1977) 1378–1382,
Zeichner, M., Stern, R.

40. “The Dynamics of Chromatography”, J.C. Giddings (Marcel Dekker, N.Y. 1965) Part 1, Principles and theory.

41. Effect of sample volume on peak width in high-performance gel filtration chromatography. J. Chromatogr. 324
(1985) 422–427, Hagel. L.

42. “Process chromatography. A practical guide”, G.K. Sofer and L.-E. Nyström (eds) (Academic Press, London
1989) pp 36–41.

43. “Process chromatography. A practical guide”, G.K. Sofer and L.-E. Nyström (eds) (Academic Press, London
1989) pp 55–66.

44. Purification of plasmid DNA by fast protein liquid chromatography on Superose 6 preparative grade. Anal.
Biochem. 177 (1989) 378-382, McClung, J.K., Gonzales, R.A.

100
45. Universal calibration of gel permeation chromatography and determination of molecular shape in solution. Anal.
Biochem. 162 (1987) 47–64, Potschka, M.

46. Size-exclusion chromatography of DNA restriction fragments. Fragment length determinations and a comparison
with the behaviour of proteins in size-exclusion chromatography. J. Chromatogr. 467 (1989) 217–226, Ellegren,
H., Laas, T.

47. Size-exclusion chromatography and universal calibration of gel columns. Anal. Biochem. 177 (1989) 50–56, Le
Maire, M., Viel, A., Møller, J.

48. Improved preparation of the integral membrane proteins of human red cells, with special reference to the glucose
transporter. Biochim. Biophys. Acta 855 (1986) 345–356, Lundahl, P., Griejer, E., Cardell, S. et al.

49. Purification and characterization of membrane-bound phospholipase C specific for phosphoinositides from
human platelets. J. Biol. Chem. 263 (1988) 11459–11465, Banno, Y., Yada, Y., Nozawa, Y.

50. Sodium dodecyl sulphate-protein complexes. Changes in size or shape below the critical micelle concentration, as
monitored by high-performance agarose gel chromatography. J. Chromatogr. 476 (1989) 147–158, Mascher, E.,
Lundahl, P.

51. Molecular characterization of proteins in detergent solutions. Biochemistry 13 (1974) 2369–2376, Tanford, C.,
Nozaki, Y., Reynolds, J.A. et al.

52. Glass wool as a potential source of artifacts in chromatography. J. Chromatogr. 152 (1978) 514–516, Schwartz,
D.P.

BioPilot, FPLC, FPLCmanager, HiLoad, HiPrep, HiTrap, LKB, MicroPerpex, NAP, NICK,
Pharmacia, Sephacryl, Sephadex, Sepharose, Superdex, Superloop, Superose and Uvicord are
trademarks of Amersham Pharmacia Biotech Limited or its subsidiaries

Amersham is a trademark of Nycomed Amersham plc

Pharmacia and Drop Design are trademarks of Pharmacia & Upjohn Inc

All goods and services are sold subject to the terms and conditions of sale of the company within
the Amersham Pharmacia Biotech group which supplies them. A copy of these terms and
conditions of sale is available on request.

© Amersham Pharmacia Biotech AB 1998 - All rights reserved

Amersham Pharmacia Biotech UK Limited Amersham Place Little Chalfont Buckinghamshire

England HP7 9NA
Amersham Pharmacia Biotech AB SE-751 84 Uppsala Sweden
Amersham Pharmacia Biotech Inc 800 Centennial Avenue PO Box 1327 Piscataway
NJ 08855 USA

101
 Ordering information
Column Code No. Media Pack Code No.
size
Superdex Peptide HR 10/30 17-1453-01 Superose 6 prep grade 125 ml 17-0489-01
Superdex Peptide PE 7.5/300 17-5003-01 Superose 12 prep grade 125 ml 17-0536-01
Superdex Peptide PC 3.2/30 17-1458-01
Superdex 75 PC 3.2/30 17-0771-01 Superdex 30 prep grade 25 ml 17-0905-10
Superdex 200 PC 3.2/30 17-1089-01 Superdex 30 prep grade 150 ml 17-0905-01
Superdex 30 prep grade 1 litre 17-0905-03
Superose 6 PC 3.2/30 17-0673-01 Superdex 30 prep grade 5 litres 17-0905-04
Superose 12 PC 3.2/30 17-0674-01
Superose 6 HR 10/30 17-0537-01 Superdex 75 prep grade 25 ml 17-1044-10
Superose 12 HR 10/30 17-0538-01 Superdex 75 prep grade 150 ml 17-1044-01
Superdex 75 prep grade 1 litre 17-1044-02
Superdex 75 HR 10/30 17-1047-01 Superdex 75 prep grade 5 litres 17-1044-04
Superdex 200 HR 10/30 17-1088-01
Superdex 200 prep grade 25 ml 17-1043-10
HiLoad 16/60 Superdex 30 pg 17-1139-01 Superdex 200 prep grade 150 ml 17-1043-01
HiLoad 26/60 Superdex 30 pg 17-1140-01 Superdex 200 prep grade 1 litre 17-1043-02
HiLoad 16/60 Superdex 75 pg 17-1068-01 Superdex 200 prep grade 5 litres 17-1043-04
HiLoad 26/60 Superdex 75 pg 17-1070-01
HiLoad 16/60 Superdex 200 pg 17-1069-01 Sephacryl S-100 HR 150 ml 17-0612-10
HiLoad 26/60 Superdex 200 pg 17-1071-01 Sephacryl S-100 HR 750 ml 17-0612-01
Sephacryl S-100 HR 10 litres 17-0612-05
HiPrep 16/60 Sephacryl S-100 HR 17-1165-01 Sephacryl S-200 HR 150 ml 17-0584-10
HiPrep 26/60 Sephacryl S-100 HR 17-1194-01 Sephacryl S-200 HR 750 ml 17-0584-01
HiPrep 16/60 Sephacryl S-200 HR 17-1166-01 Sephacryl S-200 HR 10 litres 17-0584-05
HiPrep 26/60 Sephacryl S-200 HR 17-1195-01 Sephacryl S-300 HR 150 ml 17-0599-10
HiPrep 16/60 Sephacryl S-300 HR 17-1167-01 Sephacryl S-300 HR 750 ml 17-0599-01
HiPrep 26/60 Sephacryl S-300 HR 17-1196-01 Sephacryl S-300 HR 10 litres 17-0599-05
Sephacryl S-400 HR 150 ml 17-0609-10
HiTrap Desalting (5 columns) 17-1408-01 Sephacryl S-400 HR 750 ml 17-0609-01
Fast Desalting Column HR 10/10 17-0591-01 Sephacryl S-400 HR 10 litres 17-0609-05
Sephacryl S-500 HR 150 ml 17-0613-10
Sephacryl S-500 HR 750 ml 17-0613-01
Column Qty. Code No. Sephacryl S-500 HR 10 litres 17-0613-05
Sephacryl S-1000 SF 750 ml 17-0476-01
PD-10 Prepacked Disposable 30 17-0851-01
NAP-5 20 17-0853-01 Sepharose 6B 1 litre 17-0110-01
NAP 5 50 17-0853-02 Sepharose 6B 10 litres 17-0110-05
NAP-10 20 17-0854-01 Sepharose 4B 1 litre 17-0120-01
NAP 10 50 17-0854-02 Sepharose 4B 10 litres 17-0120-05
NAP-25 20 17-0852-01 Sepharose 2B 1 litre 17-0130-01
NAP 25 50 17-0852-02 Sepharose 2B 10 litres 17-0130-05
NICK Column 20 17-0855-01
NICK Column 50 17-0855-02 Sepharose CL-6B 1 litre 17-0160-01
NICK Spin Columns 20 17-0862-01 Sepharose CL-6B 10 litres 17-0160-05
NICK Spin Columns 50 17-0862-02 Sepharose CL-4B 1 litre 17-0150-01
Sepharose CL-4B 10 litres 17-0150-05
Sepharose CL-2B 1 litre 17-0140-01
Sepharose CL-2B 10 litres 17-0140-05

Sephadex G-10 100 g 17-0010-01

Sephadex G-10 500 g 17-0010-02
Sephadex G-10 5 kg 17-0010-03

Sephadex G-15 500 g 17-0020-01

Sephadex G-15 5 kg 17-0020-02
Sephadex G-15 500 g 17-0020-02
102
 Media Pack Code No. Media Pack Code No.
size size
Sephadex G-25 Fine 100 g 17-0032-01 Sephadex G-25 25 g 17-0572-01
Sephadex G-25 Fine 500 g 17-0032-02 DNA Grade SF 100 g 17-0572-02
Sephadex G-25 Fine 5 kg 17-0032-03
Sephadex G-25 Medium 100 g 17-0033-01 Sephadex G-50 25 g 17-0573-01
Sephadex G-25 Medium 500 g 17-0033-02 DNA Grade F 100 g 17-0573-02
Sephadex G-25 Medium 5 kg 17-0033-03
Sephadex G-25 Coarse 100 g 17-0034-01 Sephadex G-100 25 g 17-0045-01
Sephadex G-25 Coarse 500 g 17-0034-02 DNA Grade M 100 g 17-0045-02
Sephadex G-25 Coarse 5 kg 17-0034-03
Sephadex G-25 Superfine 100 g 17-0031-01 Sephadex G-100 25 g 17-0574-01
Sephadex G-25 Superfine 5 kg 17-0031-03 DNA Grade SF 100 g 17-0574-02

Sephadex G-50 Fine 100 g 17-0042-01 Handbook. 18-1022-18

Sephadex G-50 Fine 500 g 17-0042-02 Gel filtration, Principles
Sephadex G-50 Fine 5 kg 17-0042-03 and Methods
Sephadex G-50 Medium 100 g 17-0043-01
Sephadex G-50 Medium 500 g 17-0043-02
Sephadex G-50 Medium 5 kg 17-0043-03
Sephadex G-50 Coarse 100 g 17-0044-01
Sephadex G-50 Coarse 500 g 17-0044-02
Standards Pack Code No.
Sephadex G-50 Coarse 5 kg 17-0044-03 size
Sephadex G-50 Superfine 100 g 17-0041-01
Sephadex G-50 Superfine 5 kg 17-0041-03 Gel Filtration LMW
Calibration kit 1 kit 17-0442-01
Sephadex G-75 100 g 17-0050-01 Gel Filtration HMW
Sephadex G-75 500 g 17-0050-02 Calibration kit 1 kit 17-0441-01
Sephadex G-75 5 kg 17-0050-03 Blue Dextran 2000 10 g 17-0360-01
Sephadex G-75 Superfine 100 g 17-0051-01
Sephadex G-75 Superfine 5 kg 17-0051-03

Sephadex G-100 100 g 17-0060-01

Sephadex G-100 500 g 17-0060-02
Sephadex G-100 5 kg 17-0060-03
Sephadex G-100 Superfine 100 g 17-0061-01
Sephadex G-100 Superfine 5 kg 17-0061-03

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 pancreas
chymotrypsinogen A 25 000 20.9 bovine pancreas
ovalbumin 43 000 30.5 hen egg
albumin 67 000 35.5 bovine serum
Blue Dextran 2000

High Molecular Weight Gel Filtration Calibration Kit

Aldolase* 158 000 48.1 rabbit muscle
catalase 232 000 52.2 bovine liver
ferritin* 440 000 61.0 horse spleen
thyroglobulin 669 000 85.0 bovine thyroid
Blue Dextran 2000
Each Kit contains 50 mg of each protein and 50 mg of Blue Dextran 2000.

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Printed in Sweden by Rahms i Lund 9809.