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Gel filtration – Principles and Methods

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First published Dec. 2000.

Gel filtration
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Gel filtration
Principles and Methods
Contents
Introduction............................................................................................................................................... 7
Chapter 1.
Gel filtration in practice.......................................................................................................................... 9
Introduction...................................................................................................................................................................... 9
Purification by gel filtration....................................................................................................................................... 9
Group separation.............................................................................................................................................. 10
High-resolution fractionation...................................................................................................................... 11
Rapid purity check and screening............................................................................................................. 12
Resolution in gel filtration.............................................................................................................................. 12
Sample volume and column dimensions............................................................................................... 12
Media selection............................................................................................................................................................ 14
Sample and buffer preparation........................................................................................................................... 17
Sample buffer composition.......................................................................................................................... 17
Sample concentration and viscosity........................................................................................................ 17
Sample volume................................................................................................................................................... 19
Buffer composition . ........................................................................................................................................ 19
Denaturing (chaotropic) agents and detergents................................................................................ 19
Column and media preparation.......................................................................................................................... 20
Sample application.................................................................................................................................................... 21
Elution and flow rates............................................................................................................................................... 21
Controlling flow.................................................................................................................................................. 23
Method development for high resolution fractionation........................................................................... 23
Maintenance of gel filtration columns.............................................................................................................. 24
Equipment selection.................................................................................................................................................. 24
Scaling up....................................................................................................................................................................... 24
BioProcess Media for large-scale production............................................................................................... 25
Troubleshooting........................................................................................................................................................... 26
Chapter 2.
Superdex: the first choice for high resolution, short run times, and high recovery.................. 31
Separation options..................................................................................................................................................... 33
Separation examples................................................................................................................................................ 34
Performing a separation......................................................................................................................................... 39
First time use or after long-term storage.............................................................................................. 39
Cleaning................................................................................................................................................................. 40
Removing severe contamination............................................................................................................... 40
Media characteristics................................................................................................................................................ 41
Chemical stability.............................................................................................................................................. 41
Storage................................................................................................................................................................... 41
Chapter 3.
Superose: broad fractionation range for laboratory scale............................................................. 43
Separation options..................................................................................................................................................... 45
Separation examples................................................................................................................................................ 45
Performing a separation......................................................................................................................................... 46
First time use or after long-term storage.............................................................................................. 46
Cleaning................................................................................................................................................................. 47

2 18-1022-18 AK
 Media characteristics................................................................................................................................................ 47
Chemical stability.............................................................................................................................................. 48
Storage................................................................................................................................................................... 48
Chapter 4.
Sephacryl: fast, high recovery separations at laboratory and industrial scale......................... 49
Separation options..................................................................................................................................................... 52
Separation examples................................................................................................................................................ 53
Performing a separation......................................................................................................................................... 54
First time use or after long-term storage.............................................................................................. 54
Cleaning................................................................................................................................................................. 55
To remove severe contamination.............................................................................................................. 55
Media characteristics................................................................................................................................................ 55
Chemical stability.............................................................................................................................................. 56
Storage................................................................................................................................................................... 56
Chapter 5.
Sephadex: desalting, buffer exchange and sample clean up......................................................... 57
Separation options..................................................................................................................................................... 58
Separation examples................................................................................................................................................ 62
Performing a separation......................................................................................................................................... 63
General considerations............................................................................................................................................ 63
Small-scale desalting of samples.............................................................................................................. 63
Desalting larger sample volumes using HiTrap and HiPrep columns...................................... 63
Buffer preparation............................................................................................................................................ 64
Sample preparation......................................................................................................................................... 64
Buffer exchange................................................................................................................................................ 64
HiTrap Desalting columns...................................................................................................................................... 64
Manual purification with a syringe........................................................................................................... 65
Simple desalting with ÄKTAprime plus.............................................................................................................. 66
Desalting on a gravity-feed PD-10 column.................................................................................................... 67
Buffer Preparation............................................................................................................................................ 67
Optimization of desalting.............................................................................................................................. 67
Scale-up and processing larger sample volumes....................................................................................... 68
Increasing sample loading capacity from 1.5 ml up to 7.5 ml..................................................... 69
Increasing sample loading capacity from 15 ml up to 60 ml....................................................... 69
For sample volumes greater than 60 ml................................................................................................ 70
Media characteristics................................................................................................................................................ 70
Column Packing................................................................................................................................................. 71
Cleaning................................................................................................................................................................. 71
Chemical stability.............................................................................................................................................. 71
Storage................................................................................................................................................................... 71
Chapter 6.
Sephadex LH-20 – gel filtration in presence of organic solvents................................................... 73
Media characteristics................................................................................................................................................ 73
Separation examples................................................................................................................................................ 73
Packing a column....................................................................................................................................................... 75
Performing a separation......................................................................................................................................... 76
Cleaning.......................................................................................................................................................................... 76
Chemical stability........................................................................................................................................................ 77
Storage............................................................................................................................................................................ 77
Transferring Sephadex LH-20 from aqueous solution to organic solvents.................................... 77

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Chapter 7
Gel filtration in theory........................................................................................................................... 79
Defining the process................................................................................................................................................. 79
Selectivity curves and media selection............................................................................................................ 81
Resolution....................................................................................................................................................................... 82
Chapter 8.
Gel filtration in a purification strategy............................................................................................... 85
The purification strategy according to CIPP................................................................................................... 85
Gel filtration as a polishing step................................................................................................................. 86
Purification of humanized IgG4 monoclonal antibody..................................................................... 87
Appendix 1.
Column packing and preparation........................................................................................................ 89
Columns for packing gel filtration media........................................................................................................ 89
Checking column efficiency................................................................................................................................... 90
Column packing for high resolution fractionation using
Superdex prep grade and Sephacryl High Resolution.............................................................................. 91
Column packing for group separations using Sephadex........................................................................ 93
Controlling flow............................................................................................................................................................ 95
Appendix 2.
Sephadex and Darcy’s law.................................................................................................................... 96
Appendix 3.
Sample preparation............................................................................................................................... 97
Sample clarification................................................................................................................................................... 97
Centrifugation..................................................................................................................................................... 97
Filtration................................................................................................................................................................. 97
Desalting............................................................................................................................................................... 97
Denaturation....................................................................................................................................................... 98
Precipitation and resolubilization........................................................................................................................ 98
Ammonium sulfate precipitation.............................................................................................................. 99
Removal of lipoproteins................................................................................................................................101
Appendix 4.
Selection of purification equipment..................................................................................................102
Appendix 5.
Converting from linear flow (cm/h) to volumetric flow rates (ml/min) and vice versa.................103
From linear flow (cm/h) to volumetric flow rate (ml/min) . ..........................................................103
From volumetric flow rate (ml/min) to linear flow (cm/hour)......................................................103
From ml/min to using a syringe...............................................................................................................104
Appendix 6.
Conversion data....................................................................................................................................105
Proteins..........................................................................................................................................................................105
Nucleic Acids...............................................................................................................................................................105
Column pressures.....................................................................................................................................................105
Appendix 7.
Amino acids table ................................................................................................................................106
Appendix 8 .
Analysis and characterization...........................................................................................................108
Protein detection and quantification...............................................................................................................108
Purity check and protein characterization...................................................................................................108

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 Purity.....................................................................................................................................................................108
Characterization..............................................................................................................................................109
Appendix 9.
Storage of biological samples............................................................................................................110
General recommendations..................................................................................................................................110
Common storage conditions for purified proteins....................................................................................110
Appendix 10.
Molecular weight estimation and molecular weight distribution analysis...............................111
Performing a molecular weight determination..........................................................................................113
Product index.........................................................................................................................................115
Related literature.................................................................................................................................116
Ordering information...........................................................................................................................117

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6 18-1022-18 AK
Introduction
Biomolecules are purified using chromatography techniques that separate them according to
differences in their specific properties, as shown in Figure 1.

Property Technique
Size Gel filtration (GF), also called size exclusion chromatography (SEC)
Hydrophobicity Hydrophobic interaction chromatography (HIC)
Reversed phase chromatography (RPC)
Charge Ion exchange chromatography (IEX)
Biorecognition (ligand specificity) Affinity chromatography (AC)
Isoelectric point Chromatofocusing (CF)

Fig 1. Schematic drawing of separation principles in chromatography purification. From left to right: GF, HIC, IEX, AC, and RPC.

Since the introduction of Sephadex™ more than 50 years ago, gel filtration has played a key role
in the purification of proteins and enzymes, polysaccharides, nucleic acids and other biological
macromolecules. Gel filtration is the simplest and mildest of all the chromatography techniques
and separates molecules on the basis of differences in size. The technique can be applied in two
distinct ways:
1. Group separations: the components of a sample are separated into two major groups
according to size range. A group separation can be used to remove high or low molecular
weight contaminants (such as phenol red from culture fluids) or for desalting and buffer
exchange.
2. High resolution fractionation of biomolecules: the components of a sample are separated
according to differences in their molecular size. High resolution fractionation can be used
to isolate one or more components, to separate monomers from aggregates, or to perform
a molecular weight distribution analysis.

Gel filtration can also facilitate the refolding of denatured proteins by careful control of changing
buffer conditions.

18-1022-18 AK 7
This handbook describes the use of gel filtration for the purification and separation of
biomolecules, with a focus on practical information for obtaining the best results. The media
available, selection criteria and examples with detailed instructions for the most common
applications are included, as well as the theoretical principles behind the technique. The first
step towards a successful separation is to select the correct medium and this handbook
focuses on the most up-to-date gel filtration media and prepacked columns.

Symbols
this symbol indicates general advice to improve procedures or recommend
action under specific situations.
this symbol denotes mandatory advice and gives a warning when special

care should be taken.
highlights chemicals, buffers and equipment.
outline of experimental protocol.

8 18-1022-18 AK
Chapter 1
Gel filtration in practice
Introduction
Gel filtration (also referred to as size exclusion chromatography, SEC) separates molecules
according to differences in size as they pass through a gel filtration medium packed in
a column. Unlike ion exchange or affinity chromatography, molecules do not bind to the
chromatography medium so buffer composition does not directly affect resolution (the degree
of separation between peaks). Consequently, a significant advantage of gel filtration is that
conditions can be varied to suit the type of sample or the requirements for further purification,
analysis or storage without altering the separation.
Gel filtration is well suited for biomolecules that may be sensitive to changes in pH,
concentration of metal ions or co-factors and harsh environmental conditions. Separations
can be performed in the presence of essential ions or cofactors, detergents, urea, guanidine
hydrochloride, at high or low ionic strength, at 37°C or in the cold room according to the
requirements of the experiment. Purified proteins can be collected in any chosen buffer.
This chapter provides general guidelines applicable to any gel filtration separation. A key step
towards successful separation is selecting the correct medium; this handbook includes guides
to the most up-to-date gel filtration media and prepacked columns. Application examples and
product-specific information are found in Chapters 2 to 6.

Purification by gel filtration

To perform a separation, gel filtration medium is packed into a column to form a packed bed.
The medium is a porous matrix of spherical particles with chemical and physical stability and
inertness (lack of reactivity and adsorptive properties). The packed bed is equilibrated with
buffer which fills the pores of the matrix and the space between the particles. The liquid inside
the pores, or stationary phase, is in equilibrium with the liquid outside the particles, or mobile
phase. Samples are eluted isocratically so there is no need to use different buffers during the
separation. However, a wash step using the running buffer is usually included at the end of a
separation to remove molecules that may have been retained on the column and to prepare
the column for a new run.
Gel filtration can be used directly after ion exchange, chromatofocusing, hydrophobic
interaction, or affinity, since the buffer composition will not generally affect the final separation.
For further details on using gel filtration in a purification strategy, refer to Chapter 8.
Figure 1.1 illustrates the separation process of gel filtration and the theory for this process is
described in Chapter 7.

18-1022-18 AK 9
Fig 1.1. Process of gel filtration (A) Schematic picture of a bead with an electron microscopic enlargement. (B) Schematic
drawing of sample molecules diffusing into bead pores. (C) Graphical description of separation I. Sample is applied on
the column, II. The smallest molecule (yellow) is more delayed than the largest molecule (red). III. The largest molecule is
eluted first from the column. Band broadening causes significant dilution of the protein zones during chromatography.
(D) Schematic chromatogram.

Group separation
Gel filtration is used in group separation mode to remove small molecules from a group of
larger molecules and as a fast, simple solution for buffer exchange. Small molecules such
as excess salt or free labels are easily separated from larger molecules. Samples can be
prepared for storage or for other chromatography techniques and assays. Gel filtration in
group separation mode is often used in protein purification schemes for desalting and buffer
exchange. Sephadex G-10, G-25 and G-50 are used for group separations. Large sample
volumes, up to 30% of the total column volume (packed bed), can be applied at high flow rates
using broad, short columns. Figure 1.2 shows the chromatogram (elution profile) of a typical
group separation. Large molecules are eluted in or just after the void volume, Vo, as they pass
through the column at the same speed as the flow of buffer. For a well-packed column the void
volume is equivalent to approximately 30% of the total column volume. Small molecules such

10 18-1022-18 AK
as salts that have full access to the pores move down the column, but do not separate from
each other. These molecules usually elute just before one total column volume, Vt, of buffer
has passed through the column. In this case the proteins are detected by monitoring their UV
absorbance, usually at 280 nm, and the salts are detected by monitoring the conductivity of
the buffer.

Column: HiTrap™ Desalting 5 ml

Sample: (Histidine)6 protein eluted from HiTrap Chelating HP
with sodium phosphate 20 mM, sodium chloride 0.5 M,
imidazole 0.5 M, pH 7.4
Buffer: Sodium phosphate 20 mM, sodium chloride 0.15 M, pH 7.0

void volume Vo, total column volume Vt

A 280 nm
UV 280 nm
Conductivity
0.15
(Histidine)6-protein

0.10

Salt

0.05

Inject
Vo Vt

0 1 2 min

Fig 1.2. Typical chromatogram of a group separation. UV (protein) and conductivity (salt) detection enable pooling of the
desalted fractions and facilitate optimization of the separation.

Refer to Chapter 5, p 57 for detailed information on how Sephadex is used in group separation
of high and low molecular weight substances in applications like desalting, buffer exchange,
and sample clean up.
Refer to Chapter 7 for detailed information on the theory of gel filtration.

High-resolution fractionation
Gel filtration is used in fractionation mode to separate multiple components in a sample on the
basis of differences in their size. The goal may be to isolate one or more of the components, or
to analyze the molecular weight distribution in the sample. The best results for high resolution
fractionation will be achieved with samples that originally contain few components or with
samples that have been partially purified by other chromatography techniques to eliminate
most of the unwanted proteins of similar size.
High-resolution fractionation by gel filtration is well suited for the final polishing step in a
purification scheme. Monomers are easily separated from aggregates. Samples can be
transferred to a suitable buffer for assay or storage.

18-1022-18 AK 11
Rapid purity check and screening
Superdex™ is a high resolution gel filtration medium. Superdex 75 5/150 GL and Superdex
200 5/150 Gl are short columns with small bed volumes and are suitable for rapid protein
homogeneity analyses or purity checks. They save time when screening many samples, and
require less buffer and sample than longer columns. However, when using the same media,
shorter columns give lower resolution than longer columns.

Resolution in gel filtration

The success of gel filtration depends primarily on choosing conditions that give sufficient
selectivity and counteract peak broadening effects during the separation. After selection
of gel filtration medium, sample volume and column dimensions are the two most critical
parameters that will affect the resolution of the separation.
The final resolution is influenced by many factors, see Table 1.1. The molecular weight range
over which a gel filtration medium can separate molecules is referred to as the selectivity of
the medium (see fractionation range guide for gel filtration media on page 16). Resolution is a
function of the selectivity of the medium and the efficiency of that medium to produce narrow
peaks (minimal peak broadening), as illustrated in Chapter 7, Figure 7.7.

Table 1.1. Factors that influence resolution

Medium-related factors Particle size

Particle uniformity
Match between pore size and analyte size
Column-related factors Bed height
Column packing quality
Experimental-related factors Flow rate
Sample volume
Viscosity

Sample volume and column dimensions

The sample volume can be expressed as a percentage of the total column volume (packed bed).
Smaller sample volumes help to avoid overlap if closely spaced peaks are eluted. Figure 1.3
illustrates how sample volume can influence a high resolution fractionation.

For group separations, use sample volumes up to 30% of the total column volume.
For high resolution fractionation, a sample volume from 0.5% to 4% of the total column
volume is recommended, depending on the type of medium used. For most applications
the sample volume should not exceed 2% to achieve maximum resolution. Depending
on the nature of the specific sample, it may be possible to load larger sample volumes,
particularly if the peaks of interest are well resolved. This can only be determined by
experimentation.

12 18-1022-18 AK
A) A 280 nm Column: Superdex 200 HR 10/30 (Vt: 24 ml)
0.25 Vo 25 µl Vt
Sample: Mr Conc. (mg/ml)
Thyroglobulin 669 000 3
0.20
Ferritin 440 000 0.7
IgG 150 000 3
0.15 Transferrin 81 000 3
Ovalbumin 43 000 3
0.10 Myoglobin 17 600 2
Vitamin B12 1 355 0.5
0.05 Total 15.2
Sample load: A) 25 µl (0.1% × Vt)
0.00 B) 250 µl (1% × Vt)
0. 0 5. 0 10. 0 15.0 20. 0 2 5.0 min C) 1000 µl (4.2% × Vt)
A 280 nm Buffer: 0.05 M sodium phosphate,
B)
Vo 250 µl Vt 0.15 M NaCl, pH 7.0
0.15
Flow rate: 1.0 ml/min (76.4 cm/h)

0.10

0.05

0.00
0. 0 5. 0 10. 0 15.0 20. 0 2 5.0 min

C) A 280 nm
Vo 1000 µl Vt

0.10

0.05

0.00
0. 0 5. 0 10. 0 15.0 20. 0 2 5.0 min

Fig 1.3. Influence of sample volume on resolution (Superdex 200 HR 10/30 is replaced with Superdex 200 10/300 GL).

The ratio of sample volume to column volume influences resolution, as shown in

Figure 1.4, where higher ratios give lower resolution. Column volumes are normally
selected according to the sample volumes to be processed. Since larger sample
volumes may require significantly larger column volumes, it may be beneficial to repeat
the separation several times on a smaller column and pool the fractions of interest or
concentrate the sample (see Appendix 3 on sample preparation).
For analytical separations and separations of complex samples, start with a sample
volume of 0.5% of the total column volume. Sample volumes of less than 0.5% dot not
normally improve resolution.
Concentrating samples can increase the capacity of a gel filtration separation. Avoid
concentrations above 70 mg/ml protein as viscosity effects may interfere with the
separation.
Sample dilution is inevitable since diffusion occurs as sample passes through the
column. To minimize sample dilution, use a sample volume that gives the resolution
required between the peaks of interest.
18-1022-18 AK 13
Column: HiLoad™ 16/60 Superdex 200 prep grade
Sample: Solution of transferrin (Mr 81 000) and
IgG (Mr 160 000) by equal weight
Sample concentration: 8 mg/ml
Buffer: 50 mM NaPO4, 0.1 M NaCl, pH 7.2
Flow rate: 1 ml/min (30 cm/h)

1.5
Resolution, Rs

1.0

0.5

0
0 1 2 3 4 5
Sample volume (% of column volume)

Fig 1.4. Influence of ratio of sample volume to column volume on the resolution of transferrin and IgG on prepacked
HiLoad 16/60 Superdex 200 prep grade. Resolution is defined in Chapter 7.

The height of the packed bed affects both resolution and the time taken for elution. The
resolution in gel filtration increases with the square root of bed height. Doubling the bed
height gives an increase in resolution equivalent to √2 = 1.4 (40%). For high resolution and
fractionation, long columns will give the best results. Sufficient bed height together with a low
flow rate allows time for ‘intermediate’ molecules to diffuse in and out of the matrix and give
sufficient resolution.
If a very long column is necessary, the effective bed height can be increased by using
columns, containing the same media, coupled in series.
Refer to Chapter 7 for detailed information on the theory of gel filtration.

Media selection
Today’s gel filtration media cover a molecular weight range from 100 to 80 000 000, separating
biomolecules from peptides to very large proteins and protein complexes.
The selectivity of a gel filtration medium depends solely on its pore size distribution and is
described by a selectivity curve. Gel filtration media are supplied with information about selectivity,
as shown for Superdex in Figure 1.5. The curve is a plot of the partition coefficient Kav against
the log of the molecular weight for a set of standard proteins (for calculation of Kav, see Chapter 7
Gel filtration in theory).

14 18-1022-18 AK
K av
1.00
Superdex peptide

Superdex 75

Superdex 200

Superdex 30 prep grade

0.75
Superdex 75 prep grade

Superdex 200 prep grade

0.50

0.25

101 102 103 104 105 106

M r logarithmic scale
Fig 1.5. Selectivity curves for Superdex.

Selectivity curves are almost linear in the range Kav = 0.1 to Kav = 0.7 and can be used to
determine the fractionation range of a gel filtration medium (Fig 1.6).

1.0

0.7

K av

Exclusion limit
0.1

log M r
Fractionation range

Fig 1.6. Defining fractionation range and exclusion limit from a selectivity curve.

The fractionation range defines the range of molecular weights that have partial access
to the pores of the matrix; that is molecules within this range should be separable by high
resolution fractionation. The exclusion limit for a gel filtration medium, also determined from
the selectivity curve, indicates the size of the molecules that are excluded from the pores of the
matrix and therefore elute in the void volume.
The steeper the selectivity curve, the higher the resolution that can be achieved.
When choosing a medium, consider two main factors:
1. The aim of the experiment (high resolution fractionation or group separation).
2. The molecular weights of the target proteins and contaminants to be separated.

18-1022-18 AK 15
The final scale of purification should also be considered. Figure 1.7 gives some guidance to
media selection. All media are available in prepacked columns, which is recommended if you
have little experience in column packing.

Mr 10 2 10 3 10 4 10 5 10 6 10 7 10 8 Resolution

Superdex Peptide

Superdex 75

Superdex 200

Superdex 30 prep grade

Superdex 75 prep grade

Superdex 200 prep grade

High resolution fractionation

Superose™ 6
Superose 12

Superose 6 prep grade

Superose 12 prep grade

Sephacryl™ S-100 HR

Sephacryl S-200 HR

Sephacryl S-300 HR

Sephacryl S-400 HR

Sephacryl S-500 HR

Sephacryl S-1000 SF
Group separation/Desalting

Sephadex G-10 Exclusion limit

Sephadex G-25 SF
Sephadex G-25 F Exclusion limit
Sephadex G-25 M
Sephadex G-50 F Exclusion limit

Sephadex LH-20

Fig 1.7. Gel filtration media fractionation range guide.

Superdex is the first choice for high resolution, short run times, and high recovery.
Superdex prep grade and Sephacryl are suitable for fast, high recovery separations at
laboratory and industrial scale.

Superdex, Sephacryl, or Superose are high resolution media with a wide variety of fractionation
ranges. In cases when two media have similar fractionation ranges, select the medium with
the steepest selectivity curve (see chapter 2, 3, and 4 for the respective medium) for the best
resolution of all the sample components. If a specific component is of interest, select the
medium where the log of molecular weight for the target component falls in the middle of the
selectivity curve.

16 18-1022-18 AK
 Sephadex is recommended for rapid group separations such as desalting and buffer
exchange. Sephadex is used at laboratory and production scale, before, between or
after other chromatography purification steps.

For group separations, select gel filtration media that elute high molecular weight molecules
at the void volume to minimize peak broadening or dilution and reduce time in the column. The
lowest molecular weight substances should appear by the time one column volume of buffer
has passed through the column.

Table 1.2. Sephadex media properties

Medium Cut-off Application examples

Sephadex G-10 700 Desalting of peptides
Sephadex G-25 1500 Desalting of proteins and oligonucleotides
Sephadex G-50 5000 Removal of free labels from labeled macromolecules

Sample and buffer preparation

Removal of particles in the sample is extremely important for gel filtration. Clarifying a sample
before applying it to a column will avoid the risk of blockage, reduce the need for stringent
washing procedures and extend the life of the medium.

Samples must be clear and free from particulate matter, especially when working with

bead sizes of 34 μm or less.
Appendix 3 contains an overview of sample preparation techniques. For small sample
volumes a syringe-tip filter of cellulose acetate or PVDF can be sufficient.

Sample buffer composition

The pH, ionic strength and composition of the sample buffer will not significantly affect
resolution as long as these parameters do not alter the size or stability of the proteins to be
separated and are not outside the stability range of the gel filtration medium. The sample
does not have to be in exactly the same buffer as that used to equilibrate and run through the
column. Sample is exchanged into the running buffer during the separation, an added benefit
of gel filtration.

Sample concentration and viscosity

Gel filtration is independent of sample mass and hence sample concentration, as can be seen
in Figure 1.8. High resolution can be maintained despite high sample concentration and, with
the appropriate medium, high flow rates.

18-1022-18 AK 17
Column: XK 16/70 (140 ml)
Medium: Superdex 200 prep grade
Sample: Solution of transferrin (Mr 81 000) and IgG (Mr 160 000) by equal weight
Sample volume: 0.8% × Vt
Buffer: 0.05 sodium phosphate, 0.1 M sodium chloride, pH 7.2
Flow rate: 1 ml/min (30 cm/h)
0 24 48 72 96 120 mg/ml sample
1.5

1.0
Resolution, R S

0.5

0.0
0.0 0.2 0.4 0.6 0.8 1.0 mg sample/ml
packed bed
Fig 1.8. Influence of sample concentration on the resolution of transferrin and IgG on Superdex 200 prep grade.

The solubility or the viscosity of the sample may however limit the concentration that can be used.
A critical variable is the viscosity of the sample relative to the running buffer, as shown by the
change in elution profiles of hemoglobin and NaCl at different sample viscosities in Figure 1.9.
High sample viscosity causes instability of the separation and an irregular flow pattern. This
leads to very broad and skewed peaks, and the back pressure might increase.

Low viscosity

Intermediate viscosity

High viscosity

Elution volume
Fig 1.9. Deteriorating separation caused by increasing viscosity. Elution diagrams obtained when hemoglobin (blue)
and NaCl (red) were separated. Experimental conditions were identical except that the viscosities were altered by the
addition of increasing amounts of dextran. Note that lowering flow rate will not improve the separation.

18 18-1022-18 AK
 Samples should generally not exceed 70 mg/ml protein. Dilute viscous samples, but not
more than necessary to keep the sample volume low. Remember that viscosity varies
with temperature.

Sample volume
Sample volume is one of the most important parameters in gel filtration. Refer to page 12 for
more information.

Buffer composition
Buffer composition will generally not directly influence the resolution unless the buffer affects
the shape or biological activity of the molecules. Extremes of pH and ionic strength and the
presence of denaturing agents or detergents can cause conformational changes, dissociation
or association of protein complexes.
Select buffer conditions that are compatible with protein stability and activity. The product
of interest will be collected in this buffer. Use a buffer concentration that maintains buffering
capacity and constant pH. Use from 25 mM up to 150 mM NaCl to avoid nonspecific ionic
interactions with the matrix which can be seen as delays in peak elution. Note that some
proteins may precipitate in low ionic strength solutions. Volatile buffers such as ammonium
acetate, or ammonium bicarbonate should be used if the separated product will be lyophilized.
Use high quality water and chemicals. Solutions should be filtered through 0.45 μm
or 0.22 μm filters before use. It is essential to degas buffers before any gel filtration
separation since air bubbles can significantly affect performance. Buffers will be
automatically degassed if they are filtered under vacuum.
When working with a new sample, try these conditions first: 0.05 M sodium phosphate,
0.15 M NaCl, pH 7.0 or select the buffer into which the product should be eluted for the
next step (e.g., further purification, analysis, or storage).
Avoid extreme changes in pH or other conditions that may cause inactivation or even

precipitation. If the sample precipitates in the gel filtration column, the column will be
blocked, possibly irreversibly, and the sample may be lost.

Denaturing (chaotropic) agents and detergents

Denaturing agents such as guanidine hydrochloride or urea can be used for initial solubilization
of a sample as well as in gel filtration buffers to maintain solubility. However, since the proteins
will denature, chaotropics should be avoided unless denaturation is specifically desired.
Superdex and Sephacryl are in general more suitable than classical media such as Sepharose™
or Sephadex for working under dissociating or denaturing conditions or at extreme pH values.
Detergents are useful as solubilizing agents for proteins with low aqueous solubility, such as
membrane components, and will not affect the separation. Sometimes, denaturing agents
or detergents are necessary to maintain the solubility of the sample. Such additives must be
present all the time, both in the running buffer and the sample buffer.
If high concentrations of additives are needed, use lower flow rates to avoid excessive
pressure since they may increase the viscosity of the buffer.
If proteins precipitate, elute later than expected, or are poorly resolved during gel filtration,
it is recommended to add a suitable concentration of a denaturing agent or detergent to the
running buffer.

18-1022-18 AK 19
 Urea or guanidine hydrochloride is very useful for molecular weight determination.
The presence of these denaturing agents in the running buffer maintains proteins
and polypeptides in an extended configuration. For accurate molecular weight
determination the calibration standards must also be run in the same buffer.
Note that selectivity curves are usually determined using globular proteins and do not

reflect the behavior of denatured samples.
Gel filtration can be used to exchange the detergent environment of a protein. For
example, a protein solubilized in SDS could be transferred to a milder detergent such as
Triton™ X-100 without losing solubility.

Column and media preparation

To perform a separation, gel filtration medium is packed into a column 30–60 cm in height for
high-resolution fractionation and up to 10 cm in height for group separations. Rapid screening
experiments can be performed on 15 cm columns. The volume of the packed bed is determined
by the sample volumes that will be applied.
Efficient column packing is essential, particularly for high resolution fractionation. The efficiency
of a packed column defines its ability to produce narrow symmetrical peaks during elution.
Column efficiency is particularly important in gel filtration in which separation takes place
as only a single column volume of buffer passes through the column. The uniformity of the
packed bed and the particles influences the uniformity of the flow profile and hence affects the
shape and width of the peaks. High-performance gel filtration media with high bed uniformity
(smaller and more uniform particles) give decreased peak widths and better resolution.
Efficiency is defined in terms of theoretical plates per meter (N).
N = 5.54 (Ve/W½)2 × 1000/L
where
Ve = peak elution (retention) volume
W½ = peak width at half peak height
L = bed height (mm)
Ve and W½ are in same units
Refer to Chapter 7 Gel filtration in theory and Appendix 1 for further information on column
efficiency and column packing.
Prepacked columns are highly recommended for the best performance and
reproducible results.
Efficiency can be improved by using a smaller particle size. However, using a smaller
particle size may create an increase in back pressure so that flow rate must be
decreased and run time lengthened.
Buffers, media or prepacked columns must have the same temperature before use.
Rapid changes in temperature, for example removing packed columns from a cold
room and applying buffer at room temperature, can cause air bubbles in the packing
and affect the separation.
Storage solutions and preservatives should be washed away thoroughly before using any
gel filtration medium. Equilibrate the column with 1–2 column volumes of buffer before starting
a separation.

20 18-1022-18 AK
Sample application
A liquid chromatography system should be used for high-resolution separation. For group
separations it is possible to use manual purification. Samples can be applied by gravity feed to
prepacked columns such as PD-10 Desalting.

Elution and flow rates

Samples are eluted isocratically from a gel filtration column, using a single buffer system.
After sample application the entire separation takes place as one column volume of buffer
(equivalent to the volume of the packed bed) passes through the column.
The goal for any separation is to achieve the highest possible resolution in the shortest possible
time. Figures 1.10, 1.11, and 1.12 shows that resolution decreases as flow rate increases. Each
separation must be optimized to provide the best balance between these two parameters. Put
simply, maximum resolution is obtained with a long column and a low flow rate whereas the
fastest run is obtained with a short column and a high flow rate. Suitable flow rates for high
resolution fractionation or group separation are usually supplied with each product.
The advantage of a higher flow rate (and consequently a faster separation) may outweigh the
loss of resolution in the separation.

Column: Superdex 200 HR 10/30 (Vt: 24 ml)

Sample: Mr Conc. (mg/ml)
Thyroglobulin 669 000 3
Ferritin 440 000 0.7
IgG 150 000 3
Transferrin 81 000 3
Ovalbumin 43 000 3
Myoglobin 17 600 2
Vitamin B12 1 355 0.5
Total 15.2
Buffer: 0.05 M sodium phosphate, 0.15 M NaCl, pH 7.0
Flow rate: 1) 0.25 ml/min (19.1 cm/h)
2) 1.0 ml/min (76.4 cm/h)

A 280 nm 25 µl, 0.25 ml/min (19 cm/h) A 280 nm 25 µl, 1.0 ml/min (76 cm/h)
0.30
Vo Vt 0.25 Vo Vt

0.25
0.20
0.20
0.15
0.15
0.10
0.10

0.05
0.05

0.00 0.00
0.0 25.0 50.0 75.0 100. 0 min 0.0 5.0 10.0 15. 0 2 0. 0 2 5.0 min

Fig 1.10. Influence of flow rate on resolution (Superdex 200 HR 10/30 is replaced with Superdex 200 10/300 GL).

18-1022-18 AK 21
Column: HiLoad 16/60 Superdex 30 prep grade
Sample: IGF-1 containing monomers and dimers
Sample load: 1 ml (0.8% × Vt)
Sample concentration: a) 1.25 mg/ml
b) 5 mg/ml
Buffer: 50 mM sodium acetate, 0.1 M NaCl, pH 5.0

2.0

1.5
a
Resolution, Rs

b
1.0

0.5

0
0 20 40 60 80
Flow, cm/h
Fig 1.11. Resolution between two different concentrations of IGF-1 containing monomers and dimers at different flow rates.

Columns: HiPrep™ 16/10 Sephacryl S-100 HR

HiPrep 16/10 Sephacryl S-200 HR
HiPrep 16/10 Sephacryl S-300 HR
Sample: IgG, ovalbumin, cytochrome C, 1:2:1
Sample load: 2.4 ml (2% × Vt)
Total sample load: 8 mg
Buffer: 50 mM NaPO4, 0.15 M NaCl, 0.02% NaN3, pH 7.0

3 HiPrep 16/60

2
Resolution, R s

S-100
S-200

1 S-300

0
0.0 0.2 0.4 0.6 0.8 1.0 1.2
Flow, ml/min

Fig 1.12. Influence on resolution between IgG, ovalbumin and cytochrome C using different gel filtration media.

If peaks are well separated at a low flow rate, increase the flow rate or shorten the
column to save time. Alternatively, increase the sample volume and benefit from a
higher capacity without significant loss of resolution.
For group separations such as desalting, monitor the elution of protein at A280 and follow the
elution of the salt peak using a conductivity monitor. Adjust flow rate and sample volume
to balance speed of separation against an acceptable level of salt in the final sample.
Recommended flow rates are given in the instructions supplied with each product.

22 18-1022-18 AK
Flow rate is measured in volume terms, for example ml/min, but when comparing results
between columns of different sizes it is useful to use the linear flow, cm/hour. A conversion
table is available in Appendix 5. Results obtained at the same linear flow on different sized
columns will be comparable as far as the effects of flow rate are concerned.
Selecting a smaller particle size of the same medium (if available) can also help to achieve the
correct balance between flow rate and resolution. Smaller particles of the same medium can
increase column efficiency, improve resolution to allow the use of higher flow rates. However,
smaller particles can also result in increased back pressure and this factor may become
restrictive if the intention is to scale up the separation.
Include a wash step at the end of a run to remove any molecules that may have been retained on
the column, to prevent cross-contamination and to prepare the column for a new separation.

Controlling flow
Accurate, reproducible control of the flow rate is not only essential for good resolution, but also
for reliability in routine preparative work and repeated experiments.
Use a chromatography system (rather than a peristaltic pump or gravity feed) to fully
utilize the high rigidity and excellent flow properties of Sephacryl, Superose, or Superdex
for high resolution fractionation.
Always pump the buffer to a column rather than drawing the buffer through the column
with the pump below. This reduces the risk of bubble formation as a result of suction. If
you have packed the column yourself, always use a lower flow rate for separation than
the flow rate used for column packing.
Use a syringe or a pump for work with small prepacked columns such as HiTrap Desalting.
Gravity feed with PD-10 Desalting for group separations of small sample volumes.
Gel filtration columns must not run dry. Ensure that there is sufficient buffer for long,

unattended runs or that the pump is programmed to stop the flow after a suitable time.
Columns that run dry must be repacked since the packed bed has been destroyed.

Method development for high resolution fractionation

Prepacked columns are delivered with standard running conditions that give satisfactory results
in most situations. If optimization is needed, follow these steps (given in order of priority):
1. Select the medium that will give the best resolution of the target protein(s), see gel filtration
media fractionation range guide, page 16.
2. To ensure reproducibility and high resolution, select a prepacked column that is best suited
to the volume of sample that needs to be processed (see Chapters 2 to 4 for details of
prepacked columns containing Superdex, Sephacryl, or Superose).
3. Select the highest flow rate that maintains resolution and minimizes separation time. Check
recommended flow rates supplied in the instructions for the specific medium and column.
4. Determine the maximum sample volume that can be loaded without reducing resolution.
Higher flow rates and viscous buffers yield higher operating pressures (remember that buffer
viscosity increases when running at 4°C). Check the maximum operating pressure of the
packed column and set the upper pressure limit on the chromatography system accordingly.
If greater resolution is required, increase the bed height by connecting two columns
containing the same medium in series. Alternatively, try a medium with the same or
similar fractionation range, but with a smaller particle size.
To process larger sample volumes, see scaling up below.

18-1022-18 AK 23
Maintenance of gel filtration columns
When a gel filtration medium has been in use for some time, it may be necessary to remove
precipitated proteins or other contaminants that can build up on the column. Cleaning may be
necessary when; a colored band can be seen at top of the column, a space occurs between the
upper adaptor and the bed surface, there is a loss in resolution, or a significant increase in back
pressure. Detailed cleaning procedures for each gel filtration medium are given in Chapters 2 to
6. In all cases, prevention is better than cure and routine cleaning is recommended.
If an increase in back pressure is observed, either on the pressure monitor or by seeing the
surface of the medium move downwards, check that the problem is actually caused by the
column before starting the cleaning procedure. Disconnect one piece of equipment at a time
(starting at the fraction collector), start the pump and check the pressure after each piece is
disconnected. Clogged on-line filters is a common cause of increased back pressure. Check
back pressure at the same stage during each run, since the value can vary within a run, for
example during sample injection or when changing to a different buffer.
Always use filtered buffers and samples to reduce the need for additional column
maintenance. See Appendix 3 for further details on sample preparation.
Always use well degassed buffers to avoid the formation of air bubbles in the packed
column during a run.
Buffers, prepacked columns and samples should be kept at the same temperature to
prevent air bubbles forming in the column.
Filter cleaning solutions before use and always re-equilibrate the column with 2–3
column volumes of buffer before the next separation.

Equipment selection
Appendix 4 provides a guide to the selection of suitable systems for gel filtration separation.

Scaling up
After establishing a high-resolution or group separation on a small column, larger columns can
be packed to process larger sample volumes in a single step. General guidelines for scaling up
are shown in Table 1.3.

Table 1.3 General guidelines for scaling up gel filtration separations

Maintain Increase
Bed height Column diameter
Linear flow rate Volumetric flow rate
Sample composition Sample volume

To scale up a gel filtration separation, follow this workflow:

1. Optimize the separation at small scale (see method development page 23).
2. Maintain the sample concentration and the ratio of sample to column volume.
3. Increase the column volume by increasing the cross-sectional area of the column.
4. Maintain the bed height.
5. Run the separation at the same linear flow rate as used on the smaller column (see Appendix 5).
Refer to Appendix 1 for column selection and column packing.

24 18-1022-18 AK
Different factors related to the equipment may affect performance after scale-up. If a larger
column has a less efficient flow distribution system, or a larger system introduces dead
volumes, peak broadening may occur. This will cause extra dilution of the product fraction or
even loss of resolution if the application is sensitive to variations in efficiency.
For media such as Superdex or Superose it is usually recommended to select a larger particle
size for scale-up. For high-resolution fractionation, pack a small column containing the larger
particles and repeat the separation to facilitate any optimization that may be needed to
achieve the same resolution on the larger column.
Scaling up on Sephadex G-25, even to production scale, is a straightforward and well-established
process. Well-known examples of commercial applications include buffer exchange in
processes for removing endotoxins from albumin, and preparative steps during the production
of vaccines. Figure 1.13 shows an example of a large scale buffer exchange step used during
the production of albumin and IgG from human plasma.

Column: BPSS 400/600, 75 l

Sample: 10 l human plasma
1: sample application
2: eluent application
Eluent: 0.025 M sodium acetate
Flow: 240 l/h

A 280
Conductivity

1 2 1 2 1 2 1 2 1 2

Elution volume
Fig 1.13. Chromatogram of the buffer exchange step on Sephadex G-25 Coarse during production of albumin and IgG
from human plasma.

BioProcess™ Media for large-scale production

Specific BioProcess Media have been designed for each chromatographic stage in a process
from Capture to Polishing. Use BioProcess Media for every stage results in a systematic approach
to method development. Column packing methods are established for a wide range of scales.
The same BioProcess Media can be used for development work, pilot studies, and routine
production.
High flow rates, high capacity and high recovery contribute to the overall economy of an
industrial process.
All BioProcess Media can be cleaned- and sanitized-in-place.
Regulatory Support Files contain details of performance, stability, extractable compounds
and analytical methods available. The essential information in these files gives an invaluable
starting point for process validation, as well as support for clinical and marketing applications
submitted to regulatory authorities.

18-1022-18 AK 25
Large capacity production integrated with clear ordering and delivery routines mean BioProcess
media are available in the right quantity, at the right place, at the right time. Future supplies of
BioProcess media are assured, making them a safe investment for long-term production.
BioProcess Media is produced following validated methods and are tested under strict control,
fulfilling performance specifications. A certificate of analysis is available with each order. Our
media safety stock agreements offer the right quantity of media, manufactured to specified
quality levels, and delivered at the right time.

Troubleshooting
This section focuses on practical problems that may occur during gel filtration. Figure 1.14
indicate how a chromatogram may deviate from ideal behavior during a separation. Table 1.4
on the following pages contains suggestions of possible causes and their remedies.

Satisfactory separation.
Well resolved, symmetrical peaks.

Vo Vt

Poor resolution.
Review factors affecting resolution (see page 12), including
media selection, particle size, sample volume: column volume
and flow rate.

Vo Vt

Leading peaks.
Asymmetric peaks: sample elutes before void volume
indicates channelling in column bed. Leading peaks can also
be due an overpacked column (packed at too high a pressure
or flow rate). The column may need to be repacked.

Vo

Tailing peaks.
Asymmetric peaks: sample application uneven. Check the top
of column if possible. Ensure the medium is evenly packed
and that sample is applied without disturbing the packed
bed. Tailing peaks can also be due to underpacking of the
column (packed at too low a pressure or flow rate).
Vo

Late elution.
Peaks seen after one column volume of buffer has passed
through the column. Always include a wash step between
runs to ensure removal of late eluting molecules.

Vo Vt

Fig 1.14. Normal chromatograms and chromatograms deviating from ideal behavior.

26 18-1022-18 AK
Table 1.4. Troubleshooting guide for gel filtration

Situation Cause Remedy

Peak of interest is Sample volume is too high. Decrease sample volume and apply carefully.
poorly resolved from Sample is too viscous. Dilute with buffer, but check maximum sample volume.
other major peaks. Maintain protein concentration below 70 mg/ml
Sample contains particles Re-equilibrate column, filter sample and repeat.
Column not mounted vertically. Adjust column position. Column may need to be
repacked’.
Column is poorly packed. Check column efficiency (See Appendix 1).
Repack if needed*. Use prepacked columns.
Column is dirty. Clean and re-equilibrate.
Incorrect medium. Check selectivity curve. Check for adsorption
effects. Consider effects of denaturing agents or
detergents if present.
Large dead volumes. Minimize dead volumes in tubings and connections.
Column too short. See Appendix 1 for recommended bed heights.
Flow rate too high. Check recommended flow rates. Reduce flow.
Protein does not Ionic interactions between protein and Maintain ionic strength of buffers above 0.05 M
elute as expected. matrix. (preferably include 0.15 M NaCl).
Hydrophobic interactions between Reduce salt concentration to minimize hydrophobic
protein and matrix. interaction. Increase pH. Add suitable detergent or
organic solvent, e.g., 5% isopropanol.
Sample has not been filtered properly. Clean the column, filter the sample and repeat.
Sample has changed during storage. Prepare fresh samples.
Column is not equilibrated sufficiently. Repeat or prolong the equilibration step.
Proteins or lipids have precipitated on Clean the column or use a new column.
the column.
Column is overloaded with sample. Decrease the sample load.
Microbial growth has occurred in the Microbial growth rarely occurs in columns during
column. use. To prevent infection of packed columns, store in
20% ethanol when possible.
Protein elutes later Hydrophobic and/or ionic interactions Reduce salt concentration to minimize hydrophobic
than expected or between protein and matrix. interaction. Increase pH. Add suitable detergent or
even after running a organic solvent e.g., 5% isopropanol.
total column volume. Increase salt concentration (up to 150 mM) to
minimize ionic interaction.
Peaks elute late Column is dirty. Clean and re-equilibrate.
and are very broad.
Protein elutes earlier Channeling in the column. Repack column using thinner slurry of medium.
than expected (before Avoid introduction of air bubbles*.
the void volume).
Molecular weight or Protein has changed during storage Prepare fresh samples.
shape is not as expected.
Ionic interactions between proteins Maintain ionic strength of buffers above 0.05 M
and matrix. (preferably include 0.15 M NaCl).
Hydrophobic interactions between Reduce salt concentration to minimize hydrophobic
protein and matrix. interaction. Increase pH. Add suitable detergent or
organic solvent e.g., 5% isopropanol.
Precipitation of protein in the column If possible, clean the column, exchange or clean the
filter and/or at the top of the bed. filter or use a new column.
Leading or very rounded Column overloaded. Decrease sample load and repeat.
peaks in chromatogram.
Tailing peaks. Column is ‘under packed’. Check column efficiency (see Appendix 1). Repack
using a higher flow rate. Use prepacked columns*.

18-1022-18 AK 27
Situation Cause Remedy
Leading peaks. Column is ‘over packed’. Check column efficiency (see Appendix 1). Repack
using a lower flow rate. Use prepacked columns*.
Medium/beads appear Bed support end piece is loose or Replace or tighten.
in eluent. broken.
Column operated at too high pressure. Do not exceed recommended operating pressure for
medium or column.
Low recovery of Protein may be unstable or inactive in Determine the pH and salt stability of the protein.
activity but normal the buffer
recovery of protein.
Enzyme separated from co-factor Test by pooling aliquots from the reactions and
or similar. repeat the assay.
Lower yield than expected. Protein may have been degraded Add protease inhibitors to the sample and buffers to
by proteases. prevent proteolytic digestion. Fun sample through a
medium such as Benzamidine 4 Fast Flow (high sub)
to remove trypsin-like serine proteases.
Adsorption to filter during sample Use another type of filter.
preparation.
Sample precipitates. May be caused by removal of salts or unsuitable
buffer conditions.
Hydrophobic proteins. Use denaturing agents, polarity reducing agents,
or detergents.
Nonspecific adsorption.
More sample is recovered Protein is co-eluting with other Optimize conditions to improve resolution. Check
than expected. substances. buffer conditions used for assay before and after the
run. Check selection of medium.
More activity is Different assay conditions have Use the same assay conditions for all the assays in
recovered than was been used before and after the the purification scheme.
applied to the column. chromatography step.
Removal of inhibitors during separation.
Reduced or poor flow Presence of lipoproteins or protein Remove lipoproteins and aggregates during sample
through the column. aggregates. preparation (see Appendix 3).
Protein precipitation in the column Modify the eluent to maintain stability.
caused by removal of stabilizing agents
during separation.
Blocked column filter. If possible, replace the filter or use a new column.
Always filter samples and buffer before use.
Blocked end-piece or adaptor or tubing. If possible, remove and clean or use a new column.
Precipitated proteins. Clean the column using recommended methods or
use a new column.
Bed compressed. If possible, repack the column or use a new column*.
Microbial growth. Microbial growth rarely occurs in columns during
use. To prevent infection of packed columns, store in
20% ethanol when possible.

Fines (Sephadex). Decant fines before column packing. Avoid using

magnetic stirrers that can break the particles.
Back pressure increases Turbid sample. Improve sample preparation (see Appendix 3).
during a run or during Improve sample solubility by the addition of ethylene
successive runs. glycol, detergents, or organic solvents.
Precipitation of protein in the column Clean using recommended methods. If possible,
filter and/or at the top of the bed. exchange or clean filter or use a new column.
Include any additives that were sued for initial
sample solubilization in the running buffer.

28 18-1022-18 AK
Situation Cause Remedy
Air bubbles in the bed. Column packed or stored at cool Remove small bubbles by passing degassed buffer
temperature and then warmed up. through the column. 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. Repack column if possible (see Appendix 1*).
Buffers not properly degassed. Buffers must be degassed thoroughly.
Cracks in the bed. Large air leak in column. Check all connections for leaks. Repack the column if
possible (see Appendix 1).
Distorted bands as sample Air bubble at the top of the column or in If possible, re-install the adaptor taking care to avoid
runs into the bed. the inlet adaptor. air bubbles.
Particles in buffer or sample. Filter or centrifuge the sample. Protect buffers from
dust.
Blocked or damaged net in upper If possible, dismantle the adaptor, clean or replace
adaptor. the net.
Keep particles out of samples and eluents.
Distorted bands as sample Column poorly packed. Suspension too thick or too thin. Bed packed at a
passes down the bed. temperature different from run.
Bed insufficiently packed (too low packing pressure,
too short equilibration). Column packed at too high
pressure.
* Not all prepacked columns can be repacked.

18-1022-18 AK 29
30 18-1022-18 AK
Chapter 2
Superdex: the first choice for high resolution,
short run times, and high recovery

Superdex are gel filtration media with a unique composite medium of dextran and agarose.
This matrix combines the excellent gel filtration properties of cross-linked dextran with the
physical and chemical stabilities of highly cross-linked agarose, to produce a separation medium
with outstanding selectivity and high resolution. In addition, its low nonspecific interaction
permits high recovery of biological material. Together these properties make Superdex the first
choice gel filtration media for all applications from laboratory to process scale.

Mr 10 2 10 3 10 4 10 5 10 6

Superdex Peptide

Superdex 75

Superdex 200

Superdex 30 prep grade

Superdex 75 prep grade

Superdex 200 prep grade

Fig 2.1. Fractionation ranges for Superdex.

Fig 2.2. Prepacked HiLoad Superdex prep grade columns.

Superdex is the first choice for a high resolution fractionation with short run times and good
recovery. Selectivity curves and pressure-flow relationship curves for Superdex are shown in
Figures 2.3 and 2.4. A typical linear flow is up to 75 cm/h. The media is available in two versions,
Superdex and Superdex prep grade. The difference is the particle sizes.

18-1022-18 AK 31
K av
1.00

Superdex peptide

Superdex 75

0.75 Superdex 200

Superdex 30 prep grade

Superdex 75 prep grade

Superdex 200 prep grade

0.50

0.25

101 102 103 104 105 106

M r logarithmic scale

Fig 2.3. Selectivity curves for Superdex (13 μm) and Superdex prep grade (34 μm) media.

4
Pressure (bar)

3
0 0
/6 /6
26 16
2

20 40 60 80 10 0 1 20 14 0
Flow rate (cm/h)
Fig 2.4. 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 by cross-sectional area
of column (2 cm2 for XK 16, 5.3 cm2 for XK 26).See Appendix 5 for more information about flow rate calculations.

Superdex is a composite medium based on highly cross linked porous agarose particles to
which dextran has been covalently bonded, as illustrated in Figure 2.5. The result is media with
high physical and chemical stability, mainly due to the highly cross-linked agarose matrix, and
excellent gel filtration properties determined mainly by the dextran chains. The mechanical
rigidity of Superdex allows even relatively viscous eluents, such as 8 M urea, to be run at practical
flow rates. The media can withstand high flow rates during equilibration or cleaning thereby
shortening overall cycle times. This stability makes Superdex prep grade very suitable for use in
industrial processes where high flow rates and fast, effective cleaning-in- place protocols are
required. Under normal chromatography conditions nonspecific interactions between proteins
and Superdex are negligible when using buffers with ionic strengths in the range 0.15 M to 1.5 M.

Crosslinked Agarose
Dextran

Fig 2. 5. A schematic section of a Superdex particle. In Superdex the dextran chains are covalently linked to a highly
cross-linked agarose matrix.

32 18-1022-18 AK
Separation options
Superdex is produced in two different mean particle sizes (13 μm and 34 μm) and four different
selectivities (Superdex Peptide, Superdex 30, Superdex 75 and Superdex 200, see Table 2.1).
Use the 13 μm particles of Superdex Peptide, Superdex 75 and Superdex 200 in
prepacked columns for highest resolution analytical separations with smaller sample
volumes.
Use the 34 μm particles of Superdex prep grade (available in prepacked columns or as
bulk media) for preparative applications.

Table 2.1. Separation options with Superdex medium

Fractionation Sample . Maximum

range, Mr loading operating back Recommended
Product* (globular proteins) capacity† pressure operation flow‡
Superdex Peptide PC 3.2/30 1 × 102–7 × 103 25–250 µl 1.8 MPa, 18 bar, <0.15 ml/min
260 psi
Superdex Peptide 10/300 GL 1 × 102–7 × 103 25–250 µl 1.8 MPa, 18 bar, <1.2 ml/min
260 psi
HiLoad 16/60 Superdex 30 pg§ <1 × 104 ≤5 ml 0.3 MPa, 3 bar, ≤1.6 ml/min
42 psi
HiLoad 26/60 Superdex 30 pg§ <1 × 104 ≤13 ml 0.3 MPa, 3 bar, ≤4.4 ml/min
42 psi
Superdex 30 pg§ (Bulk media) <1 × 104 0.5–4% of total Column- 10–50 cm/h
column volume dependent
Superdex 75 PC 3.2/30 3 × 103–7 × 104 <50 µl 2.4 MPa, 24 bar, <0.1 ml/min
350 psi
Superdex 75 5/150 GL 3 × 103–7 × 104 4–50 µl 1.8 MPa, 18 bar, <0.6 ml/min
260 psi
Superdex 75 10/300 GL 3 × 103–7 × 104 25–250 µl 1.8 MPa, 18 bar, <1.5 ml/min
260 psi
HiLoad 16/60 Superdex 75 pg§ 3 × 103–7 × 104 ≤5 ml 0.3 MPa, 3 bar, ≤1.6 ml/min
42 psi
HiLoad 26/60 Superdex 75 pg§ 3 × 103–7 × 104 ≤13 ml 0.3 MPa, 3 bar, ≤4.4 ml/min
42 psi
Superdex 75 pg§(Bulk media) 3 × 103–7 × 104 0.5–4% of total Column- 10–50 cm/h
column volume dependent
Superdex 200 PC 3.2/20 1 × 104–6 × 105 <50 µl 1.5 MPa, 15 bar, <0.1 ml/min
220 psi
Superdex 200 5/150 GL 1 × 104–6 × 105 4–50 µl 1.5 MPa, 15 bar, <0.6 ml/min
220 psi
Superdex 200 10/300 GL 1 × 104–6 × 105 25–250 µl 1.5 MPa, 15 bar, 0.25–0.75 ml/min
220 psi
HiLoad 16/60 Superdex 200 pg§ 1 × 104–6 × 105 ≤5 ml 0.3 MPa, 3 bar, ≤1.6 ml/min
42 psi
HiLoad 26/60 Superdex 200 pg§ 1 × 104–6 × 105 ≤13 ml 0.3 MPa, 3 bar, ≤4.4 ml/min
42 psi
Superdex 200 pg§ (Bulk media) 1 × 104–6 × 105 0.5–4% of total Column- 10–50 cm/h
column volume dependent
* PC and 10/300 GL columns are packed with Superdex and HiLoad are packed with Superdex prep grade.
†
For maximum resolution, apply as small sample volume as possible. Note that sample volumes less than 0.5% normally do
not improve resolution.
‡
See Appendix 5 to convert linear flow (cm/hour) to volumetric flow rates (ml/min) and vice versa.
§
prep grade.

18-1022-18 AK 33
 Start with Superdex 200 when the molecular weight of the protein of interest is unknown.
Superdex 200 or Superdex 200 prep grade are especially suitable for the separation of
monoclonal antibodies from dimers and from contaminants of lower molecular weight,
for example albumin and transferrin.
Start with Superdex Peptide or Superdex 30 prep grade for separations of peptides,
oligonucleotides and small proteins below Mr 10 000.
Exposure to temperatures outside the range 4°C to 40°C will destroy the efficiency of the
packed bed and the column will need to be repacked.

Separation examples
Figures 2.6 to 2.15 illustrate examples of separations performed on Superdex Peptide,
Superdex 75 and Superdex 200 run on ÄKTA™ design systems. Detection can also be made by
light scattering (Fig 2.9). This method reveals the molecular mass in an unambiguous way.

Column: Superdex Peptide 10/300 GL

Sample: 1. Cytochrome c (Mr 12 384) 1 mg/ml
2. Aprotinin (Mr 6 512) 2 mg/ml
3. Vitamin B12 (Mr 1 355) 0.1 mg/ml
4. (Gly)3 (Mr 189) 0.1 mg/ml
5. Gly (Mr 75) 7.8 mg/ml
Sample load: 50 µl
Buffer: 50 mM phosphate, 0.15 M NaCl, pH 7.0
Flow rate: 0.5 ml/min, room temperature
A215 nm

mAU 3

600

500

400

300

1
2

200 5

100

V0 Vt
0

0 5.0 10.0 15.0 20.0 25.0 ml

Fig 2.6. Separation of standard peptides on Superdex Peptide 10/300 GL.

34 18-1022-18 AK
Column: Superdex 200 HR 10/30
Sample: Mr Conc. (mg/ml)
Thyroglobulin 669 000 3
Ferritin 440 000 0.7
IgG 150 000 3
Transferrin 81 000 3
Ovalbumin 43 000 3
Myoglobin 17 600 2
Vitamin B12 1 355 0.5
Total 15.2
Total sample amount: 0.38 mg
Sample load: 25 ml
Buffer: 0.05 M sodium phosphate, 0.15 M NaCl, pH 7.0
Flow rate: 0.25 ml/min (19 cm/h)

A 280 nm 2 7
0.30

Vo Vt

0.20

1
3 4
5
6
0.10

0
0 5.0 10.0 15.0 20.0 25.0
Volume (ml)
Fig 2.7. Separation of standard proteins (Superdex 200 HR 10/30 is replaced with Superdex 10/300 GL).

Column: Superdex 75 10/300 GL

Sample: rec Cys-prot
Sample load: 200 μl
Buffer: 0.05 M Tris HCl, 1 mM EDTA, 0.15 M NaCl, pH 8.4
Flow rate: 0.5 ml/min
A) B) mAU
mAU
Dimer 250 Monomer
1500
200
1000 Monomer 150
100
500 50
0 0
0.0 5.0 10.0 15.0 20.0 25.0 ml 0.0 5.0 10.0 15.0 20.0 25.0 ml

C)

Lanes
1. is the original dimer monomer sample; the dimer content is
Mr 20 000
high, which also is reflected in the chromatogram.
2. is the dimer fraction
3. corresponds to the monomer fraction from A, respectively.
4. shows the monomer peaks from B.

Lanes 1 to 4 were run under non-reducing conditions.

S 1 2 3 4

Fig 2.8. (A) Dimer-monomer separation of a recombinant cystein-containing protein (recCys-prot) on Superdex 75 10/300 GL.
(B) purification of the dimer fraction reduced with DTE. (C) Coomassie™ stained SDS-PAGE gel. Lane S is LMW-SDS Marker Kit
(17-0446-01),

18-1022-18 AK 35
 A)
A280
1.0

200 µl
0.8 100 µl
40 µl
Relative scale

0.6

0.4

0.2

0.0
0 5 10 15 20 25
Time (min)
B)
A280
1.0
200 µl
100 µl
0.8 40 µl
Relative scale

0.6

0.4

0.2

0.0
0 5 10 15 20 25
Time (min)

Fig 2.9. Analysis of ovalbumin aggregation on 2 Superdex 200 5/150 columns in series. (A) Detection with A280. (B) Detection
with light scattering. Peak 1 is dimer/trimer, peak 2 is the monomer. Note the strong scattering signal from minute amounts
of impurities in the void fraction.
Column: (A) Superdex 200 HR 10/30
A) B) (B) HiLoad 16/60 Superdex
200 prep grade
A 280 VO VC A280 VO VC Bed volumes: (A) 24 ml
0.60
(B) 122 ml
Sample: Mouse monoclonal IgG1
0.60
10 × concentrated cell
culture supernatant
Sample volume: (A) 200 ml, 0.8% × Vt
(B) 1.0 ml, 0.8% × Vt
0.40 lgG 1
lgG 1 Buffer: 50 mM NaH2PO4,
0.40 0.15 M NaCl, pH 7.0
Flow: (A) 1.0 ml/min (76 cm/h)
(b) 0.5 ml/min (15 cm/h)

0.20
0.20

5.0 15.0 25.0 50 100 150

Vol. (ml) Vol. (ml)
Fig 2.10. Scale-up (five times) of a mouse monoclonal IgG1 separation from (A) Superdex 200 HR 10/30 (replace by
Superdex 200 10/300 GL) to (B) HiLoad 16/60 Superdex 200 prep grade. Superdex 200 prep grade medium (mean
particle size 34 μm) has a similar selectivity to Superdex 200 (mean particle size 13 μm).

36 18-1022-18 AK
Column: HiLoad 16/60 Superdex 30 prep grade
Sample: 50 ml mix of five synthetic peptides in 1% TFA
1. Mr 3 894
2. Mr 3 134
3. Mr 2 365
4. Mr 1 596
5. Mr 827
Buffer: 20 mM Tris-HCl, 0.25 M NaCI, pH 8.5
Flow rate: 1 ml/min (30 cm/h)

Absorbance TFA
0.005

1
2 3

4 5

0 80 160 240 320

V olume (ml)

Fig 2.11. Separation of test substances on HiLoad 16/60 Superdex 30 prep grade.

Columns: A) HiLoad 16/60 Superdex 75 prep grade

B) HiLoad 16/60 Superdex 200 prep grade
Sample: 1. Myoglobin 1.5 mg/ml, Mr 17 000
2. Ovalbumin 4 mg/ml, Mr 43 000
3. Albumin 5 mg/ml, Mr 67 000
4. IgG 0.2 mg/ml, Mr 158 000
5. Ferritin 0.24 mg/ml, Mr 440 000
Sample load: 0.5 ml
Buffer: 0.05 M phosphate buffer, 0.15 M NaCl, 0.01% sodium azide, pH 7.0
Flow rate: 1.5 ml/min (45 cm/h)

A) B)
A 280 nm A 280 nm
HiLoad 16/60 HiLoad 16/60
0. 2 4 and 5 Superdex 75 prep grade 0. 2 Superdex 200 prep grade

3 2

3 2
0. 1 0. 1
1 5
1

Vo 4

0 0
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
Ti me (min) Ti me (min)
Fig 2.12. Comparison of the selectivity of Superdex 75 prep grade and Superdex 200 prep grade for model proteins.
Superdex 75 prep grade (A) gives excellent resolution of the three proteins in the Mr range 17 000 to 67 000 while the two
largest proteins elute together in the void volume. Superdex 200 prep grade (B) resolves the two largest proteins completely.
The three smaller proteins are not resolved quite as well as the larger ones or as in (A). The void volume (Vo) peak at
28 minutes in (B) is caused by protein aggregates.

18-1022-18 AK 37
Columns: HiLoad Superdex 200 prep grade
Column volumes, V t: A) ≈ 120 ml (16/60)
B) ≈ 320 ml (26/60)
Sample: Mouse monoclonal cell supernatant, IgG2b incl. 1% Fetal Calf Serum
Sample pretreatment: Concentration ≈ 40×
Sample load: A) 1.2 ml (1% × Vt)
B) 3.2 ml (1% × Vt)
Buffer: 50 mM NaH2PO4, 0.15 NaCI, pH 7.0
Flow rate: A) 1.6 ml/min (50 cm/h)
B) 4.4 ml/min (50 cm/h)
(max. recommended flow rates)

A) B)
A 280 nm A 280 nm

HiLoad 16/60 HiLoad 26/60

0.60
Superdex 200 prep grade 0.60 Superdex 200 prep grade

IgG2b IgG2b
0.40
0.40

0.20 0.20

0.00 0.00

0 20.0 40.0 60.0 80.0 Time (min) 0 20.0 40.0 60.0 80.0 Time (min)

Fig 2.13. Purification of mouse monoclonal IgG2b from cell supernatant using (A) HiLoad 16/60 Superdex 200 prep grade,
column volume 120 ml and (B) HiLoad 26/60 Superdex 200 prep grade, column volume 320 ml. Almost identical separations
are the result, even using prepacked columns of different sizes.

Column: Superdex 75 5/150 GL

Sample: Trypsin (Mr 23 800) 11mg/ml
Aprotinin (Mr 6 500) 3 mg/ml
Trypsin 11 mg/ml and Aprotinin 3mg/ml
Sample volume: 10 μl
Buffer: PBS, pH 7.4
Flow rate: 0.3 ml/min
mAU
Trypsin and
700 Aprotinin

600
Trypsin
500

400

300

200
Aprotinin
Autoprotolytic
100 fragments

0.0 0.5 1.0 1.5 2.0 2.5 3.0 ml

Fig 2.14. Monitoring of protein complex formation between trypsin and aprotinin.

38 18-1022-18 AK
Column: Superdex 200 5/150 GL
Sample: Integral membrane protein (Mr 60 000) from E. coli
Sample load: 10 μl
Buffers (including 0.1 or 0.3 M NaCl): 0.02 M sodium acetate, 0.03% dodecyl maltoside, 0.5 mM TCEP, pH 5.2
0.02 M HEPES, 0.03% dodecyl maltoside, 0.5 mM TCEP, pH 7.5
0.02 M CAPSO, 0.03% dodecyl maltoside, 0.5 mM TCEP, pH 9.5
Flow rate: 0.35 ml/min
Detection: 280 nm

A) pH 5.2 B) pHSample:
7.5 14 ug (10ul) EM35 C) pHSample:
9.5 9.4 ug (10 ul) EM35
Buffer: 20 mM Na-acetate pH 5.2 100 mM NaCl Buffer: 20 mM HEPES pH 7.5 100 mM NaCl Buffer: 20 mM CAPSO pH 9.5 100 mM NaCl
mAU mAU mAU 1.76
0.1 M NaCl 12.0 0.1 M NaCl 0.1 M NaCl
15.0
20.0 10.0

8.0
15.0 10.0
6.0

10.0 4.0
1.29
5.0
2.0
5.0
0.0
0.0
0.0 -2.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 ml 0.0 0.5 1.0 1.5 2.0 2.5 3.0 ml 0.0 0.5 1.0 1.5 2.0 2.5 3.0 ml

D) pHSample:
5.2 16 ug (10 ul) EM35 E) pHSample:
7.5 9.4 ug (10 ul) EM35 F) pHSample:
9.5 7.2 ug (10 ul) EM35
Buffer: 20 mM Na-acetate pH 5.2 300 mM NaCl Buffer: 20 mM HEPES pH 7.5 300 mM NaCl Buffer: 20 mM CAPSO pH 9.5 300 mM NaCl
mAU mAU mAU 1.78
40.0 16.0 1.78 14.0
0.3 M NaCl 0.3 M NaCl 0.3 M NaCl
35.0 14.0 12.0

30.0 12.0
10.0
25.0 10.0
8.0
20.0 8.0
1.29 6.0
6.0
15.0
4.0 1.3
4.0
10.0
2.0
2.0
5.0
0.0
0.0
0.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 ml 0.0 0.5 1.0 1.5 2.0 2.5 3.0 ml 0.0 0.5 1.0 1.5 2.0 2.5 3.0 ml

Fig 2.15. Screening of pH and ionic strength conditions for optimal homogeneity and stability of a detergent-protein
complex. The chromatograms (A–F) represent the results from the different screening conditions.

Performing a separation
Buffer: 0.05 M sodium phosphate, 0.15 M NaCl pH 7, or select the buffer in which the sample
should be stored or solubilized for the next step.

Use 0.15 M NaCl, or a buffer with equivalent ionic strength, to avoid pH-dependent ionic
interactions with the matrix. At very low ionic strength, the presence of a small number of
negatively charged groups on the medium may cause retardation of basic proteins.
The sample should be fully dissolved. Centrifuge or filter to remove particulate material
(see Appendix 3). Always use degassed buffers and maintain a constant temperature
during the run to avoid introducing air into the column.
Set an appropriate pressure limit on the chromatography system to avoid damage to the
column packing.

First time use or after long-term storage

1. Equilibrate the column with 1 column volume of buffer, but containing 0.05 M NaCl
at 30 cm/h (0.4 ml/min for 10/300 GL, 1 ml/min for HiLoad 16/60 or 2.6 ml/min for
HiLoad 26/60).
2. Equilibrate with 2 column volumes of buffer containing 0.15 M NaCl at 50 cm/h
(0.65 ml/min for 10/300 GL, 1.6 ml/min for HiLoad 16/60 or 4.3 ml/min for HiLoad 26/60).

18-1022-18 AK 39
 3. Reduce linear flow to 30 cm/h. Apply a sample volume equivalent to 0.5–4% of the
column volume (up to 0.25 ml for 10/300 GL, 1.2 ml for HiLoad 16/60 or 3.2 ml for
HiLoad 26/60). Note that the smaller the sample volume the better the resolution.
4. Elute with 1 column volume of buffer.
5. Before applying a new sample, re-equilibrate column with 1 column volume of buffer at
50 cm/h and until the baseline monitored at A280 is stable.

Column performance should be checked at regular intervals by determining the theoretical plate
number per meter and peak symmetry. Prepacked columns are supplied with recommended
values. See page 90 for how to check column efficiency.
See page 23 for advice on optimizing the separation.

Exposure to temperatures outside the range 4°C to 40°C will destroy the efficiency of a

packed bed and the column will need to be repacked.

Cleaning
1. Wash with 1 column volume of 0.5 M NaOH at a flow of 25 cm/h (0.33 ml/min for
10/300 GL, 0.8 ml/min for HiLoad 16/60 or 2.2 ml/min for HiLoad 26/60) to remove
most nonspecifically adsorbed proteins.
2. Wash with 1 column volume of distilled water at 25 cm/h.
3. Re-equilibrate with 2 column volumes of buffer at a flow of 50 cm/hr (0.4 ml/min for
10/300 GL, 1.6 ml/min HiLoad 16/60 or 4.3 ml/min for HiLoad 26/60) or until the
baseline monitored at A280 and the pH of the eluent are stable.
Further equilibration may be necessary if the buffer contains detergent.

Routine cleaning after every 10–20 separations is recommended, but the frequency of
cleaning will also depend on the nature of the samples being applied.

Removing severe contamination

1. Reverse the flow and wash at a linear flow of 25 cm/h at room temperature.
2. Wash with 4 column volumes of 1 M NaOH (to remove hydrophobic proteins or
lipoproteins) followed by 4 column volumes of distilled water.
3. Wash with 0.5 column volume of 30% isopropanol to remove lipids and very
hydrophobic proteins, followed by 2 column volumes of distilled water.
4. Equilibrate the column with at least 5 column volumes of buffer, or until the baseline
monitored at A280 and the pH of the eluent are stable, before beginning a new
separation.
For extreme cases of contamination, check the instructions supplied with the product.

40 18-1022-18 AK
Media characteristics
Matrix: Spherical composite of cross-linked agarose and dextran.

Table 2.3 Superdex media characteristics

Product Efficiency* pH stability† Particle size Mean particle size

Superdex Peptide ≥ 30 000 m -1
Long term: 1–14 12–14 µm 13 µm
Short term: 1–14
Superdex 75 ≥ 30 000 m-1 Long term: 3–12 12–14 µm 13 µm
Short term: 1–14
Superdex 200 ≥ 30 000 m-1 Long term: 3–12 12–14 µm 13 µm
Short term: 1–14
Superdex 30 prep grade ≥ 13 000 m-1 Long term: 3–12 24–44 µm 34 µm
Short term: 1–14
Superdex 75 prep grade ≥ 13 000 m-1 Long term: 3–12 24–44 µm 34 µm
Short term: 1–14
Superdex 200 prep grade ≥ 13 000 m-1 Long term: 3–12 24–44 µm 34 µm
Short term: 1–14
* Theoretical plates per meter (prepacked columns only).
†
Long term pH stability refers to the pH interval where the medium is stable over a long period of time without adverse side
effects on its chromatography performance. Short term pH stability refers to the pH interval for regeneration, cleaning-in-
place and sanitization procedures. All ranges are estimates based on the experience and knowledge within GE Healthcare.

Chemical stability
Superdex is stable in all commonly used aqueous buffers, pH 3–12, and additives such as
detergents (1% SDS), denaturing agents (8 M urea or 6 M guanidine hydrochloride).
The following solutions can be used for cleaning: up to 30% acetonitrile, up to 1 M sodium hydroxide,
up to 70% ethanol (Superdex 30 prep grade), up to 24% ethanol (Superdex 75 prep grade and
Superdex 200 prep grade), up to 1 M acetic acid, up to 30% isopropanol or up to 0.1 M HCl
(Superdex 30 prep grade).

Storage
Store unused media 4°C to 30°C in 20% ethanol. Do not freeze.
Columns can be left connected to a chromatography system with a low flow rate (0.01 ml/min)
of buffer passing through the column to prevent bacterial growth or the introduction of air into
the column which destroys the packing.
For long term storage, wash with 4 column volumes of distilled water followed by 4 column
volumes of 20% ethanol. Store at 4°C to 30°C.
Degas the ethanol/water mixture thoroughly and use a low flow rate, checking the back pressure
as the column equilibrates.
Avoid changes in temperature which may cause air bubbles in the packing.

18-1022-18 AK 41
42 18-1022-18 AK
Chapter 3
Superose: broad fractionation range for
laboratory scale
Superose are media with high physical and chemical stability based on highly cross-linked
porous agarose particles. Typical fractionation ranges for Superose are shown in Figure 3.1. The
mechanical rigidity of Superose allows even relatively viscous eluents, such as 8 M urea, to be
run at practical flow rates. Under normal chromatography conditions nonspecific interactions
between proteins and Superose are negligible when using buffers with ionic strengths in the
range 0.15 M to 1.5 M.

Mr 10 2 10 3 10 4 10 5 10 6 10 7 10 8

Superose 6

Superose 12
Superose 6 prep grade

Superose 12 prep grade

Fig 3.1. Fractionation ranges of Superose.

Some hydrophobic interactions have been noted, particularly for compounds such as smaller
hydrophobic and/or aromatic peptides, membrane proteins and/or lipoproteins which may elute
later than predicted. However, in some applications, these interactions can be an advantage for
increasing the resolution of the separation.
Typical selectivity curves for Superose are shown in Figure 3.2.
K av
1.0

Superose 6 prep grade

0.8

0.6

Superose 12
0.4

Superose 6

0.2

Superose 12 prep grade

0
3 4 5 6
10 10 10 10 Mr
Fig 3.2. Selectivity curves of Superose for globular proteins.

18-1022-18 AK 43
Figure 3.3 gives a comparison of the different selectivities of Superose 6 and Superose 12
prepacked columns.

Column: Superose 6 10/300 GL

Sample: Proteins from Gel Filtration Calibration Kits LMW and HMW (GE Healthcare)
T = thyroglobulin (Mr 669 000)
F = ferritin (Mr 440 000)
Ald = aldolase (Mr 158 000)
O = ovalbumin (Mr 43 000)
CA = carbonic anhydrase (Mr 29 000)
R = RNase A (Mr 13 700)
Apr = aprotinin (Mr 6 500)
Sample load: 100 μl
Eluent: 0.050 M Phosphate, 0.15 M NaCl, ph 7.2
Flow rate: 0.5 ml/min
System: ÄKTAexplorer™ 10
Detection: 280 nm
A) mAU Apr 1.00
Kav

mAU Kav
700 CA Apr 1.00
0.90
R
700 CA 0.90
0.80
Aprotinin
600 O R RNase A
0.80
0.70
600 Ald O Aprotinin Carb. anh
RNase A
500 Ald
0.70
0.60 Ovalb
Carb. anh
500 0.60
0.50 Ovalb Aldolase
400 Ferritin
Aldolase
400 F 0.50
0.40
T Ferritin
300 F 0.40
0.30
Thyrogl
T
300 0.30
Thyrogl
0.20
200
0.20
200 0.10
100 0.10
0.00
100 10
3
10
5
10
7

0 0.00 Mr logarithmic scale

3 5 7
10 10 10
0 0 5 10 15 20 25 ml Mr logarithmic scale

0 5 10 15 20 25 ml

Column: Superose 12 10/300 GL

Sample: Proteins from Gel Filtration Calibration Kits LMW and HMW (GE Healthcare)
F = ferritin (Mr 440 000)
Ald = aldolase (Mr 158 000)
O = ovalbumin (Mr 43 000)
CA = carbonic anhydrase (Mr 29 000)
R = RNase A (Mr 13 700)
Apr = aprotinin (Mr 6 500)
Sample load: 100 μl
Eluent: 0.050 M Phosphate, 0.15 M NaCl, ph 7.2
Flow rate: 0.5 ml/min
System: ÄKTAexplorer 10
Detection: 280 nm
mAU Kav
B) mAU
R 1.00
Kav
R 1.00
700 O CA Apr 0.90

700 O CA Apr 0.90

0.80
600 Ald 0.80
0.70
600 Ald
0.70
500 0.60
Aprotinin
500 0.60 RNase A
0.50 Aprotinin
400 RNase A anh
Carb.
0.50
400 0.40
Ovalb
Carb. anh
300 0.40
Ovalb
F 0.30 Aldolase
300
F 0.30 Aldolase
200 0.20 Ferritin
200 0.20
0.10
Ferritin

100 0.10
100 0.00
3 5 7
0.00 10 10 10
0 3 Mr logarithmic
5 scale 7
10 10 10
0 0 5 10 15 20 25 ml Mr logarithmic scale
0 5 10 15 20 25 ml

Fig 3.3. (A) Standard proteins separated on Superose 6 10/300 GL, Mr range: 5 000 to 5 000 000 and calibration curve.
(B) Standard protein separated on Superose 12 10/300 GL, Mr range 1 000 to 300 000 and calibration curve.

44 18-1022-18 AK
Separation options
Superose is produced in different particle sizes (10 μm, 13 μm and 30 μm) and with two different
selectivities, Superose 6 and Superose 12, and is available as prepacked columns and bulk packs
(Table 3.1).

Use 11 μm or 13 μm particles for analytical separations and 30 μm particles for

preparative separations
Table 3.1. Separation options with Superose media

Product Fractionation Sample loading Maximum operating . Recommended

range, Mr capacity* back pressure operation flow†
(globular
proteins)
Superose 6 10/300 GL 5 × 103–5 × 106 25–250 µl 1.5 MPa, 15 bar, 217 psi 0.1-0.5 ml/min
Superose 6 PC 3.2/30 5 × 10 –5 × 10
3 6
<50 µl 1.2 MPa, 12 bar, 175 psi 0.01-0.1 ml/min
Superose 6 prep grade 5 × 103–5 × 106 0.5–4% of total Column-dependent 10-20 cm/h
(Bulk media) column volume
Superose 12 10/300 GL 1 × 103–3 × 106 25-250 µl 3.0 MPa, 30 bar, 435 psi 0.5-1 ml/min
Superose 12 PC 3.2/30 1 × 10 –3 × 10
3 6
<50 µl 2.4 MPa, 24 bar, 350 psi 0.04 ml/min
Superose 12 prep grade 1 × 103–3 × 106 0.5–4% of total Column-dependent 10-20 cm/h
(Bulk media) column volume
* For maximum resolution, apply as small sample volume as possible. Note that sample volumes less than 0.5% normally
do not improve resolution.
†
See Appendix 5 to convert linear flow (cm/hour) to volumetric flow rates (ml/min) and vice versa.

Separation examples
Figures 3.4 and 3.5 show examples of separations performed on Superose media.

Column: Superose 12 PC 3.2/30

Sample: 0.75 µl human tears
Buffer: 0.02 M sodium phosphate, 0.5 M NaCl, pH 5.3
Flow: 40 µl/min

A 280 nm

0.025

0.020

0.015

0.010

0.005

0.000
0 20 40 60 Time (min)
Fig 3.4. Microfractionation of 0.75 μl of human tears.

18-1022-18 AK 45
Column: 2× Superose 6 HR 10/30 in series Column: Superose 6 HR 10/30
Sample: 10 µg Hae III cleaved pBR 322 Sample: fX-174 RF DNA-Hae III digest, 10 µg
Buffer: 0.05 M Tris-HCl, 1 mM EDTA, pH 8.0 Buffer: 0.05 M Tris-HCl, pH 8.0
Flow: 0.1 ml/min Flow: 0.4 ml/min

A) B)
A 254 nm A 254 nm
0.05 434 0.05 1353
458 1078
504 872
540 603
587

267
192
184
234
213

123 310
124 281
271

234
104
89 80
194
64 57
51

21 18
11 8
11 7 72

2 3 4 5 Time (h) 30 60 Time (min)

Fig 3.5. Separation of DNA fragments (Superose 6 HR 10/30 is replaced with Superose 6 10/300 GL). Peak figures correspond
to number of base pairs.

Performing a separation
Buffer: 0.05 M sodium phosphate, 0.15 M NaCl pH 7, or select the buffer in which the sample
should be stored or solubilized for the next step.

Use 0.15 M NaCl or a buffer with equivalent ionic strength to avoid pH dependent nonionic
interactions with the matrix. At very low ionic strength, the presence of a small number of negatively
charged groups may cause retardation of basic proteins and exclusion of acidic proteins.
The sample should be fully dissolved. Centrifuge or filter to remove particulate material
(see Appendix 3). Always use degassed buffers and maintain a constant temperature
during the run to avoid introducing air into the column.
Set an appropriate pressure limit on the chromatography system to avoid damage to the
column packing.

First time use or after long-term storage

Superose 6
Superose 6 swells slightly when transferred from ethanol to water. To avoid local high back
pressure, take the following steps during initial equilibration:
a) 12 ml distilled water at 0.2 ml/min at room temperature
b) 38 ml distilled water at 0.5 ml/min at room temperature
c) 50 ml eluent at 0.5 ml/min at room temperature
Ensure that the back pressure over the column does not exceed 1.2 MPa during equilibration.
Superose 12
a) 50 ml distilled water at 0.2 to 0.5 ml/min at room temperature
b) 50 ml eluent at 0.5 ml/min at room temperature
Ensure that the back pressure over the column does not exceed 2 MPa during equilibration.

46 18-1022-18 AK
Note: Precipitation of the sample may block the filter and cause compression of the gel.
Therefore, the advise is to never set the pressure limit control to more than 0.2 MPa above the
actual operating pressure.
Column performance should be checked at regular intervals by determining the theoretical plate
number per meter and peak symmetry. Prepacked columns are supplied with recommended
values. See page 90 for how to check column efficiency.
See page 23 for advice on optimizing the separation.
Exposure to temperatures outside the range 4°C to 40°C will destroy the efficiency of a

packed bed and the column will need to be repacked.

Cleaning
1. Wash with 1 column volume 0.5 M NaOH at 40 cm/h (0.5 ml/min for 10/300 GL columns).
2. Rinse immediately with 1 column volume of distilled water or buffer at 40 cm/h.
3. Continue to re-equilibrate with 2 column volumes of buffer or until the baseline and the
eluent pH are stable.
For extreme cases of contamination, check the instructions supplied with the product.

In special cases, 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 done with extreme care to avoid serious loss of
resolution. Note that Precision Columns should not be opened.
Superose prep grade may be autoclaved repeatedly at 121°C, pH 7 for 30 minutes without
significantly affecting its chromatography properties. The medium must be removed from the
column as autoclaving can damage column components (Note that Precision Columns cannot
be repacked).

Media characteristics
Matrix: Cross-linked agarose.

Superose prep grade shows less tendency towards hydrophobic interactions than Superose
in prepacked columns. Superose 6 shows less tendency towards hydrophobic interactions
than Superose 12.

Table 3.2 Superose media characteristics

Product Efficiency* pH stability† Particle size Mean particle size

Superose 6 ≥ 30 000 m -1
Long term: 3–12 11–15 µm 13 µm
Short term: 1–14
Superose 6 prep grade ‡
Long term: 3–12 20–40 µm 30 µm
Short term: 1–14
Superose 12 ≥ 40 000 m-1 Long term: 3–12 8–12 µm 10 µm
Short term: 1–14
Superose 12 prep grade ‡
Long term: 3–12 20–40 µm 30 µm
Short term: 1–14
* Theoretical plates per meter (prepacked columns only).
†
Long term pH stability refers to the pH interval where the medium is stable over a long period of time without adverse side
effects on its chromatography performance. Short term pH stability refers to the pH interval for regeneration, cleaning-in-
place and sanitization procedures. All ranges are estimates based on the experience and knowledge within GE Healthcare.
‡
A minimum column efficiency of 10 000 m-1 should be expected for a well-packed column.

18-1022-18 AK 47
Chemical stability
Superose is stable in all commonly used aqueous buffers and additives such as detergents
(1% SDS), denaturing agents (8 M urea or 6 M guanidine hydrochloride) and 30% acetonitrile.

Storage
Store unused media 4°C to 30°C in 20% ethanol. Do not freeze.
Columns can be left connected to a chromatography system with a low flow rate (0.01 ml/min)
of buffer passing through the column to prevent bacterial growth or the introduction of air into
the column which would destroy the packing.
For long term storage, wash with 2 column volumes of distilled water followed by 2 column
volumes of 20% ethanol. Store at 4°C to 30°C.
Degas the ethanol/water mixture thoroughly and use a low flow rate, checking the back
pressure as the column equilibrates.
Avoid changes in temperature which may cause air bubbles in the packing.

48 18-1022-18 AK
Chapter 4
Sephacryl: fast, high recovery separations
at laboratory and industrial scale

Sephacryl High Resolution (HR) media provide a useful alternative to Superdex prep grade for
applications that require a slightly broader fractionation range, as shown in Figure 4.1. High
chemical stability and tolerance of high flow rates make Sephacryl well suited for industrial use.

Mr 10 2 10 3 10 4 10 5 10 6 10 7 10 8

Sephacryl S-100 HR

Sephacryl S-200 HR

Sephacryl S-300 HR

Sephacryl S-400 HR

Sephacryl S-500 HR

Sephacryl S-1000 SF

Fig 4.1. Fractionation ranges for Sephacryl HR.

Fig 4.2. Sephacryl is available as loose media and in prepacked columns.

Figure 4.3 shows comparisons of the different selectivities of Sephacryl HR.

Typical selectivity and pressure-flow relationship curves for Sephacryl are shown in
Figures 4.4 and 4.5.

18-1022-18 AK 49
Column: A) HiPrep 16/60 Sephacryl S-100 HR
B) HiPrep 16/60 Sephacryl S-200 HR
C) HiPrep 16/60 Sephacryl S-300 HR
Sample: 500 µl of a mixture comprising IgG (Mr 160 000), BSA (Mr 67 000),
β-lactoglobulin (Mr 35 000), cytochrome C (Mr 12 400), and cytidine (Mr 240)
Buffer: 0.05 M sodium phosphate, 0.15 M NaCl, pH 7.0
Flow rate: 0.8 ml/min (24 cm/h)
A) HiPrep 16/60 Sephacryl S-100 HR B) HiPrep 16/60 Sephacryl S-200 HR
A) HiPrep 16/60 Sephacryl S-100 HR B) HiPrep
A28016/60
nm
Sephacryl S-200 HR
A280 nm
A280 nm
A280 nm
0.3 0.3
BSA
0.3 0.3
IgG BSA
Cytochrome C BSA
IgG
0.2
Cytochrome C Cytidine
0.2 BSA Cytochrome C
0.2 0.2 IgG Cytochrome C Cytidine
β-lactoglobulin Cytidine IgG β-lactoglobulin Cytidine
0.1 β-lactoglobulin 0.1 β-lactoglobulin
0.1 0.1

20 40 60 80 100 120 Vol. (ml) 20 40 60 80 100 120 Vol. (ml)

20 40 60 80 100 120 Vol. (ml) 20 40 60 80 100 120 Vol. (ml)
C) HiPrep 16/60 Sephacryl S-300 HR
C) HiPrep 16/60
A280 nm
Sephacryl S-300 HR
A280 nm

BSA
0.2 Cytochrome C
BSA Cytidine
0.2 Cytochrome C
IgG β-lactoglobulin Cytidine
0.1 IgG β-lactoglobulin
0.1

20 40 60 80 100 120 Vol. (ml)

20 40 60 80 100 120 Vol. (ml)

Column: D) HiPrep 16/60 Sephacryl S-400 HR

E) HiPrep 16/60 Sephacryl S-500 HR
Sample: D)1.2 ml of a sample containing three dextrans; Mr > 20 × 106,
Mr 270 000, and Mr 12 000
E)1.2 ml of a sample containing three dextrans, Mr > 20 × 106,
Mr 1.8 × 106, and Mr 25 000
Buffer: 0.25 M NaCl
Flow rate: 0.5 ml/min (15 cm/h)

D) HiPrep 16/60 Sephacryl S-400 HR E) HiPrep 16/60 Sephacryl S-500 HR

D) HiPrep 16/60 Sephacryl S-400 HR E) HiPrep
Refractive 16/60 Sephacryl S-500 HR
index
Refractive mV
Refractive
index index
100.0
mV
Refractive mV Mr 25 000
index Mr 2 × 107 100.0
50.0 Mr 12 000 Mr 25 000
mV 7 80.0
Mr 2 × 10
50.0 Mr 12 000 Mr 2 × 107
80.0
60.0
40.0
50.0 Mr 270 000
Mr 2 × 107 Mr 1.8 × 106
30.0 60.0
40.0 Mr 270 000 40.0
Mr 1.8 × 106
20.0
30.0 40.0
20.0
10.0
20.0 Mr 58 (NaCl) Mr 58 (NaCl)
20.0
0.0
10.0 Mr 58 (NaCl) 0.0 Mr 58 (NaCl)
0.0 0 20 40 60 80 100 120 Vol. (ml) 0.0 0 20 40 60 80 100 Vol. (ml)

0 20 40 60 80 100 120 Vol. (ml) 0 20 40 60 80 100 Vol. (ml)

Fig 4.3. Comparison of the selectivity of the five different prepacked HiPrep Sephacryl HR columns.

50 18-1022-18 AK
 Globular proteins* Proteins† Dextran standards*
Kav Kav Kav

0.8 0.8 0.8

S-
50
0H
S-4

R
S-
40

00
S-
0
S-3

HR

40

HR
0.6 0.6 0.6

0H
0H 0

R
S-2
S-1

R
00
00 H

HR
R

0.4 0.4 0.4

0.2 0.2 0.2

104 105 106 104 105 106 104 105 106

Mr logarithmic scale Mr logarithmic scale Mr logarithmic scale

* In 0.05 M phosphate buffer, 0.15 M NaCl, pH 7.0

†
In 6 M guanidine hydrochloride

Fig 4.4. Selectivity curves for Sephacryl HR chromatography media.

A) Column diameter 1.6 cm B) Column diameter 2.6 cm

MPa MPa
0.3 0.3

HR
00
S-3
0.2 00
HR 0.2 0 HR
S-2 0 HR 20
S-30 S-
HR
00
S-1 0 HR
R S-40
00 H
0.1 S-1
R
0.1 S-500
HR
S-500 H

S-400 HR

25 50 75 100 25 50 75 100
Linear flow rate (cm/h) Linear flow rate (cm/h)

Fig 4.5. Pressure drop as a function of flow rate for Sephacryl HR. Bed height approximately 60 cm, distilled water,
temperature 25°C. To calculate the volumetric flow rate, multiply the linear flow by the cross-sectional area of the column
(2 cm2 for XK 16 or 5.3 cm2 for XK 26).

Sephacryl HR is a composite medium prepared by covalently cross-linking allyl dextran with

N,N’-methylene bisacrylamide to form a hydrophilic matrix of high mechanical strength. The
porosity of the medium, determined by the dextran component, has been controlled to yield
five different selectivities. The mechanical rigidity of Sephacryl HR allows even relatively viscous
eluents, such as 8 M urea, to be run at practical flow rates. Under normal chromatography
conditions (A280, 0.05 M phosphate, 0.15 M NaCl, pH 7.0) Sephacryl S-100 HR gave yields of at
least 96% of the following substances: Blue Dextran 2000, ferritin, catalase, aldolase, BSA,
ovalbumin, β-lactoglobulin A+B, chymotrypsinogen A, myoglobin, lysozyme, ribonuclease A, and
cytochrome C. An ionic strength of at least 0.15 M is recommended for best results.

18-1022-18 AK 51
Separation options
Five Sephacryl HR chromatography media are available as prepacked columns and in bulk
packs (Table 4.1).
Table 4.1. Separation options with Sephacryl media

Product Fractionation Fractionation Sample . Maximum . Recommended

range, Mr range, Mr loading operating . operation flow†
(globular (dextrans) capacity* back pressure
proteins)
HiPrep 16/60 1 × 103–1 × 105 ≤ 5 ml 0.15 MPa, 5 bar, 0.5 ml/min
Sephacryl S-100 HR 21 psi
HiPrep 26/60 1 × 103–1 × 105 ≤ 13 ml 0.15 MPa, 5 bar, 1.3 ml/min
Sephacryl S-100 HR 21 psi
Sephacryl S-100 HR 1 × 103–1 × 105 0.5–4% of total Column- 10–35 cm/h
(Bulk media) column volume dependent
HiPrep 16/60 5 × 103–2.5 × 105 1 × 103–8 × 104 ≤ 5 ml 0.15 MPa, 5 bar, 0.5 ml/min
Sephacryl S-200 HR 21 psi
HiPrep 26/60 5 × 103–2.5 × 105 1 × 103–8 × 104 ≤ 13 ml 0.15 MPa, 5 bar, 1.3 ml/min
Sephacryl S-200 HR 21 psi
Sephacryl S-200 HR 5 × 103–2.5 × 105 1 × 103–8 × 104 0.5–4% of total Column- 10–35 cm/h
(Bulk media) column volume dependent
HiPrep 16/60 1 × 104–1.5 × 106 2 × 103–4 × 105 ≤ 5 ml 0.15 MPa, 5 bar, 0.5 ml/min
Sephacryl S-300 HR 21 psi
HiPrep 26/60 1 × 104–1.5 × 106 2 × 103–4 × 105 ≤ 13 ml 0.15 MPa, 5 bar, 1.3 ml/min
Sephacryl S-300 HR 21 psi
Sephacryl S-300 HR 1 × 104–1.5 × 106 2 × 103–4 × 105 0.5–4% of total Column- 10–35 cm/h
(Bulk media) column volume dependent
HiPrep 16/60 2 × 104–8 × 106 1 × 104–2 × 106 ≤ 5 ml 0.15 MPa, 5 bar, 0.5 ml/min
Sephacryl S-400 HR 21 psi
HiPrep 26/60 2 × 104–8 × 106 1 × 104–2 × 106 ≤ 13 ml 0.15 MPa, 5 bar, 1.3 ml/min
Sephacryl S-400 HR 21 psi
Sephacryl S-400 HR 2 × 104–8 × 106 1 × 104–2 × 106 0.5–4% of total Column- 10–35 cm/h
(Bulk media) column volume dependent
HiPrep 16/60 4 × 104–2 × 107 ≤ 5 ml 0.15 MPa, 5 bar, 0.5 ml/min
Sephacryl S-500 HR 21 psi
HiPrep 26/60 4 × 104–2 × 107 ≤ 13 ml 0.15 MPa, 5 bar, 1.3 ml/min
Sephacryl S-500 HR 21 psi
Sephacryl S-500 HR 4 × 104–2 × 107 0.5–4% of total Column- 10–35 cm/h
(Bulk media) column volume dependent
Sephacryl S-1000 SF 0.5–4% of total not determined 2–30 cm/h
(Superfine) (Bulk media) column volume
* For maximum resolution, apply as small sample volume as possible. Note that sample volumes less than 0.5% normally do
not improve resolution.
†
See Appendix 5 to convert linear flow (cm/hour) to volumetric flow rates (ml/min) and vice versa.

52 18-1022-18 AK
Separation examples
Figures 4.6 to 4.8 illustrate examples of separations performed on Sephacryl media.

Column: HiPrep 26/60 Sephacryl S-100 HR

Sample: 1 ml of a mixture containing bovine insulin chain A (Mr 2532) and chain B (Mr 3496),
0.5 mg/ml of each
Buffer: 0.05 M sodium phosphate, 0.15 M NaCl, pH 7.0
A 280rate:
Flow nm 2.0 ml/min (22 cm/h)

0.03

Chain A
0.02

Chain B

0.01

0.00
0 40 80 120 160 200 240 Vol. (ml)

Fig 4.6. Separation of insulin chains on HiPrep 26/60 Sephacryl S-100 HR.

Column: BP 113/120 containing Sephacryl S-200 HR, bed height 100 cm

Sample: IgG fraction from previous ion exchange step (sample volume equivalent to 1% of V t.
(Vt =column volume)
Buffer: 0.05 M Tris-HCl, 0.15 NaCl, pH 7.5
Flow rate: 7.5 cm/h
A 280 nm
1 2
Immunoglobulin

Elution volume

Fig 4.7. Purification of monoclonal antibodies on Sephacryl S-200 HR. Inset shows analysis by gradient the immunoglobulin
pool. Lane 1, native sample; lane 2, sample reduced with 2-mercaptoethanol.

18-1022-18 AK 53
Medium: Sephacryl S-400 HR
Column: K 26/70, packed bed 2.6 × 61 cm
Sample: Integral membrane proteins prepared from human erythrocytes solubilized in
0.1 M phosphate, 100 mM SDS, 1 mM EDTA, 1 mM DTE, pH 7.4
Sample volume: 2 ml (2 mg/ml)
Buffer: 0.1 M phosphate, 50 mM SDS, 1 mM EDTA, 1 mM DTE, pH 7.4
Flow rate: 1 ml/min (11 cm/h)
A 280 nm
1. Large phospoholipid vesicles (LPLV)
2. Small phospholipid vesicles (SPLV)

0.6 1
2
0.4

0.2

50 100 150 200 250

Elution volume (ml)

Fig 4.8. Gel filtration on Sephacryl S-400 HR quickly separates phospholipid vesicles (liposomes) into large (LPLV) and small
(SPLV) phospholipid vesicles. Data provided by E. Greijer and P. Lundahl, Dept. of Biochemistry, Biomedical Centre, University
of Uppsala, Sweden.

Performing a separation
Buffer: 0.05 M sodium phosphate, 0.15 M NaCl, pH 7.2 or select the buffer in which the
sample should be stored or solubilized for the next step.
Use 0.15 M NaCl, or a buffer with equivalent ionic strength, to avoid pH dependent nonionic
interactions with the matrix. At very low ionic strength, the presence of a small number of negatively
charged groups may cause retardation of basic proteins and exclusion of acidic proteins.

The sample should be fully dissolved. Centrifuge or filter to remove particulate material
(see Appendix 3). Always use degassed buffers and maintain a constant temperature
during the run to avoid introducing air into the column.
Set an appropriate pressure limit on the chromatography system to avoid damage to the
column packing.

First time use or after long-term storage

1. Equilibrate the column with at least 0.5 column volume of distilled water at 15 cm/h
(0.5 ml/min for 16/60 column or 1.3 ml/min for 26/60).
2. Equilibrate with 2 column volumes of buffer at 30 cm/h (1.0 ml/min for 16/60 column or
2.6 ml/min for 26/60).
3. Reduce flow to 15 cm/h and, for best resolution, apply a sample volume equivalent to
1% of the column volume (1.2 ml for 16/60 column or 3.2 ml for 26/60). Sample volumes
between 0.5% and 4% can be applied.
4. Elute with 1 column volume of buffer.
5. Before applying a new sample re-equilibrate column with 1 column volume of buffer at
30 cm/h until the baseline monitored at A280 is stable.

Column performance should be checked at regular intervals by determining the

theoretical plate number per meter and peak symmetry. Prepacked columns are supplied
with recommended values. See page 90 on how to check column efficiency.

54 18-1022-18 AK
See page 23 for advice on optimizing the separation.
Exposure to temperatures outside the range 4°C to 40°C will destroy the efficiency of a

packed bed and the column will need to be repacked.

Cleaning
1. Wash with 0.5 column volume of 0.2 M NaOH at a flow of 15 cm/h (0.5 ml/min for
column 16/60 or 1.3 ml/min for 26/60) to remove most nonspecifically adsorbed proteins.
2. Re-equilibrate immediately with 2 column volumes of buffer or until the baseline
monitored at A280 and the pH of the eluent are stable.
Further equilibration may be necessary if the buffer contains detergent.

Routine cleaning after every 10–20 separations is recommended, but the frequency of
cleaning will also depend on the nature of the samples being applied.

If required Sephacryl HR may be autoclaved repeatedly at 121°C, pH 7, for 30 minutes without

significantly affecting its chromatography properties. The medium must be removed from the
column as autoclaving can damage column components (Note that HiPrep columns cannot
be repacked).

To remove severe contamination

Reverse the flow and wash at a flow rate of 10 cm/h (0.3 ml/min for column 16/60 or
0.8 ml/min for 26/60) at room temperature using the following solutions:
1. Wash with 0.25 column volumes of 0.5 M NaOH (to remove hydrophobic proteins or
lipoproteins) followed by 4 column volumes of distilled water.
2. Wash with 0.5 column volume of 30% isopropanol (to remove lipids and very
hydrophobic proteins), followed by 2 column volumes of distilled water.
For extreme cases of contamination, check the instructions supplied with the product.

Reversing flow through a column packed with Sephacryl media should only be

considered under cases of severe contamination. There is a risk that reversing the
flow may cause channeling through the packed bed leading to poor resolution, loss of
efficiency and the need to repack the column. Professionally packed columns are less
likely to be affected, but extreme care must be taken.

Media characteristics
Matrix: Cross-linked copolymer of allyl dextran and N,N’-methylene bisacrylamide.

18-1022-18 AK 55
Table 4.2 Sephacryl media characteristics

Product Efficiency: theoretical plates per pH stability* Particle . Mean

meter (prepacked columns only) size particle size
Sephacryl S-100 HR ≥ 5 000 m-1 Long term: 3–11 25–75 µm 47 µm
Short term: 2–13
Sephacryl S-200 HR ≥ 5 000 m-1 Long term: 3–11 25–75 µm 47 µm
Short term: 2–13
Sephacryl S-300 HR ≥ 5 000 m-1 Long term: 3–11 25–75 µm 47 µm
Short term: 2–13
Sephacryl S-400 HR 9 000 m-1 Long term: 3–11 25–75 µm 47 µm
Short term: 2–13
Sephacryl S-500 HR 9 000 m-1 Long term: 3–11 25–75 µm 47 µm
Short term: 2–13
Sephacryl S-1000 SF †
Long term: 3–11 40–105 µm 65 µm
(Superfine) Short term: 2–13
* Long term pH stability refers to the pH interval where the medium is stable over a long period of time without adverse side
effects on its chromatography performance. Short term pH stability refers to the pH interval for regeneration, cleaning-in-
place and sanitization procedures. All ranges are estimates based on the experience and knowledge within GE Healthcare.
Not determined.
†

Chemical stability
Sephacryl HR is stable in all commonly used aqueous buffers and additives such as detergents
(1% SDS), denaturing agents (8 M urea or 6 M guanidine hydrochloride). The medium is also
stable in 30% acetonitrile, 0.5 M sodium hydroxide, up to 24% ethanol, up to 1 M acetic acid and
up to 30% isopropanol.

Storage
Store unused media 4°C to 30°C in 20% ethanol. Do not freeze.
Columns can be left connected to a chromatography system with a low flow rate (0.01 ml/min)
of buffer passing through the column to prevent bacterial growth or the introduction of air into
the column which would destroy the packing.
For long-term storage, wash with 4 column volumes of distilled water followed by 4 column
volumes of 20% ethanol. Store at 4°C to 30°C.
Degas the ethanol/water mixture thoroughly and use a low flow rate, checking the back
pressure as the column equilibrates.
Avoid changes in temperature which may cause air bubbles in the packing.

56 18-1022-18 AK
Chapter 5
Sephadex: desalting, buffer exchange
and sample clean up

Gel filtration based on Sephadex enables group separation of biomolecules that are above the
exclusion limit of the medium, from contaminants such as salts, dyes, and radioactive labels.
Sephadex is prepared by cross-linking dextran with epichlorohydrin.
The different types of Sephadex vary in their degree of cross-linking and hence in their degree of
swelling and selectivity for specific molecular sizes (see Media characteristics on page 70).
Sephadex G-10 is well suited for the separation of biomolecules such as peptides (Mr >700) from
smaller molecules (Mr <100).
Sephadex G-50 is suitable for the separation of molecules Mr >30 000 from molecules Mr <1 500
such as labeled protein or DNA from unconjugated dyes. The medium is often used to remove
small nucleotides from longer chain nucleic acids.
Sephadex G-25 is recommended for the majority of group separations involving globular
proteins. These media are excellent for removing salt and other small contaminants away from
molecules that are greater than Mr 5000. Using different particle sizes enables columns to be
packed according to application requirements, see Table 5.1. The particle size determines the
flow rates and the maximum sample volumes that can be applied. For example, smaller particles
give higher column efficiency (narrow, symmetrical peaks), but may need to be run more slowly
as they create higher operating pressures.

Table 5.1. Sephadex G-25 media

Sephadex G-25 Application

Superfine For highest column efficiency (highest resolution), but operating pressures
increase
Fine For laboratory-scale separations
Coarse and Medium Use when a high flowrate at a low operating pressure is essential, e.g., in large-
scale applications
Coarse For batch procedures

Fig 5.1. Prepacked Sephadex G-25 columns: (A) HiPrep 26/10 Desalting, (B) HiTrap Desalting 5 ml.

18-1022-18 AK 57
 Use desalting/buffer exchange when needed, before purification, between purification
steps, and/or after purification. These are very fast methods compared to dialysis, but
remember that each extra step can reduce yield and that desalting often dilutes the
sample (centrifugation protocols do not dilute samples).
Use Sephadex G-25 products to remove salts and other low molecular weight compounds
from proteins with Mr > 5000 and Sephadex G-10 products for proteins with Mr > 700.
Desalting provides several advantages over dialysis. Dialysis is generally a slow technique that
requires large volumes of buffer and carries the risk that material and target protein activity will
be lost during handling. When desalting, sample volumes of up to 30% of the total volume of the
desalting column can be processed. The high speed and capacity of the separation allows even
relatively large sample volumes to be processed rapidly and efficiently in the laboratory, as illustrated
in Figure 5.2. Sample concentration does not influence the separation as long as the concentration
of proteins does not exceed approximately 70 mg/ml when using normal aqueous buffers, and
provided that the target protein is stable and soluble at the concentration used. Use 100 mM
ammonium acetate or 100 mM ammonium hydrogen carbonate if volatile buffers are required.
When desalting is the first chromatography step, the sample should first be clarified;
centrifugation and/or filtration is recommended.
A) B)
RS RS

3.5 3.5

3 HiPrep 26/10 Desalting 3 HiPrep 26/10 Desalting

Flow: 9 ml/min (100 cm/h) Sample volume: 15 ml
2.5 2.5

2 2

1.5 1.5

1 1

0.5 0.5

0 0
0 2 4 6 8 10 12 14 16 18 0 5 10 15 20 25 30 35
Sample volume (ml) Flow (ml/min)

Fig 5.2. (A) Influence of sample volume on resolution. (B) Influence of flow rate on resolution.

Desalting columns are used not only to remove low molecular weight contaminants such as salt,
but also for buffer exchange before and after different chromatography techniques and for the
rapid removal of reagents to terminate a reaction. Examples of group separations include:
• removal of phenol red from culture fluids prior to anion exchange chromatography or nucleic
acid preparations
• removal of unincorporated nucleotides during DNA sequencing
• removal of free low molecular weight labels
• termination of reactions between macromolecules and low molecular weight reactants
• removal of products, cofactors or inhibitors from enzymes
• removal of unreacted radiolabels such as [α-32P] ATP from nucleic acid labeling reactions

Separation options
For group separations the medium should be selected so that the high molecular weight
molecules are eluted at the void volume with minimum peak broadening or dilution and
minimum time on the column. The lowest molecular weight substances should appear by the
time one column volume of buffer has passed through the column.
58 18-1022-18 AK
Sephadex media are available in prepacked columns (also spin columns), microplates, and as
bulk medium (Table 5.2).

Table 5.2. Group separation options with Sephadex media

Columns and . Chromatography Loaded Eluted Dilution Operation

96-well plates medium volume (ml) volume (ml) factor
PD SpinTrap™ G-25 Sephadex G-25 0.07–0.13 0.07–0.13* No dilution Centrifuge
Medium
PD MultiTrap™ G-25 Sephadex G-25 0.07–0.13 0.07–0.13* No dilution Centrifuge
Medium
PD MiniTrap™ G-25 Sephadex G-25 0.2–0.5 0.1–0.5 No dilution Centrifuge
Medium
0.1-0.5 1.0 2-10 Gravity flow
PD MidiTrap™ G-25 Sephadex G-25 0.75-1.0 0.5–1.0 No dilution Centrifuge
Medium
0.5-1.0 1.5 1.5-3 Gravity flow
PD-10 Desalting columns Sephadex G-25 1.75-2.5 1.0–2.5 No dilution Centrifuge
Medium
1.0-2.5 3.5 1.5-3.5 Gravity flow
PD MiniTrap G-10 Sephadex G-10 0.1-0.3 0.5 1.7-5 Gravity flow
Medium
PD MidiTrap G-10 Sephadex G-10 0.4-1.0 1.2 1.2-3.0 Gravity flow
Medium
HiTrap Desalting Sephadex G-25 0.25 1.0 4 (approx) Syringe/pump/system
Superfine
0.5 1.5 3 (approx) Syringe/pump/system
1.0 2.0 2 (approx) Syringe/pump/system
1.5 (max.) 2.0 1.3 (approx) Syringe/pump/system
2× HiTrap Desalting Sephadex G-25 3.0 (max.) 4.0–5.0 1.3–1.7 Syringe/pump/system
Superfine
3× HiTrap Desalting Sephadex G-25 4.5 (max.) 6.0–7.0 1.3–1.7 Syringe/pump/system
Superfine
HiPrep 26/10 Sephadex G-25 10 10–15 1.0–1.5 Pump/system
Fine
15 (max.) 15–20 1.0–1.3 Pump/system
2× HiPrep 26/10 Sephadex G-25 30 (max.) 30–40 1.0–1.3 Pump/system
Fine
3× HiPrep 26/10 Sephadex G-25 45 (max.) 45–55 1.0–1.2 Pump/system
Fine
4× HiPrep 26/10 Sephadex G-25 60 (max.) 60–70 1.0–1.2 Pump/system
Fine

Contains Sephadex G-25 Medium

Contains Sephadex G-10 Medium
Contains Sephadex G-25 Superfine
Contains Sephadex G-25 Fine
* Applied volume = eluted volume; For sample volumes less than 100 μl it is recommended to apply a stacker volume of 30 μl
equilibration buffer after the sample has fully absorbed.

For convenience and reliable performance, use prepacked Sephadex columns such as
HiTrap Desalting 5 ml and HiPrep 26/10 Desalting.
Always use disposable columns if there is a risk of biological or radioactive
contamination or when any possibility of carryover between samples is unacceptable.
The type of equipment available and the sample volume to be processed also govern the
choice of prepacked column, as shown in Figure 5.3.

18-1022-18 AK 59
 Is the Mr of your target Will you use an
YES YES
protein >5000? automated
purification
system such as
ÄKTAdesign?

Syringe or
peristaltic pump

Gravity flow

NO NO

Centrifugation

1
7.5 ml; up to five columns in series
2
60 ml; up to four columns in series
3
For volumes outside those specified, dilute
sample to nearest volume range

Contains Sephadex G-25 Medium

Contains Sephadex G-25 Fine
Gravity flow
Contains Sephadex G-25 Superfine
(Mr > 700)
Contains Sephadex G-10 Medium

Fig 5.3. Selecting prepacked columns for desalting and buffer exchange.

60 18-1022-18 AK
 0.1–1.5 ml HiTrap Desalting
(7.5 ml)1
What is your
sample volume?
2.5–15 ml HiPrep 26/10 Desalting
(60 ml)2

What is your 0.1–1.5 ml HiTrap Desalting

sample volume? (7.5 ml)1

0.1–0.5 ml PD MiniTrap G-25

What is your 0.5–1.0 ml PD MidiTrap G-25

sample volume?

1.0–2.5 ml PD-10 Desalting

70–130 µl3 PD SpinTrap G-25

0.2–0.5 ml3 PD MiniTrap G-25 +

MiniSpin Adapter
What is your
sample volume?
0.75–1.0 ml3 PD MidiTrap G-25 +
MidiSpin Adapter

1.75–2.5 ml3 PD-10 Desalting

+ PD-10 Spin Adapter

70–130 µl PD MultiTrap G-25

(96-well plate)

100–300 µl3 PD MiniTrap G-10

What is your
sample volume?
400 µl–1.0 ml3 PD MidiTrap G-10

18-1022-18 AK 61
Separation examples
Figures 5.4 and 5.5 show examples of separations performed on Sephadex media.

Sample: (Histidine)6-protein eluted from HiTrap Chelating HP with sodium phosphate 20 mM,
sodium chloride 0.5 M, imidazole 0.5 M, pH 7.4
Column: HiTrap Desalting 5 ml
Buffer: Sodium phosphate 20 mM, sodium chloride 0.15 M, pH 7.0

A 280 nm
UV 280 nm
Conductivity
0.15
(Histidine) 6 protein

0.10

Salt

0.05

Inject
Vo Vt

0 1 2 min

Fig 5.4. Desalting a (Histidine)6 fusion protein using HiTrap Desalting 5 ml on ÄKTAprime™. The UV (protein) and conductivity
(salt) traces enable pooling of the desalted fractions and facilitate optimization of the separation.

Column: HiPrep 26/10 Desalting

Sample: 2 mg/ml BSA, 0.07 mg/ml N-Hydroxysuccinimide (NHS) in 50 mM sodium phosphate,
0.15 M NaCl, pH 7.0. Filtered through a 0.45 µm filter
Sample volume: 13 ml
Buffer: 50 mM sodium phosphate, 0.15 M NaCl, pH 7.0
Flow rate: 31 ml/min (350 cm/h)
A 280 nm NHS

BSA

0.0 1.0 2.0 Time (min)

Fig 5.5. Reproducible removal of N-hydroxysuccinimide from bovine serum albumin.

62 18-1022-18 AK
Performing a separation
Desalting and buffer exchange can take less than 5 minutes per sample with greater than 95%
recovery for most proteins.
To prevent possible ionic interactions the presence of a low salt concentration (25 mM NaCl)
is recommended during desalting and in the final sample buffer. Volatile buffers such as
100 mM ammonium acetate or 100 mM ammonium hydrogen carbonate can be used if
it is necessary to avoid the presence of NaCl.
The sample should be fully dissolved. Centrifuge or filter to remove particulate material
(see Appendix 3). Always use degassed buffers to avoid introducing air into the column.
Sample concentration up to 70 mg/ml protein should not influence the separation when
using normal aqueous buffers.
If possible use a chromatography system with a UV and a conductivity monitor to facilitate
optimization of the sample loading. The elution of the protein peak at A280 and the appearance
of the salt peak can be followed exactly and different separations can be easily compared, as
shown in Figure 5.6.
If conductivity cannot be monitored and recovery of completely desalted sample is the major
requirement, apply sample volumes of between 15 and 20% of the total column volume.
A 280 nm Conductivity (mS/cm)

0.25 A 280 Conductivity

0.20
10.0

0.15

0.10
5.0
0.05

0.00
0.0 1.0 2.0 Ti me (min)

Fig 5.6. Buffer exchange of mouse plasma on HiPrep 26/10 Desalting.

General considerations
Small-scale desalting of samples
For sample volumes ranging from 0.2 to 2.5 ml, it is possible to run multiple samples in parallel
with PD-10 Desalting, PD MidiTrap G-25, and PD MiniTrap G-25 columns. Two different protocols
are available for these columns: one for manual use on the laboratory bench and one for use
together with a standard centrifuge in combination with a Spin Adapter. For smaller proteins
(Mr > 700), PD MiniTrap G-10 and PD MidiTrap G-10 columns may be used.
For smaller sample volumes in the range of 70 to 130 μl, multiple samples can be run on
PD SpinTrap G-25 spin columns together with a microcentrifuge or PD MultiTrap G-25 96-well
plate using centrifugation for extraction. Although possible to perform, using PD MultiTrap G-25
with vacuum is not recommended due to reduced reproducibility compared with operation using
centrifugation.

Desalting larger sample volumes using HiTrap and HiPrep columns

Connect up to three HiTrap Desalting columns in series to increase the sample volume capacity.
For example, two columns allow a sample volume of 3 ml, and three columns allow a sample
volume of 4.5 ml (Table 5.2).
18-1022-18 AK 63
Connect up to four HiPrep 26/10 Desalting columns in series to increase the sample volume
capacity. For example, two columns allow a sample volume of 30 ml, and four columns allow a
sample volume of 60 ml. Even with four columns in series, the sample can be processed in 20 to
30 min without back pressure problems.

Buffer preparation
For substances carrying charged groups, an eluent containing a buffer salt is recommended.
A salt concentration of at least 150 mM is recommended to prevent possible ionic interactions
with the chromatography medium. Sodium chloride is often used for this purpose. Often a buffer
with 25 to 50 mM concentration of the buffering substance is sufficient. At salt concentrations
above 1 M, hydrophobic substances may be retarded or may bind to the chromatography medium.
At even higher salt concentrations, > 1.5 M ammonium sulfate, the column packing shrinks.

Sample preparation
Sample concentration does not influence the separation as long as the viscosity does not differ
by more than a factor of 1.5 from that of the buffer used. This corresponds to a maximum
concentration of 70 mg/ml for proteins, when normal, aqueous buffers are used. The sample
should be fully solubilized. Centrifuge or filter (0.45 μm filter) immediately before loading to
remove particulate material if necessary.

Buffer exchange
Protein solubility often depends on pH and/or ionic strength (salt concentration), and the
exchange of buffer may therefore result in precipitation of the protein. Also, protein activity can
be lost if the change of pH takes it outside of the range where the protein is active. Samples
that have been obtained after purification will usually be free from particles, unless the purified
protein or a contaminant has been aggregated.
The protocols in the following sections describe desalting and buffer exchange using different
formats of prepacked columns.

HiTrap Desalting columns

Fig 5.7. HiTrap Desalting column allows easy and efficient group separations with a syringe, pump, or chromatography system.

HiTrap Desalting is a 5 ml column (Fig 5.7) packed with the gel filtration medium Sephadex G-25
Superfine, which is based on cross-linked dextran beads. The fractionation range for globular
proteins is between Mr 1000 and 5000, with an exclusion limit of approximately Mr 5000. This
ensures group separations of proteins/peptides larger than Mr 5000 from molecules with a
molecular weight less than Mr 1000.

64 18-1022-18 AK
HiTrap Desalting can be used with aqueous solutions in the pH range 2 to 13. The prepacked
medium is stable in all commonly used buffers, solutions of urea (8 M), guanidine hydrochloride (6 M),
and all nonionic and ionic detergents. Lower alcohols (methanol, ethanol, propanol) can be used
in the buffer or the sample, but we recommend that the concentration be kept below 25% v/v.
Prolonged exposure (hours) to pH below 2 or above 13, or to oxidizing agents, should be avoided.
The recommended range of sample volumes is 0.1 to 1.5 ml when complete removal of low
molecular weight components is desired. The separation is not affected by the flow rate, in
the range of 1 to 10 ml/min. The maximum recommended flow rate is 15 ml/min. Separations
are easily performed with a syringe, pump, or chromatography system. Up to three columns
can be connected in series, allowing larger sample volumes to be handled. To avoid cross-
contamination, use the column only with the same type of sample.

Manual purification with a syringe

A) B) C)

Fig 5.8. Using HiTrap columns with a syringe. (A) Prepare buffers and sample. Remove the column’s top cap and twist
off the end. (B) Equilibrate the column, load the sample and begin collecting fractions. (C) Wash and elute, continuing to
collect fractions.

1. Fill the syringe with binding buffer. Remove the stopper and connect the column to the
syringe (use the connector supplied) “drop to drop” to avoid introducing air into the
column.
2. Remove the snap-off end at the column outlet.
3. Equilibrate the column with 5 column volumes of binding buffer.
4. Apply the pretreated sample using a syringe fitted to the Luer connector on the column.
For best results, use a flow rate of 0.2 to 1 ml/min (1 ml column) and 0.5 to 5 ml/min (5 ml
column) during sample application*.
5. Wash with 5 to 10 column volumes of binding buffer or until no material appears in
the effluent. Maintain a flow rate of 1 to 2 ml/min (1 ml column) and 5 to 10 ml/min (5
ml column) for washing. Optional: collect the flowthrough (in 1 ml fractions for the 1
ml column and 2 ml fractions for the 5 ml column) and reserve until the procedure has
been successfully completed. Retain a sample for analysis by SDS-PAGE to measure the
efficiency of protein binding to the medium.
6. Elute with 5 to 10 column volumes of elution buffer. Maintain a flow rate of 0.2 to 1 ml/min
( ml column) and 0.5 to 5 ml/min (5 ml column) for elution.
7. After elution, regenerate the column by washing it with 3 to 5 column volumes of binding
buffer. The column is now ready for a new purification.
* 1 ml/min corresponds to approximately 30 drops/min when using a syringe with a HiTrap 1 ml column; 5 ml/min
corresponds to approximately 120 drops/min when using a HiTrap 5 ml column

For large sample volumes, a simple peristaltic pump can be used to apply sample and buffers.

18-1022-18 AK 65
Simple desalting with ÄKTAprime plus
ÄKTAprime plus contains pre-programmed templates for individual HiTrap Desalting 5 ml and
HiPrep 26/10 Desalting columns.

Fig 5.9. ÄKTAprime plus.

Prepare at least 500 ml of each buffer.

1. Follow instructions supplied on the ÄKTAprime plus cue card to connect the column
and load the system with binding buffer.
2. Select the Application Template.
3. Start the method.
4. Enter the sample volume and press OK to start.

A) B)

C) D)

Fig 5.10. Typical procedures using ÄKTAprime plus. (A) Prepare the buffers. (B) Connect the column. (C) Prepare the fraction
collector. (D) Load the sample.

66 18-1022-18 AK
Desalting on a gravity-feed PD-10 column
Buffer Preparation

1. Remove top cap and pour off the excess liquid.

2. Cut off the bottom tip.
3. Place column in the Desalting Workmate supplied onto the plastic tray and equilibrate
with 25 ml buffer. Discard the eluent.
4. Add a total sample volume of 2.5 ml. If the sample volume is less than 2.5 ml, add buffer
to reach a final volume of 2.5 ml. Discard the eluent.
5. Add 3.5 ml buffer to elute high molecular weight components and collect the eluent.

Using the standard procedure described above protein yield is typically greater than 95%
with less than 4% salt (low molecular weight) contamination. The dilution factor is 1:4.
Sephadex G-10 can be packed into empty PD-10 columns and run in the same manner
as PD-10 Desalting columns.

Optimization of desalting

1. When possible select a prepacked column that is best suited to the volume of sample
that needs to be desalted (see Separation Options). For the majority of separations the
instructions supplied ensure satisfactory results and very little optimization should be
necessary.
2. Ensure that buffer conditions are optimal for the separation.
3. Select the highest flow rate recommended. Figure 5.11 shows an example of the
influence of flow rate on group separation.
4. Determine the maximum sample volume that can be loaded. Figure 5.12 shows an
example of the influence of sample volume on group separation.

Column: HiTrap Desalting 5 ml

Sample: Bovine serum albumin, 2 mg/ml in 0.5 M NaCl, 0.05 M sodium phosphate, pH 7.0
Buffer: 0.05 M sodium phosphate, 0.15 M NaCl, pH 7.0
Sample volume: 0.8 ml
Flow rate: 1.7, 3.3, 6.7, 10.0, 13.3, 16.7, 20.0 ml/min
A 280 nm Conductivity (mS/cm)
3.3 ml/min 75
0.40
6.7
10.0
0.30 13.3
1.2

0.20 1.0
Relative resolution

0.8

0.10 0.6

0. 4
BSA NaCl
0.2
0.00 0
0.0
0 2 4 6 8 0 10 20
ml ml/min

Fig 5.11. Influence of flow rate on separation using a HiTrap Desalting column.

18-1022-18 AK 67
Column: HiTrap Desalting 5 ml
Sample: Bovine serum albumin, 2 mg/ml in 0.5 M NaCl, 0.05 M sodium phosphate, pH 7.0
Buffer: 0.05 M sodium phosphate, 0.15 M NaCl, pH 7.0
Sample volume: 0.8, 1.3, 1.7, 2.2 ml
Flow rate: 5 ml/min
A 280 nm Volume collected: 1.5 + × ml
Conductivity (mS/cm)
75
0.40

2.2 ml
0.30
1.7 ml
0.8 ml sample
30
1.3 ml

% N aCl contamination
1.3 ml sample
0.20
1.7 ml sample
0.8 ml 2.2 ml sample
20

0.10
10

0.00 0
2.0 2.5 3.0 3.0
0 2 4 6 8 ml Volume collected: 1.5 + × ml

Fig 5.12. Influence of sample volume on separation using a HiTrap Desalting column.

As the sample volume increases (up to a maximum of 30% of the total column volume) the
dilution factor decreases and there may be a slight increase in the amount of salt remaining in
the sample after elution.
Sample volumes up to 30% of the total column volume give a separation with minimal
sample dilution. Larger sample volumes can be applied, but resolution will be reduced.

Scale-up and processing larger sample volumes

Connecting columns in series increases the effective column volume and so increases sample
loading capacity. Table 5.3 shows the sample loading capacities and dilution factors when using
prepacked desalting columns alone or in series, see also Figure 5.13 for HiTrap application examples.

Table 5.3. Selection guide for desalting/buffer exchange columns

Column Loaded . Eluted . Dilution . Operation

volume (ml) volume (ml) factor
HiPrep 26/10 Desalting 10 10–15 1–1.3 pump
15 (max) 15–20 1–1.5 pump
2 × HiPrep 26/10 Desalting 30 (max) 30–40 1–1.3 pump
3 × HiPrep 26/10 Desalting 45 (max) 40–55 1–1.2 pump
4 × HiPrep 26/10 Desalting 60 (max) 60–70 1–1.2 pump
HiTrap Desalting 0.25 1.0 4 syringe/pump
0.5 1.5 3 syringe/pump
1.0 2.0 2 syringe/pump
1.5 (max) 2.0 1.3 syringe/pump
2 × HiTrap Desalting 3.0 4–5 1.3–1.7 syringe/pump
3 × HiTrap Desalting 4.5 (max) 6–7 1.3–1.7 syringe/pump
PD-10 Desalting columns 1.5 3.5 2.3 gravity
2.0 3.5 1.7 gravity
2.5 3.5 1.4 gravity

68 18-1022-18 AK
Increasing sample loading capacity from 1.5 ml up to 7.5 ml
Column: HiTrap Desalting, 1 × 5 ml, 3 × 5 ml, 5 × 5 ml
Sample: 2 mg/ml BSA in 50 mM sodium phosphate, 0.5 M sodium chloride, pH 7.0
Sample volume: 28% × Vt (1.4, 4.3 and 7.1 ml respectively)
Buffer: 50 mM sodium phosphate, 0.15 M sodium chloride, pH 7.0
Flow rate: 5 ml/min

B) HiTrap Desalting 3 × 5 ml in series

A) HiTrap Desalting 1 × 5 ml
A 280 nm Conductivity (mS/cm)
A 280 nm Conductivity (mS/cm)
BSA
BSA NaCl
0.40 NaCl 0.40
50 50

0.30 0.30
40 40
0.20 0.20

30 30
0.10 0.10

0.00 20 0.00 20
0 2 .0 4. 0 6 .0 ml 0 5 .0 10.0 15. 0 20. 0 ml

C) HiTrap Desalting 5 × 5 ml in series

A 280 nm Conductivity (mS/cm)
BSA
NaCl
0.40 50

0.30
40

0.20

30
0.10

0.00 20
0 10.0 20.0 30. 0 ml

Fig 5.13. Scale-up using HiTrap columns connected in series.

Increasing sample loading capacity from 15 ml up to 60 ml

Connect HiPrep 26/10 Desalting columns in series. With 2 columns, a sample volume of 30 ml
can be applied, and 4 columns allow a sample volume of 60 ml (Fig 5.14). Even with four columns
in series, high flow rates can be maintained without causing back pressure difficulties so that up
to 60 ml of sample can be processed in 20–30 minutes.

Fig 5.14. Four HiPrep 26/10 Desalting columns connected in series.

18-1022-18 AK 69
For sample volumes greater than 60 ml
Select a suitable particle size of Sephadex G-25, rehydrate and pack into a short, wide column
to facilitate high flow rates and rapid recovery of desalted materials. See Appendix 1 for details
on column packing. The particle size determines the flow rates and sample volumes that can
be applied, as shown in Figure 5.15.

100 200
maximum flow rate
% of column volume

cm/h flow velocity

(linear flow rate)
maximum sample volume

Superfine Fine Medium Coarse

increasing particle size

Fig 5.15 Sephadex G-25: recommended sample volumes and flow rates vary with particle size.

• Use Superfine grade with a bed height of approximately 15 cm when requiring the highest
efficiencies.
• Use Fine grade with an approximate bed height of 15 cm for laboratory scale separations.
• Use Medium and Coarse grades for preparative processes where a high flow rate at a low
operating pressure is essential. Pack in a column less than 50 cm in bed height. The Coarse
grade is suitable for batch procedures.

Media characteristics
Sephadex is prepared by cross-linking dextran with epichlorohydrin. Variations in the degree
of cross-linking create the different Sephadex media and influence their degree of swelling and
their selectivity for specific molecular sizes.

Table 5.4. Sephadex media characteristics

Product Fractionation pH stability* Bed volume Maximum Particle size in

range, Mr ml/g dry operating 0.15 M sodium
(globular proteins) Sephadex flow chloride
Sephadex G-10 < 7 × 102 Long term: 2–13 2–3 Darcy’s law† 55–165 µm
Short term: 2–13
Sephadex G-25 Coarse 1 × 103–5 × 103 Long term: 2–13 4–6 Darcy’s law† 87–510 µm
Short term: 2–13
Sephadex G-25 Medium 1 × 103–5 × 103 Long term: 2–13 4–6 Darcy’s law† 38–235 µm
Short term: 2–13
Sephadex G-25 Fine 1 × 103–5 × 103 Long term: 2–13 4–6 Darcy’s law† 17–132 µm
Short term: 2–13
Sephadex G-25 Superfine 1 × 103–5 × 103 Long term: 2–13 4–6 Darcy’s law† 15–88 µm
Short term: 2–13
Sephadex G-50 Fine 1 × 103–3 × 104 Long term: 2–10 9–11 Darcy’s law† 34-208 µm
Short term: 2–13
* Long term pH stability refers to the pH interval where the medium is stable over a long period of time without adverse side
effects on its chromatography performance. Short term pH stability refers to the pH interval for regeneration, cleaning-in-
place and sanitization procedures. All ranges are estimated based on the experience and knowledge within GE Healthcare.
†
In practice this means that the pressure/flow considerations that must be made when using other gel filtration media do
not apply to Sephadex. Doubling flow rate will double column pressure. See Appendix 2 for an explanation of Darcy’s law.

70 18-1022-18 AK
Column Packing
See Appendix 1.

Cleaning
PD-10, SpinTrap, MultiTrap, MiniTrap, MidiTrap, and HiTrap Desalting columns are disposable,
but, depending on the type of sample and if cross-contamination is not a concern, they can be
re-used a few times.
For HiPrep 26/10 Desalting columns proceed as follows:

1. Wash the column with 2 column volumes of 0.2 M sodium hydroxide or a solution of a
non ionic detergent (typically 0.1–0.5% Triton X-100 dissolved in distilled water or 0.1 M
acetic acid) at a flow rate of 10 ml/min. Ensure that the pressure drop does not exceed
0.15 MPa (1.5 bar, 22 psi).
2. Wash the column with 5 column volumes of distilled water at a flow rate of 15 ml/min.
3. Before use, re-equilibrate the column with at least 5 column volumes of buffer until the
UV base line and pH are stable.

To remove precipitated proteins and peptides, fill the column with 1 mg pepsin/ml in 0.1 M
acetic acid, 0.5 M NaCl and leave at room temperature overnight or 1 hour at 37ºC. Repeat the
normal cleaning procedure above.

Chemical stability
Sephadex is stable in all commonly used aqueous buffers and additives such as ionic and
non-ionic detergents, denaturing agents (8 M urea or 6 M guanidine hydrochloride). The media
are stable in short chain alcohols such as ethanol, methanol and propanol, but concentrations
above 25% should not normally be used. Note that Sephadex shrinks in alcohol solutions.

Storage
Store unused media 4°C to 30°C in 20% ethanol. Do not freeze.
Wash used media with 2 column volumes of distilled water followed by 2 column volumes of
20% ethanol. Store at 4°C to 30°C.
Alternatively, wash with 2 column volumes of distilled water followed by 2 column volumes
0.01 M NaOH. Sodium hydroxide solution is bacteriostatic, easily disposed of and does not
shrink the medium.
Degas the ethanol/water mixture thoroughly and use a low flow rate, checking the back
pressure as the column equilibrates.
Avoid changes in temperature which may cause air bubbles in the packing.

18-1022-18 AK 71
72 18-1022-18 AK
Chapter 6
Sephadex LH-20 – gel filtration in presence
of organic solvents
Sephadex LH-20 is specifically designed for the separation and purification of natural products
that require the presence of organic solvents to maintain their solubility, including molecules
such as steroids, terpenoids, lipids and low molecular weight peptides (up to 35 amino acid
residues). Compounds are usually separated by a form of liquid/liquid partitioning or absorption
chromatography. Sephadex LH-20 can have a very high selectivity for aromatic compounds in
certain solvents and can be used at analytical or industrial scale for the preparation of closely
related species.
Sephadex LH-20 is made from hydroxypropylated dextran beads that have been cross-linked to
yield a polysaccharide network. The medium can be swollen in water and organic solvents.
Sephadex LH-20 is suitable for an initial purification before polishing by ion exchange or
reversed phase chromatography, or for a final polishing step, for example during the preparation
of diastereoisomers.
Depending upon the chosen solvents, Sephadex LH-20 can also separate components
by partitioning between the matrix and the organic solvent. Sephadex LH-20 exhibits
both hydrophilic and hydrophobic properties, the combination of which can offer unique
chromatography selectivity for certain applications.
Sephadex has been used for gel filtration in organic solvents, for example
dimethylformamide may be used with Sephadex G-10 and mixtures of water with the
shorter chain alcohols may be used with Sephadex G-10, G-25 and G-50.

Media characteristics
Table 6.1

Product Fractionation range Sample loading Maximum operating Maximum .

(globular proteins) capacity* back pressure operating flow
Sephadex LH-20 < 5 × 103 (exclusion 2% of column Solvent-dependent 12 cm/min (720 cm/h,
limit will depend on volume bed height 14 cm,
the solvent) 15 MPa back pressure)
* If Sephadex LH-20 is used in adsorption mode then the sample volume is unlimited until reaching the point of column saturation.

Separation examples
An HIV-1 reverse transcriptase inhibitor has been isolated from Phyllanthus niruri, a natural
medicine that has been used for many years to combat edema and jaundice. The active component
that inhibits HIV-1 reverse transcriptase has been identified as repandusinic acid A monosodium
salt, a small tannin-like molecule. The structure of the free acid is shown in Figure 6.1.

18-1022-18 AK 73
 HO OH HO HO

HO OH

O OH
6
O O O
5
O O OH
4 O 1

3 2 OH
O
OH
6' 7' O
HOOC HO OH
5''
4' 6''
5'
3' 4''
H H OH
1''
2'
HOOC H
2'' 3''
1'
O
O
Fig 6.1. Structure of free acid form of repandusinic acid A.

Table 6.2 shows the recovery of active inhibitor from an analytical separation on Sephadex LH-20.
Table 6.2 Summary of data for the isolation of repandusinic acid A from P. niruri

Purification step Yield (mg) ID50* (µg/ml) Specific activity . Total activity .
(× 102 IU/mg) (× 103 IU)†
H2O extract 6600 50 4 2640
MeOH insoluble 22 500 20 10 22 500
Sephadex LH-20 247 3.0–3.6 56–67 11 616
fr. 4–11‡
Cellulose
Fr 1 189 7.8 26 484
Fr 2 24 5.0 40 96
Fr 3 18 2.4 83 150
Fr 4 9 3.4 58 52
Fr 5 14 1.8 111 156
RA (pure substance) 5.9 0.3 668 394
* ID50 indicates the effectiveness of inhibitors expressed as concentrations which cause 50% inhibition of HIV-1-RT. Crude
HIV-1-RT was used in this experiment
†
IU are arbitrary inhibitory activity units obtained by dividing the total weight of the fraction at each step by the weight of
each fraction required to achieve 50% inhibition of [3H]dTTP incorporation into the polymer in the HIV-1-RT assay.
‡
Fractions 4–10 and fraction 11 were combined because both fractions had the inhibitory activity

Figure 6.2 shows Sephadex LH-20 used at a preparative scale for the separation of
2-acetamidobenzoic acid and 4-acetamidobenzoic acid. In this separation the hydrophilicity
and hydrophobicity of the medium provide a unique chromatography selectivity resulting in high
resolution of closely related species. The molecules differ only by the position of the acetamide
moiety on the benzene ring.

74 18-1022-18 AK
Column: Sephadex LH-20, 2.5 × 200 cm
Sample: Mixture of 2- and 4-acetamidobenzoic acid
Eluent: Acetone
Flow rate: 8 ml/min
Detection: Refractive Index
Yield: 250 mg 2-acetamidobenzoic acid
254 mg 4-acetamidobenzoic acid

RI 1 2

1. C OH

NH C CH 3

2. CH 3 C HN C OH

O O
250 mg

254 mg

6 8 Time (h)

Fig 6.2. Separation of 2- and 4-acetamidobenzoic acid on Sephadex LH-20.

Packing a column
Sephadex LH-20 should be packed in a solvent resistant column selected from Table 6.3
according to the column volume required for the separation.

Table 6.3. Solvent resistant columns

Column Volume (ml) Bed height (cm)

SR 25/454 73–220 15–45
SR 25/100 343–490 70–100

Simple steps to clarify a sample before applying it to a column will avoid the risk of
blockage, reduce the need for stringent washing procedures and extend the life of the
chromatography medium. Filter or centrifuge all solvents and samples before use.

1. Refer to Table 6.4, page 77, to calculate the amount of dry medium required as the
extent of swelling depends upon the solvent system. Swell Sephadex LH-20 for at least
3 hours at room temperature in the solvent to be used for the separation.
2. Prepare a slurry 75:25 settled medium:solvent and decant any fine particles of medium.
3. Equilibrate all materials to room temperature.
4. Resuspend and pour the slurry into the column in one continuous step (using a glass rod
will help to eliminate air bubbles).
5. Fill the column reservoir to the top with solvent. Seal, attach to a pump and open the
column outlet.
6. Pack at 300 cm/h until the bed has reached a constant height. Stop the flow, empty and
remove the packing reservoir.
7. Carefully fill the column with solvent and insert a wetted adaptor into the column.
Ensure no air bubbles are trapped under the net and adjust the adaptor O-ring to give a
sliding seal against the column wall.

18-1022-18 AK 75
 8. Connect all tubings, ensuring that there are no air bubbles in the flow path.
9. Slowly slide down the adaptor so that any air in the tubings is displaced by solvent and
lock the adaptor into position on the surface of the medium.
10. Open the column outlet and continue packing until the packed bed is stable and a final
adjustment of the top adaptor can be made.

Note: In solvents such as chloroform Sephadex LH-20 is less dense than the solvent and the
medium will float. Pour the medium into the column and drain until the second adaptor
can be inserted. Lock the adaptor in position at the surface of the medium and direct
the flow of chloroform upwards. The bed will be packed against the top adaptor and the
lower adaptor can be pushed slowly upwards towards the lower surface of the medium.
Close the column outlet when moving the adaptor to avoid compressing the bed.

Performing a separation
Start at a linear flow of 1 cm/h to check resolution. Low flow rate gives better the resolution.

1. Equilibrate the column with at least 2 column volumes of the solvent until a stable
baseline is achieved.
2. Apply a sample volume equivalent to 1–2% of the total column volume.
3. Elute in 1 column volume. Re-equilibration is not needed between runs with the
same solvent.

Cleaning
Wash the column with 2–3 column volumes of the solvent, followed by re-equilibration in a new
solvent if changing the separation conditions.

76 18-1022-18 AK
Table 6.4. Approximate values for packed bed volumes of Sephadex LH-20 swollen in different solvents

Solvent Approx. bed volume .

(ml/g dry Sephadex LH-20)
Dimethyl sulfoxide 4.4–4.6
Pyridine 4.2–4.4
Water 4.0–4.4
Dimethylformamide 4.0–4.4
Methanol 3.9–4.3
Saline 3.8–4.2
Ethylene dichloride 3.8–4.1
Chloroform* 3.8–4.1
Propanol 3.7–4.0
Ethanol †
3.6–3.9
Isobutanol 3.6–3.9
Formamide 3.6–3.9
Methylene dichloride 3.6–3.9
Butanol 3.5–3.8
Isopropanol 3.3–3.6
Tetrahydrofuran 3.3–3.6
Dioxane 3.2–3.5
Acetone 2.4–2.6
Acetonitrile ‡
2.2–2.4
Carbon tetrachloride‡ 1.8–2.2
Benzene‡ 1.6–2.0
Ethyl acetate‡ 1.6–1.8
Toluene ‡
1.5–1.6
* Containing 1% ethanol.
†
Containing 1% benzene.
‡
Solvents that give a bed volume of less than 2.5 mg/ml dry Sephadex LH-20 are not generally useful.

Chemical stability
Sephadex LH-20 is stable in most aqueous and organic solvent systems. The medium is not
stable below pH 2.0 or in strong oxidizing agents.

Storage
Store dry at 4°C to 30°C. Store packed columns and used medium at 4°C to 8°C in the presence
of a bacteriostatic agent.

Transferring Sephadex LH-20 from aqueous solution to organic solvents

Transfer Sephadex LH-20 from an aqueous solution to the organic solvent by moving through
a graded series of solvent mixtures. This will ensure efficient replacement of the water by the
required solvent.
To transfer from aqueous solution or organic solvent (100% A) to a new organic solvent (100% B),
proceed as follows: transfer to 70% A:30% B then to 30% A:70% B and finally to 100% B. If A and
B are not mutually miscible, make the transfer via an intermediate solvent, for example from
water to chloroform via acetone, as shown in Figure 6.3.

18-1022-18 AK 77
 Ethanol
Dimerhylformamide
Water Dioxane
Dimethyl sulfoxide

Acetone

Chloroform, dixhloroethane, tetrahydrofuran,

n-heptane, ethyl acetate, toluene

Fig 6.3. Suggested routes for changing to organic solvents.

1. Transfer the required amount of medium to a sintered glass Buchner funnel and
remove the excess aqueous solution by gentle suction.
2. Add the next solvent and resuspend the medium by stirring gently.
3. Suck off the excess solvent and resuspend in the same solvent.
4. Repeat the process with the next solvent in the series. Perform at least two
resuspensions for each change of solvent conditions until the final solvent
composition is reached.
5. Pack the medium into solvent resistant SR 25/45 or SR 25/100 columns.

78 18-1022-18 AK
Chapter 7
Gel filtration in theory
Defining the process
Results from gel filtration are usually expressed as an elution profile or chromatogram that
shows the variation in concentration (typically in terms of UV absorbance, for proteins usually
at 280 nm) of sample components as they elute from the column in order of their molecular
size. Figure 7.1 shows a theoretical chromatogram of a high-resolution fractionation. Molecules
that do not enter the matrix are eluted together in the void volume, Vo as they pass directly
through the column at the same speed as the flow of buffer. For a well-packed column, the void
volume is equivalent to approximately 30% of the total column volume. Molecules with partial
access to the pores of the matrix elute from the column in order of decreasing size. Small
molecules such as salts that have full access to the pores move down the column, but do not
separate from each other. These molecules usually elute just before one total column volume,
Vt, of buffer has passed through the column.

high
Absorbance molecular
weight
low
molecular
sample weight
injection

intermediate
molecular weight
equilibration
Vt void volume Vo
Vo
total column volume Vt

1 cv

Fig 7.1. Theoretical chromatogram of a high resolution fractionation.

The behavior of each component can be expressed in terms of its elution volume, Ve from
the chromatogram. As shown in Figure 7.2, there are three different ways of measuring Ve,
dependent on the volume of sample applied to the column. Ve is the direct observation of the
elution properties of a certain component.

18-1022-18 AK 79
 A) A 280 nm

Sample size negligible

compared with volume of
UV absorption

packed bed.

Ve Elution volume
Sample size not negligible
B)
compared with volume of
packed bed.
UV absorption

Ve Elution volume
C)
Sample giving plateau
elution curve.
UV absorption

inflexion point

Ve Elution volume

Fig 7.2. Measurement of elution volume, Ve. (A) Sample size negligible compared with volume of packed bed. (B) Sample size
not negligible compared with volume of packed bed. (C) Sample giving plateau elution curve

Since symmetrical peaks are common in gel filtration, elution volumes are easily determined.
However, Ve will vary with the total volume of the packed bed (Vt) and the way in which the
column has been packed. The elution of a sample is best characterized by a distribution
coefficient (Kd). Kd is used for column comparison since it is independent of column dimensions
and thus allows comparison and prediction between columns whit different sizes if the same
medium and sample is used.
Kd is derived as follows:
The volume of the mobile phase (buffer) is equal to the void volume, Vo, which is the elution
volume of molecules that remain in the buffer because they are larger than the largest pores
in the matrix and therefore pass straight through the packed bed (Fig 7.3). In a well-packed
column, the void volume is approximately 30% of the total physical volume.
The volume of the stationary phase, Vs, is equal to Vi, the volume of buffer inside the matrix. This
volume is available only to very small molecules. Vi is the elution volume of molecules that distribute
freely between the mobile and stationary phases minus the void volume. Kd represents the
fraction of the stationary phase that is available for diffusion of a given molecular species:
Ve–Vo V –V
Kd = = e o
Vt–Vo–Vgel matrix Vi

Since, in practice, Vs or Vi are difficult to determine, it is more convenient to employ the term
(Vt – Vo). The estimated volume of the stationary phase will therefore include the volume of solid
material which forms the matrix.
The stationary phase volume Vs can be substituted by the term (Vt – Vo) in order to obtain a value Kav.

Kav = (Ve–Vo) / (Vt–Vo)

80 18-1022-18 AK
Void volume Vo Total column Vt – Vo
volume Vt

Fig 7.3. 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 author and publisher).

Since (Vt – Vo) includes the volume of the matrix that is inaccessible to all solute molecules,
Kav is not a true partition coefficient. However, for a given medium there is a constant ratio
of Kav:Kd which is independent of the nature of the molecule or its concentration. Kav is easily
determined and, like Kd, defines sample behavior independently of the column 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 7.4.

Absorbance
Interacting with medium
High molecular weight

Low molecular weight

molecular weight
Sample injection

Intermediate

Vo Vt Ve– Vo
K av =
Ve Vt – Vo
Vt – Vo
Ve– Vo Ve– Vo
Vi Kd = =
Vt – Vo – Vgel matrix Vi
0.5 1.0 Ve /Vo

1 2 3

0 0.5 1.0 K av

0 0.5 1.0 Kd

Fig 7.4. Relationship between several expressions used for normalizing elution behavior.

Selectivity curves and media selection

The partition coefficient Kav is related to the size of a molecule. Molecules of similar shape and
density demonstrate a sigmoidal relationship between their Kav values and the logarithms of
their molecular weights (Mr). Over a considerable range there is a virtually linear relationship
between Kav and log Mr. The selectivity of a gel filtration medium depends solely on its pore
size distribution and is described by a selectivity curve. By plotting Kav against the log of the
molecular weight for a set of standard proteins, selectivity curves are created for each gel
filtration medium, as shown in Figure 7.5.

18-1022-18 AK 81
Superdex 30 prep grade Superdex 75 prep grade and
Superdex 200 prep grade
K av K av
0.7 0.8

0.6
0.6
0.5
Dextrans
PEG

20
Peptides

0p
0.4 0.4

g
75
p g
0.3
0.2
0.2

0.1 4 5 6 7
10 10 10 10
Mr logarithmic scale

300 103 104

Mr logarithmic scale Superdex 75 prep grade and
Superdex 200 prep grade
K av
0.8

0.6 Globular
proteins

0.4 20
0
pg
75
pg

0.2

4 5 6 7
10 10 10 10
Mr logarithmic scale
Fig 7.5. Selectivity curves for Superdex 30 prep grade, Superdex 75 prep grade and Superdex 200 prep grade.

Gel filtration media should be selected so that the important components are found in the most
linear part of the selectivity curve with minimum peak broadening or dilution and minimum
time on the column. The lowest molecular weight substances should be eluted near Vt (Kav = 1).
Under ideal conditions, no molecules can be eluted with a Kav greater than 1 or less than 0.
If the Kav is greater than 1, molecules have bound nonspecifically to the

chromatographic medium.
If Kav is less than 0 after calibration then there is channeling in the chromatography bed

and the column must be repacked.
The steeper the selectivity curve, the higher the resolution that can be achieved.

Resolution
Resolution (Rs) is defined by the following expression:

Ve2–Ve1
Rs = (W1+W2)
2

Ve1 and Ve2 are the elution volumes for two adjacent peaks measured at the center of the peak.
W1 and W2 are the respective peak widths.

82 18-1022-18 AK
Ve1 and Ve2 are the elution volumes for two adjacent peaks measured at the center of the peak.
W1 and W2 are the respective peak widths. (Ve2 – Ve1) represents the distance between the
peaks and 1/2 (W1 + W2) the mean peak width of the two peaks as shown in Figure 7.6.

(Ve2 – Ve1)

W1 W2

Fig 7.6. Parameters used to define resolution (Rs).

Final resolution, the degree of separation between peaks, is influenced by many factors: the
ratio of sample volume to column volume, flow rate, column dimensions, particle size, particle
size distribution, packing density, porosity of the particle, and viscosity of the mobile phase. The
success of gel filtration depends primarily on choosing conditions that give sufficient selectivity
and counteract peak broadening effects during the separation.
Resolution is a function of the selectivity of the medium and the efficiency of that medium to
produce narrow peaks (minimal peak broadening) as illustrated in Figure 7.7.

A) B)

high efficiency

low efficiency

Fig 7.7. Resolution depends on the selectivity of the media and the counteraction of peak broadening (lower efficiency).
(A) Good resolution (blue curve) and poor resolution (red curve). (B) Excellent resolution (blue curve) and good resolution (red curve).

The homogeny of the packed bed and the particles influences the uniformity of the flow
through the column and hence affects the shape and eventual peak width. Gel filtration media
with high uniformity and narrow particle size distribution facilitate the elution of molecules in
sharp peaks.
Gel filtration media with smaller particle sizes facilitate diffusion of sample molecules in and
out of the particles by reducing the time to achieve equilibrium between mobile and stationary
phases and so improve resolution by reducing peak width.

18-1022-18 AK 83
Sample dilution is inevitable because diffusion occurs. In order to minimize sample dilution
a maximum sample volume is used within the limits set by the separation distance, that is
the resolution required between the peaks of interest. The sample can be regarded as a zone
passing down the column. Figure 7.8 shows how, if no zone broadening occurs, 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 buffer,
and longitudinal diffusion in the bed, the zones will always be broadened. Therefore the sample
volume must always be smaller than the separation volume.
Concentration

A B

Elution volume
Concentration

Elution volume
Concentration

A Ve B Ve Elution volume
Vsep

Fig 7.8. Elution curves for different sample sizes. The top diagram corresponds to the application of a small sample. The
center diagram corresponds to the maximum sample volume that gives complete separation if there is no zone broadening.
The bottom diagram corresponds to the maximum sample volume to obtain complete separation in the conditions of the
experiment. The shaded areas correspond to the elution profiles that would be obtained if there was no zone broadening.

84 18-1022-18 AK
Chapter 8
Gel filtration in a purification strategy

The three phase purification strategy of Capture, Intermediate Purification and Polishing (CIPP)
is used in both the pharmaceutical industry and in the research laboratory to ensure faster
method development, a shorter time to pure product and good economy. This chapter gives a
brief overview of this approach, which is recommended for any multistep protein purification.
The Protein Purification Handbook from GE Healthcare is recommended as a guide to planning
efficient and effective protein purification strategies.
As shown in Figure 8.1, an important first step for any purification is correct sample preparation
and this is covered in more detail in Appendix 3. Gel filtration is often used for desalting and
buffer exchange during sample preparation using Sephadex G-25, and samples volumes up to
30% of the total column volume can be applied.
In high-resolution mode, gel filtration is ideal for the final polishing steps in a purification
when sample volumes have been reduced (sample volume significantly influences speed and
resolution in gel filtration). Samples are eluted isocratically (single buffer, no gradient) and buffer
conditions can be varied to suit the sample type or the requirements for subsequent purification,
analysis or storage, since buffer composition does not directly affect resolution.
Purity

Polishing
Achieve final
high level purity
Intermediate
purification
Remove bulk
Capture impurities

Isolate, concentrate,
Preparation, and stabilize
extraction,
clarification
Step
Fig 8.1. Sample preparation and CIPP purification strategy.

The purification strategy according to CIPP

Imagine the purification has three phases: Capture, Intermediate Purification and
Polishing. Each phase may include one or more purification steps.
Assign a specific objective to each step within the purification process.
The problem associated with a particular purification step will depend greatly upon the
properties of the starting material. Thus, the objective of a purification step will vary according to
its position in the process.
In the capture phase the objectives are to isolate, concentrate and stabilize the target product.
The product should be concentrated and transferred to an environment that will conserve
potency/activity.

18-1022-18 AK 85
During the intermediate purification phase the objective is to remove most of the bulk impurities,
such as other proteins and nucleic acids, endotoxins and viruses.
In the polishing phase most impurities have already been removed except for trace amounts or
closely related substances. The objective is to achieve final purity by removing any remaining
trace impurities or closely related substances.
The optimal selection and combination of purification techniques for Capture,
Intermediate Purification and Polishing is crucial for an efficient purification.
A guide describing the main features of the most common purification techniques and their use
in the different phases in CIPP is shown in Table 8.1.

Table 8.1. Properties of different purification techniques and strategies for their use in CIPP

Typical Purification .
characteristics phase
Intermediate
Resolution

Polishing
Capacity

Capture

Sample start . Sample end .

Method conditions conditions
AC +++ +++ +++ ++ + Various binding conditions Specific elution
or or conditions
++ ++
IMAC +++ ++ +++ ++ + For purifying histidine- High concentration
tagged proteins using of imidazole,
Ni Sepharose columns:: pH > 7, 500 mM NaCl
20-40 mM imidazole, pH > 7,
500 mM NaCl; no chelators
Other proteins: low
concentration of imidazole
GF ++ + + +++ Most conditions acceptable, Buffer exchange possible
limited sample volume Diluted sample
IEX +++ +++ +++ +++ +++ Low ionic strength. High ionic strength or pH
pH depending on protein changed
and IEX type
HIC +++ ++ ++ +++ +++ High ionic strength Low ionic strength
Addition of salt required
Chromato- +++ + ++ Low ionic strength Polybuffer
focusing Low ionic strength

RPC +++ ++ + ++ Ion-pair reagents and Organic solvents (risk for

organic modifiers may be loss of biological activity)
required

Gel filtration as a polishing step

Most commonly, separations by charge, hydrophobicity or affinity will have been used in earlier
stages of a purification strategy so that high resolution gel filtration is ideal for the final polishing
step. The product can be purified and transferred into the required buffer in one step and dimers
and aggregates can be removed, as shown in Figure 8.2.
Gel filtration is also the slowest of the chromatography techniques and the size of the column
determines the volume of sample that can be applied. It is therefore most logical to use gel
filtration after techniques that reduce sample volume so that smaller columns can be used.

86 18-1022-18 AK
Column: XK 16/60 packed with Superdex 75 prep grade
Sample: partly purified ZZ-brain IGF
Sample load: 1.0 ml
Buffer: 0.3 M ammonium 0.5 ml/min
Flow: 0.5 ml/min (15 cm/h)
A280 nm monomeric
ZZ-Brain IGF
0.01

0.005
VO Vt

I I
Fraction 1 3 I 5 I I

0 1 2 3 4 Time (h)
Fig 8.2. Final polishing step: separation of dimers and multimers on Superdex 75 prep grade.

Media for polishing steps should offer highest possible resolution. Superdex is the first
choice at laboratory scale and Superdex prep grade for large-scale applications.
CIPP does not mean that there must always be three purification steps. For example, capture
and intermediate purification may be achievable in a single step, as may intermediate
purification and polishing. Similarly, purity demands may be so low that a rapid capture step is
sufficient to achieve the desired result. For purification of therapeutic proteins, a fourth or fifth
purification step may be required to fulfill the highest purity and safety demands. The number of
steps used will always depend upon the purity requirements and intended use for the protein.

Purification of humanized IgG4 monoclonal antibody

A humanized IgG4 monoclonal antibody was expressed in a myeloma cell culture and purified by
a combination of affinity chromatography and gel filtration (Fig 8.3). The antibody was captured
by affinity chromatography using MabSelect™. Gel filtration on HiLoad Superdex 200 pg column
was then used to separate the monomer from the dimer and larger polymers.

18-1022-18 AK 87
Capture by affinity chromatography Purification of the monomer (polishing step)
Sample: 1282 ml myeloma cell culture Sample: 7.5 ml of the pooled fractions from
containing humanized IgG4 MabSelect column
(~0.33 mg/ml) Column: HiLoad 26/60 Superdex 200 prep
Column: MabSelect (18 ml), XK 16/20 column grade
Binding buffer: 20 mM sodium phosphate, Buffer: 50 mM sodium phosphate,
0.15 M NaCl, pH 7.4 0.15 M NaCl, pH 7.0
Elution buffer: 100 mM sodium citrate, pH 3.0 Flow: 22.6 cm/h (2 ml/min)
Flow: 220 cm/h (7.4 ml/min) Operation: Equilibration 2 CV, Sample
Operation: Equilibration: 5 column volumes (CV) application, Isocratic elution 1 CV
binding buffer
Sample application: 1 282 ml. CV = total column volume = Vt
Wash: 10 CV binding buffer.
Elution: step gradient 100% 5 CV
elution buffer

A 280nm (mAU) A 280nm (mAU)

5000
600
Purified IgG 4
4000
500
Monomer
3000 400

300
2000
200 Dimer/multimer

1000
100

0 Waste
1357912 1620
0 1 2 3 4 5 6 7 8 910 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70

0 400 800 1200 1600 ml 0 100 200 300 400 ml

Mr

97 000 H - chain
66 000
45 000
30 000 L - chain
20 100
14 400

1 2 3 4 5 6 1 2 3 4 5

Analysis: SDS-PAGE, silver staining

Unreduced sample Reduced sample
Lanes 1. LMW Marker
1. LMW Marker 2. Pooled eluted sample from capture step
2. Crude sample from myeloma cell culture 3. Fraction 4–9 from polishing step
3. Pooled eluted sample from capture step 4. Fraction 10–12 from polishing step
4. Fraction 4–9 from polishing step 5. Fraction 14–18 from polishing step
5. Fraction 10–12 from polishing step
6. Fraction 14–18 from polishing step

Fig 8.3. Two step purification of humanized IgG4.

88 18-1022-18 AK
Appendix 1
Column packing and preparation
A well-packed column is essential for a high resolution fractionation on any gel filtration medium.
Prepacked columns from GE Healthcare will ensure reproducible results and the highest
performance. If the column volume or medium you require is not available as a prepacked
column, contact your local GE Healthcare sales representative to inquire about our column
packing services.
Packing a column is a very critical stage in any gel filtration experiment. A poorly packed column
will give rise to uneven flow, peak broadening, and loss of resolution, it can also affect achievable
flow rates. If you decide to pack a gel filtration column yourself then the guidelines in this
appendix will apply at any scale of operation.
An instructive video on a CD, is available to demonstrate how to produce a well-packed column
(see Ordering information for “Column Packing – The Movie”). It focuses particularly on the importance
of column packing for gel filtration. Gel filtration is 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 reproducible, high resolution results for a long time.

Ensure that there is sufficient buffer for long, unattended runs or that the pump is
programmed to stop the flow after a suitable time. Gel filtration columns that run
dry must be repacked.

Columns for packing gel filtration media

Empty columns from GE Healthcare are fully compatible with the high flow rates achievable
with modern media and a broad range of column dimensions is available. Ordering information
for empty columns and main accessories can be found at the back of this handbook. For a
complete listing of all spare parts refer to the www.gelifesciences.com/protein-purification

Table A1.1. Maximum bed heights (cm) and bed volumes (ml) using one adaptor or two adaptors in the various XK columns.

With one adaptor With two adaptors

Column Volume (ml) Bed height (cm) Volume (ml) Bed height (cm)
XK 16/20 5-31 2.5-15.5 0-31 0-15.5
XK 16/40 45-70 22.5-35 16-70 8-35
XK 16/70 105-130 52.5-65 76-130 38-65
XK 16/100 165-190 82.5-95 136-190 68-95
XK 26/20 5-66 1-12.5 0-66 0-12.5
XK 26/40 122-186 23-35 45-186 8.5-35
XK 26/70 281-344 53-65 204-344 38.5-65
XK 26/100 440-504 83-95 365-504 68.5-95
XK 50/20 0-274 0-14 0-274 0-14
XK 50/30 265-559 14-28 0-559 0-28
XK 50/60 794-1088 40-56 500-1088 26-56
XK 50/100 1588-1862 81-95 1274-1862 65-95

18-1022-18 AK 89
Adaptors are adjustable column end pieces that help to eliminate any disturbances to the
surface of the packed medium as sample is applied and to prevent insoluble particles from
entering and blocking the column.
Tricorn™ and XK empty columns are delivered with one adaptor, but a second adaptor can be
used instead of a column end piece if a shorter bed height is required. HiScale™ columns are
equipped with dual adaptors. A range of accessories are available for all empty columns.

Table A1.2. Bed volumes and heights for the various Tricorn columns

Bed volumes and heights

With one adaptor With two adaptors
Column Size . Volume . Bed Height . Volume . Bed height .
Tricorn Column i.d. (mm) (ml) (mm) (ml) (mm)
Tricorn 10/20 10 0.00–2.29 0–29 0.00–2.07 0–26
Tricorn 10/100 10 6.21–8.57 79–109 3.64–8.36 46–106
Tricorn 10/150 10 10.14–12.50 129–159 7.57–12.28 96–156
Tricorn 10/200 10 14.07–16.42 179–209 11.50–16.21 146–206
Tricorn 10/300 10 21.92–24.28 279–309 19.35–24.06 246–306
Tricorn 10/600 10 45.48–47.84 579–609 42.91–47.63 546–606

Table A1.3. Bed volumes and heights for HiScale columns

Column Column volume (ml) with one adaptor

HiScale 16/20 Max bed height 20 cm, max volume 40 ml
HiScale 26/20 Max bed height 20 cm, max volume 106 ml
HiScale 16/40 Max bed height 40 cm, max volume 80 ml
HiScale 26/40 Max bed height 20 cm, max volume 212 ml
HiScale 50/20 Max bed height 20 cm, max volume 393 ml
HiScale 50/40 Max bed height 20 cm, max volume 785 ml

Longer columns (50 cm and more) can be difficult to pack under normal laboratory
conditions. As alternatives, use our column packing services or connect two or more
shorter columns (20 or 30 cm bed height) in series to achieve the required bed height.

Checking column efficiency

Column performance should be checked at regular intervals by determining the theoretical plate
number and peak symmetry. Prepacked columns are supplied with recommended values.
Typical values for column performance:
Superdex: Efficiency N>10 000, Peak symmetry As = 0.70–1.30
Sephacryl HR: Efficiency N>9 000, Peak symmetry As = 0.80–1.50

90 18-1022-18 AK
 1. Equilibrate the packed column in distilled water at a linear flow of 60 cm/h.
2. Inject acetone (10 mg/ml in water) in a volume equivalent to 0.2% of the total packed
column volume.
3. Monitor UV absorbance 280 nm from the time of injection until the acetone peak has
eluted and the signal has returned to baseline.
4. Calculate column efficiency i.e. the number of theoretical plates (N):
N = 5.54 (Ve / W1/2)2 × 1000/L Absorbance

where
Ve = peak elution (retention) volume
W1/2 = peak width at half peak height
L = bed height (mm)
Ve and W1/2 are in same units w1/2

Calculate the symmetry factor (As):

As = b/a
where
a = first half peak width at 10% peak height a b
b = second half peak width at 10% peak height
Ve Volume

Fig A1.1. Determination of column efficiency by number of theoretical plates and peak symmetry.

Column packing for high resolution fractionation using Superdex prep

grade and Sephacryl High Resolution
Superdex prep grade and Sephacryl High Resolution should be packed and equilibrated at a high
flow rate using a column from the XK-series. XK columns are optimally designed for gel filtration
with a bed design that ensures a uniform liquid flow and a dead space at the column outlet
of less than 0.1% of the column volume in order to minimize dilution and to prevent remixing
of separated peaks. XK columns are manufactured from materials which do not interfere with
labile biological substances. They are easy to dismantle and reassemble for thorough cleaning,
particularly important when handling biological samples.

Ensure that the column and all components are clean and in good condition. It is particularly
importance that the nets, net fasteners and glass tube are not damaged. Use well
degassed buffers and equilibrate all materials to the temperature at which the separation
will be performed. Avoid columns with large dead volumes as this will affect resolution.

For high resolution fractionation, use bed heights between 30–60 cm. Apply sample
volumes equivalent to 1–2% of the column volume. The sample volume can be increased
up to 4% if resolution in the particular application still is good enough.

The settled medium should have a volume of 1.15 times that of the required packed
column volume, see Table A1.1 to A1.3 for examples.

18-1022-18 AK 91
 1. Sephacryl HR and Superdex prep grade are supplied swollen in a suspension containing
20% ethanol as a preservative. Suspend the medium by shaking gently and pour a
sufficient quantity into a graduated glass cylinder or beaker.
Avoid using magnetic stirrers, spatulas or glass rods since they may damage the matrix.
2. Wash the medium with 5–10 column volumes of distilled water on a glass filter and
resuspend in distilled water to a final concentration of 50% settled medium. The medium
must be thoroughly washed to remove the 20% ethanol storage solution. Residual
ethanol may interfere with subsequent procedures.
To produce a more evenly dispersed slurry of Superdex prep grade, Tween™ 20
(250 ml per 500 ml washed slurry) can be added in order to reduce surface tension.

3. Wet the bottom filter by injecting distilled water through the effluent tubing. Close the
end piece outlet. Mount filter and bottom end piece onto the column.
4. Attach the packing reservoir tightly to the column.

For XK 16 and XK 26 columns using a second column instead of a packing reservoir

makes it easier to obtain a well-packed column. The second column is used with
Packing Connector XK 16 or XK 26 as appropriate.

5. Mount the column and packing reservoir vertically on a laboratory stand.

6. Fill the column with distilled water to a height of 2 cm above the column end piece.
Avoid air bubbles.
7. Degas the suspended medium under vacuum and carefully pour the suspended
medium down the wall of the column using a glass rod. Avoid introducing air bubbles.
Pour everything in a single operation and fill the reservoir to the top with distilled water.
8. Connect the pump outlet to the inlet on the packing reservoir. Open the column outlet
and start the flow of buffer, see Table A1.4 for flow recommendations.

To achieve satisfactory column efficiency, Superdex prep grade must be packed in

two steps: Step 1 for 2 hours or until the bed has reached a constant height and Step
2 for 60 min. Table A1.4 shows the flow rates for each step.
Sephacryl HR can usually be packed satisfactorily using only the higher flow rate
given in Step 2 of Table A1.4. Use the two step process if the column efficiency was
unsatisfactory after the first attempt.

9. Stop the pump and remove the packing reservoir. Carefully fill the column with distilled
water to form an upward meniscus at the top and insert the adaptor. Adjust the adapter
to the surface of the packed bed.
10. Continue packing the column at the flow rate used in Step 2 for approximately
10 minutes. If the recommended flow rate cannot be obtained, use the maximum flow
rate the pump can deliver. Mark the position of the top of the packed medium, stop
the pump, close the column outlet, move the adaptor down onto to the surface of the
medium and then push the adaptor a further 3 mm into the medium. The column is
now ready to use. See Table A1.4 for maximum recommended flow rate and operating
pressure for Sephacryl HR and Superdex prep grade media.
Maximum pressures (Sephacryl HR 0.3 MPa, 0.3 bar and Superdex prep grade
5 MPa, 5 bar) should not be exceeded during packing.

Always check the specific storage instructions supplied with the product.

92 18-1022-18 AK
Table A1.4. Recommended flow rates during column packing

Step 1 . Step 2 . Step 1 . Step 2 .

Sephacryl HR . Sephacryl HR . Superdex prep Superdex prep
Column Bed height cm ml/min ml/min grade ml/min grade ml/min
XK 16/40 35 2 12–14 2 10–12
XK 16/70 65 2 12–14 2 10–12
XK16/100 95 2 12–14 2 10–12
XK 26/40 35 4 6–8 4 12
XK 26/70 65 4 6–8 4 12
XK 26/100 95 4 6–8 4 12
XK 50/20 10–15 9 12 10 20
XK 50/30 20–25 9 12 10 20
XK 50/60 55 9 12 10 20
XK 50/100 95 9 12 10 20

Column packing for group separations using Sephadex

Sephadex is supplied as a dry powder and must be allowed to swell in excess buffer before
use. After swelling, adjust with buffer to form a thick slurry from which air bubbles are removed
under vacuum. Approximately 75% settled medium is suitable. Fine particles can be decanted.
Accelerate the swelling process by using a boiling water bath (Table A1.5). This also
serves to degas the suspension. Allow the suspension to cool before use.

Table A1.5. Bed volume and swelling times for Sephadex

Approx. bed volume .

Medium (ml/1 g medium) Swelling time (h), 20°C Swelling time (h), 90°C
Sephadex G-10 2–3 3 1
Sephadex G-25 (all grades) 4–6 3 1
Sephadex G-50 Fine 9–11 3 1

Ensure that the column and all components are clean and in good condition. It is
particularly important that the nets, net fasteners and glass tube are not damaged.
Use well-degassed buffers and equilibrate all materials to the temperature at which
the separation will be performed. Keep a packed column away from locations that
are exposed to drafts or direct sunlight that can cause temperature changes and the
formation of bubbles.
For group separations, use up to 10 cm bed height. Sample volumes can be up to 30%
of the column volume. Pack a quantity of medium up to 5 times the volume of the
sample to be desalted.

Note: These instructions assume that a column with two adaptors is used for packing.
1. Weigh out the correct amount of dry Sephadex and allow the medium to swell according
to the instructions above. Avoid using magnetic stirrers, spatulas or glass rods since
they may damage the medium.
2. Wet the bottom filter by injecting distilled water through the effluent tubing. Close the
end piece outlet. Mount filter and bottom end piece onto the column.

18-1022-18 AK 93
 3. If the slurry volume is greater than the volume of the column, attach a packing reservoir
to the column.
4. Mount the column and packing reservoir vertically on a laboratory stand.
5. Fill the column with distilled water or buffer to a height of approximately 2 cm above the
column end piece. Avoid air bubbles.
6. Pour the well-mixed and well-degassed suspension in a single operation down the inside
wall using a glass rod. Avoid introducing air bubbles.
7. Connect the pump outlet to the inlet of the packing reservoir. Open the column outlet
and start the flow of buffer. Pass 2–3 column volumes of buffer through the column in
order to stabilize the bed and equilibrate completely. Use a slightly higher flow rate than
the flow rate to be used during separations.
8. Maintain the packing flow rate for at least 3 column volumes after a constant bed height
is obtained.
9. Mark the bed height on the column and close the column outlet. Remove the packing
reservoir.
10. Add buffer carefully to fill the column and form an upward meniscus.
11. Connect all tubings. Slacken the adaptor tightening mechanism and insert the adaptor
at an angle into the column so that no air is trapped under the net. Slide the adaptor
slowly down the column until the mark is reached. Note that the outlet of the adaptor
should be open and the column outlet should be closed.
12. Adjust the tightening mechanism to give a sliding seal between the column wall and
O-ring. Screw the adaptor onto the column.
13. Continue packing the column for approximately 10 minutes. Stop the pump, close the
column outlet and move the top adaptor down onto the surface of the medium. Push
the adaptor a further 3 mm into the medium. The column is now ready for equilibration.

Fig A1.2. “Column Packing — The Movie” provides a step-by-step demonstration of column packing.

94 18-1022-18 AK
 Sephadex G-10, G-25 and G-50 obey Darcy’s law, for example if the flow rate is doubled
then the column pressure will double, hence maximum values for flow or operating pressures
do not need to be considered (see Appendix 2 for an explanation of Darcy’s law).

Controlling flow
The safest and easiest way in which to control flow during column packing and chromatography
separation is to use a pump controlled within an ÄKTA design chromatography system. Accurate
and reproducible flow control is particularly important for efficient column packing and when
repeating experiments or performing routine preparative work. A peristaltic pump can be used
with Sephadex packed in smaller columns.
The maximum flow rate achievable will depend on column diameter and buffer viscosity.
Narrow columns allow a higher pressure and higher linear flow (cm/h) than wide columns.
Always connect a pump so that buffer is pumped onto the column (rather than
connecting the pump after the column and drawing buffer through the column). This
reduces the risk of bubble formation due to suction effects.
Always use a flow rate for column packing that is higher than the flow rate used for
separation.
Do not exceed the maximum recommended values for pressure or linear flow for the
medium. Exceeding these values may cause the medium to compress and reduce the
flow rate and resolution during the separation.
Do not exceed 75% of the packing flow rate during any separation.
Do not use a peristaltic pump when packing Superdex or Sephacryl media in larger
columns since they cannot achieve high enough flowrate needed to get high resolution
fractionantion.

18-1022-18 AK 95
Appendix 2
Sephadex and Darcy’s law
Sephadex G-10, G-25 and G-50 may be assumed to behave as rigid spheres in gel filtration and
therefore obey Darcy’s Law. This Law describes a general relationship for flow in porous media:
U = K × ΔP × L-1 Equation (1)
U = linear flow rate expressed in cm/h (see Appendix 5)
ΔP = pressure drop over bed expressed in cm H2O
L = bed height expressed in cm
K = constant of proportionality depending on the properties of the bed material and the buffer
Assuming a buffer with viscosity of 1 cP: U = Ko × ΔP × L-1 Equation (2)
Ko = the “specific permeability” depending on the particle size of the medium and the water regain
Note that flow is proportional to the pressure drop over the bed and, assuming a
constant pressure head, inversely proportional to the bed height. In practice this means
that the pressure/flow considerations that must be made when using other gel filtration
media do not apply to Sephadex and that a doubling of flow rate leads to a doubling
in column pressure. To a good approximation, flow rate is independent of the column
diameter.
Flow at viscosities greater than 1 cP can be obtained by using the relationship: flow rate
is inversely proportional to viscosity. High buffer viscosities can be compensated for by
increasing the operating pressure to maintain a high flow rate.
Theoretical flow (not maximum) can be calculated from equation (2) by inserting values for ΔP
and L. Specific permeabilities (K) are given in Table A2.1.

Table A2.1. Specific permeabilities of Sephadex

Sephadex type Permeability K

Sephadex G-10 19
Sephadex G-25 Superfine 9
Sephadex G-25 Fine 30
Sephadex G-25 Medium 80
Sephadex G-25 Coarse 290
Sephadex G-50 Fine 36

96 18-1022-18 AK
Appendix 3
Sample preparation
Samples for chromatographic purification should be clear and free from particulate matter.
Simple steps to clarify a sample before beginning purification will avoid clogging the column,
may reduce the need for stringent washing procedures, and can extend the life of the
chromatographic medium.
Sample extraction procedures and the selection of buffers, additives, and detergents are
determined largely by the source of the material, the stability of the target molecule, the
chromatographic techniques that will be employed, and the intended use of the product.
These subjects are dealt with in general terms in the Protein Purification Handbook and more
specifically according to target molecule in the Recombinant Protein Handbook, and Antibody
Purification Handbook, available from GE Healthcare.

Sample clarification
Centrifugation and filtration are standard laboratory techniques for sample clarification and
are used routinely when handling small samples.
It is highly recommended to centrifuge and filter any sample immediately before
chromatographic purification.

Centrifugation
Centrifugation removes lipids and particulate matter, such as cell debris. If the sample is still
not clear after centrifugation, use filter paper or a 5 μm filter as a first step and one of the filters
below as a second step filter.
For small sample volumes or proteins that adsorb to filters, centrifuge at 10 000 g for 15
minutes.
For cell lysates, centrifuge at 40 000 to 50 000 g for 30 minutes.
Serum samples can be filtered through glass wool after centrifugation to remove any
remaining lipids.

Filtration
Filtration removes particulate matter. Membrane filters that give the least amount of nonspecific
binding of proteins are composed of cellulose acetate or PVDF.
For sample preparation before chromatography, select a filter pore size in relation to the bead
size of the chromatographic medium (Table A3.1).

Table A3.1. Filter pore size recommendations

Nominal pore size of filter Particle size of chromatography medium

1 µm 90 µm and upwards
0.45 µm 34 µm
0.22 µm 3, 10, 15 µm or when extra clean samples or sterile filtration is required

Check the recovery of the target protein in a test run. Some proteins may adsorb
nonspecifically to filter surfaces.

18-1022-18 AK 97
Desalting
Detailed procedures for buffer exchange and desalting are given in Chapter 5.

Denaturation
Table A3.2. Common denaturing agents

Denaturing agent Typical conditions for use Removal/comment

Urea 2 M–8 M Remove using maintain solubility. Sephadex G-25.
Guanidine hydrochloride 3 M–6 M Remove using Sephadex G-25 or during IEX.
Triton X-100 2% Remove using Sephadex G-25 or during IEX.
Sarcosyl 1.5% Remove using Sephadex G-25 or during IEX.
N-octyl glucoside 2% Remove using Sephadex G-25 or during IEX.
Sodium dodecyl sulfate 0.1%–0.5% Exchange for non-ionic detergent during first
chromatographic step, avoid anion exchange
chromatography.
Alkaline pH >pH 9, NaOH May need to adjust pH during chromatography to
maintain solubility.
Details taken from:.
Scopes R.K., Protein Purification, Principles and Practice, Springer, (1994), J.C. Janson and L. Rydén, Protein Purification,
Principles, High Resolution Methods and Applications, 2nd ed. Wiley Inc, (1998) and other sources.

See Chapter 5, page 57.

Precipitation and resolubilization

Specific sample preparation steps may be required if the crude sample is known to contain
contaminants such as lipids, lipoproteins, or phenol red that may build up on a column or if certain
gross impurities, such as bulk protein, should be removed before any chromatographic step.
Fractional precipitation Fractional precipitation is occasionally used at laboratory scale to
remove gross impurities but is generally not required in purification of affinity-tagged proteins.
In some cases, though, precipitation can be useful as a combined protein concentration and
purification step.
Precipitation techniques separate fractions by the principle of differential solubility. For
example, because protein species differ in their degree of hydrophobicity, increased salt
concentrations can enhance hydrophobic interactions between the proteins and cause
precipitation. Fractional precipitation can be applied to remove gross impurities in three
different ways, as shown in Figure A3.1.

Clarification Supernatant
Bulk proteins and
particulate matter
precipitated

Extraction, Clarification,
Concentration Redissolve pellet* Purification
Target protein precipitated Remember: if precipitating agent is
with proteins of similar incompatible with next purification
solubility step, use Sephadex G-25 for desalting
Concentration and buffer exchange, e.g., HiTrap Desalting,
PD-10 columns, or HiPrep 26/10
Extraction, Clarification Target protein
Redissolve Desalting column (refer to Chapter 5)
Bulk proteins and precipitated
particulate matter with proteins pellet*
precipitated of similar *Remember: not all proteins are easy
to redissolve, yield may be reduced
solubility
Fig A3.1. Three ways to use precipitation.

98 18-1022-18 AK
 Precipitation techniques may be affected by temperature, pH, and sample concentration.
These parameters must be controlled to ensure reproducible results.
Most precipitation techniques are not suitable for large-scale preparation.
Examples of precipitation agents are reviewed in Table A3.3. The most common precipitation
method using ammonium sulfate is described in more detail on below.

Table A3.3. Examples of precipitation techniques

Precipitation agent Typical conditions for use Sample type Comment

Ammonium sulfate As described below. > 1 mg/ml proteins, Stabilizes proteins, no
especially denaturation; supernatant
immunoglobulins. can go directly to HIC. Helps
to reduce lipid content.
Dextran sulfate Add 0.04 ml of 10% dextran Samples with high Precipitates lipoprotein.
sulfate and 1 ml of 1 M CaCl2 levels of lipoprotein,
per ml of sample, mix 15 min, e.g., ascites.
centrifuge at 10 000 × g,
discard pellet.
Polyvinylpyrrolidine Add 3% (w/v), stir 4 h, centrifuge Samples with high Alternative to dextran sulfate.
at 17 000 × g, discard pellet. levels of lipoprotein,
e.g., ascites.
Polyethylene glycol Up to 20% (w/v) Plasma proteins. No denaturation, supernatant
(PEG, Mr > 4000) goes directly to IEX or AC,
complete removal may be
difficult. Stabilizes proteins.
Acetone (cold) Up to 80% (v/v) at 0°C. Collect May denature protein
pellet after centrifugation at irreversibly. Useful for peptide
full speed in an Eppendorf™ precipitation or concentration
centrifuge. of sample for electrophoresis.
Polyethyleneimine 0.1% (w/v) Precipitates aggregated
nucleoproteins.
Protamine sulfate 1% (w/v) Precipitates aggregated
nucleoproteins.
Streptomycin sulfate 1% (w/v) Precipitates nucleic acids.
Caprylic acid (X/15) g where X = volume Antibody concentration Precipitates bulk of proteins
of sample. should be > 1 mg/ml. from sera or ascites, leaving
immunoglobulins in solution.
Details taken from: Scopes R.K., Protein Purification, Principles and Practice, Springer, (1994), J.C. Janson and L. Rydén, Protein
Purification, Principles, High Resolution Methods and Applications, 2nd ed. Wiley Inc, (1998).

Ammonium sulfate precipitation

Ammonium sulfate precipitation is frequently used for initial sample concentration and
cleanup. As the concentration of the salt is increased, proteins will begin to “salt out.” Different
proteins salt out at different concentrations, a process that can be taken advantage of to
remove contaminating proteins from the crude extract. The salt concentration needs to
be optimized to remove contaminants and not the desired protein. An additional step with
increased salt concentration should then precipitate the target protein. If the target protein
cannot be safely precipitated and redissolved, only the first step should be employed.
HIC is often an excellent next purification step, as the sample already contains a high salt
concentration and can be applied directly to the HIC column with little or no additional
preparation. The elevated salt level enhances the interaction between the hydrophobic
components of the sample and the chromatography medium.

18-1022-18 AK 99
Solutions needed for precipitation:
Saturated ammonium sulfate solution (add 100 g ammonium sulfate to 100 ml distilled
water, stir to dissolve).
1 M Tris-HCl, pH 8.0.
Buffer for first purification step.

Some proteins may be damaged by ammonium sulfate. Take care when adding
crystalline ammonium sulfate: high local concentrations may cause contamination of
the precipitate with unwanted proteins.
For routine, reproducible purification, precipitation with ammonium sulfate should be
avoided in favor of chromatography.
In general, precipitation is rarely effective for protein concentrations below 1 mg/ml.

1. Filter (0.45 μm) or centrifuge the sample (10 000 × g at 4°C).

2. Add 1 part 1 M Tris-HCl, pH 8.0 to 10 parts sample volume to maintain pH.
3. Stir gently. Add ammonium sulfate solution, drop by drop. Add up to 50% saturation*.
Stir for 1 h.
4. Centrifuge 20 min at 10 000 × g.
5. Remove supernatant. Wash the pellet twice by resuspension in an equal volume of
ammonium sulfate solution of the same concentration (i.e., a solution that will not
redissolve the precipitated protein or cause further precipitation). Centrifuge again.
6. Dissolve pellet in a small volume of the buffer to be used for the next step.
7 Ammonium sulfate is removed during clarification/buffer exchange steps with Sephadex
G-25, using desalting columns (see Chapter 5).

* The % saturation can be adjusted either to precipitate a target molecule or to precipitate contaminants.

The quantity of ammonium sulfate required to reach a given degree of saturation varies
according to temperature. Table A3.4 shows the quantities required at 20°C.

100 18-1022-18 AK
Table A3.4. Quantities of ammonium sulfate required to reach given degrees of saturation at 20°C

Final percent saturation to be obtained

20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Starting percent Amount of ammonium sulfate to add (grams) per liter of solution at 20°C
saturation
0 113 144 176 208 242 277 314 351 390 430 472 516 561 608 657 708 761
5 85 115 146 179 212 246 282 319 358 397 439 481 526 572 621 671 723
10 57 86 117 149 182 216 251 287 325 364 405 447 491 537 584 634 685
15 28 58 88 119 151 185 219 255 293 331 371 413 456 501 548 596 647
20 0 29 59 89 121 154 188 223 260 298 337 378 421 465 511 559 609
25 0 29 60 91 123 157 191 228 265 304 344 386 429 475 522 571
30 0 30 61 92 125 160 195 232 270 309 351 393 438 485 533
35 0 30 62 94 128 163 199 236 275 316 358 402 447 495
40 0 31 63 96 130 166 202 241 281 322 365 410 457
45 0 31 64 98 132 169 206 245 286 329 373 419
50 0 32 65 99 135 172 210 250 292 335 381
55 0 33 66 101 138 175 215 256 298 343
60 0 33 67 103 140 179 219 261 305
65 0 34 69 105 143 183 224 267
70 0 34 70 107 146 186 228
75 0 35 72 110 149 190
80 0 36 73 112 152
85 0 37 75 114
90 0 37 76
95 0 38

Removal of lipoproteins
Lipoproteins and other lipid material can rapidly clog chromatography columns and it is advisable
to remove them before beginning purification. Precipitation agents such as dextran sulfate and
polyvinylpyrrolidine, described under Fractional precipitation, are recommended to remove
high levels of lipoproteins from samples such as ascites fluid.
Centrifuge samples to avoid the risk of nonspecific binding of the target molecule
to a filter.
Samples such as serum can be filtered through glass wool to remove remaining lipids.

18-1022-18 AK 101
Appendix 4
Selection of purification equipment
Simple buffer exchange and desalting steps can be performed using a syringe or peristaltic
pump together with prepacked HiTrap columns. A chromatography system is needed to deliver
accurately controlled flow rates for high resolution separations.

Syringe or
peristaltic
pump +
HiTrap
Desalting Gravity-fed
Way of working Standard ÄKTA design configurations column columns
ÄKTAmicro™ ÄKTAprime ÄKTAxpress™ ÄKTApurifier™ ÄKTA avant
plus
Simple, one step x x x x x x x
desalting, buffer
exchange
Reproducible x x x x x
performance for
routine separation
Micropreparative x
and analysis
System control x x x x
and data handling
for regulatory
requirements,
e.g., GLP
Automatic method (x) x
development and
optimization
Automatic buffer (x) x
preparation
Automatic pH (x) x
scouting
Automatic media (x) x
or column scouting
Automatic multi- (x) x
step purification
Scale-up, process x
development

ÄKTA avant ÄKTAxpress ÄKTAmicro ÄKTAprime plus ÄKTApurifier

102 18-1022-18 AK
Appendix 5
Converting from linear flow (cm/h) to
volumetric flow rates (ml/min) and vice versa
It is convenient when comparing results for columns of different sizes to express flow as linear
flow rate (cm/h). However, flow is usually measured in volumetric flow rate (ml/min). To convert
between linear flow and volumetric flow rate use one of the formulas below:

From linear flow (cm/h) to volumetric flow rate (ml/min)

Volumetric flow rate (ml/min) = Linear flow (cm/h) × column cross-sectional area (cm2)
60

= Y × π × d
2

60 4
where
Y = linear flow in cm/h
d = column inner diameter in cm
Example:
What is the volumetric flow rate in an XK 16/70 column (i.d. 1.6 cm) when the linear flow is 150 cm/h?
Y = linear flow = 150 cm/h
d = inner diameter of the column = 1.6 cm

Volumetric flow rate = 150 × p × 1.6 × 1.6 ml/min

60 × 4
= 5.03 ml/min

From volumetric flow rate (ml/min) to linear flow (cm/hour)

Linear flow (cm/h) = Volumetric flow rate (ml/min) × 60
column cross-sectional area (cm )
2

= Z × 60 × 4
π × d2
where
Z = volumetric flow rate in ml/min
d = column inner diameter in cm
Example:
What is the linear flow in a Tricorn 5/50 column (i.d. 0.5 cm) when the volumetric flow rate is
1 ml/min?
Z = Volumetric flow rate = 1 ml/min
d = column inner diameter = 0.5 cm

Linear flow = 1 × 60 × 4 cm/h

π × 0.5 × 0.5
= 305.6 cm/h

18-1022-18 AK 103
 100

90

80

70
Linear flow (cm/h)

60

50

40

30

20

10

0
0 0.5 1.0 1.5 2.0 2.5 3.0
Volumetric flow (ml/min)

Fig A5.1. Linear flow as a function of volumetric flow: Blue curve 10 mm, red curve 16 mm, and green curve
26 mm column diameter.

From ml/min to using a syringe

1 ml/min = approximately 30 drops/min on a HiTrap 1 ml column
5 ml/min = approximately 120 drops/min on a HiTrap 5 ml column

104 18-1022-18 AK
Appendix 6
Conversion data
Proteins
Protein size and amount conversion
Mass (g/mol) 1 µg protein 1 nmol protein
10 000 100 pmol; 6 × 1013 molecules 10 µg
50 000 20 pmol; 1.2 × 10 molecules
13
50 µg
100 000 10 pmol; 6.0 × 1012 molecules 100 µg
150 000 6.7 pmol; 4.0 × 10 molecules
12
150 µg

Absorbance coefficient for proteins

Protein A280 for 1 mg/ml
IgG 1.35
IgM 1.20
IgA 1.30
Protein A 0.17
Avidin 1.50
Streptavidin 3.40
Bovine Serum Albumin 0.70

Nucleic Acids
Approximate molecular weights of nucleic acids
M.W. of ssRNA = (# nucleotides × 320.5) + 159.0
M.W. of ssDNA = (# nucleotides × 303.7) + 79.0
M.W. of dsDNA = (# nucleotides × 607.4) + 157.9
Absorbance units to nucleic acid concentration conversion
1 A280 dsDNA = 50 µg/ml
1 A280 ssDNA = 37 µg/ml
1 A280 ssRNA = 40 µg/ml

Column pressures
The maximum operating back pressure refers to the pressure above which the column contents
may begin to compress.
Pressure units may be expressed in megaPascal (MPa), bar, or pounds per square inch (psi) and
can be converted as follows: 1 MPa = 10 bar = 145 psi.

18-1022-18 AK 105
Appendix 7
Amino acids table
Amino acid Three-letter code Single-letter code Structure
HOOC
CH3
Alanine Ala A
H2N

HOOC NH2
Arginine Arg R CH2CH2CH2NHC
H2N NH

HOOC
Asparagine Asn N CH2CONH2
H2N

HOOC
Aspartic Acid Asp D CH2COOH
H2N

HOOC
Cysteine Cys C CH2SH
H2N

HOOC
Glutamic Acid Glu E CH2CH2COOH
H2N

HOOC
Glutamine Gln Q CH2CH2CONH2
H2N

HOOC
Glycine Gly G H
H2N

HOOC N
Histidine His H CH2
NH
H2N

HOOC
Isoleucine Ile I CH(CH3)CH2CH3
H2N

HOOC CH3
Leucine Leu L CH2CH
H2N CH3

HOOC
Lysine Lys K CH2CH2CH2CH2NH2
H2N

HOOC
Methionine Met M CH2CH2SCH3
H2N

HOOC
Phenylalanine Phe F CH2
H2N

Proline Pro P HOOC

NH

HOOC
Serine Ser S CH2OH
H2N

HOOC

Threonine Thr T CHCH3

H2N OH
HOOC

Tryptophan Trp W CH2

H2N
NH
HOOC
Tyrosine Tyr Y CH2 OH
H2N

HOOC
Valine Val V CH(CH3)2
H2N

106 18-1022-18 AK
 Middle unit residue (-H20) Side-chain
charge at Hydrophilic Uncharged Hydrophilic
Formula Mr Formula Mr neutral pH (nonpolar) (polar) (polar)

C3H7NO2 89.1 C3H5NO 71.1 Neutral •

C6H14N4O2 174.2 C6H12N4O 156.2 Basic (+ve) •

C4H8N2O3 132.1 C4H6N2O 114.1 Neutral •

C4H7NO4 133.1 C4H5NO3 115.1 Acidic (+ve) •

C3H7NO2S 121.2 C3H5NOS 103.2 Neutral •

C5H9NO4 147.1 C5H7NO3 129.1 Acidic (+ve) •

C5H10N2O3 146.1 C5H8N2O2 128.1 Neutral •

C2H5NO2 75.1 C2H3NO 57.1 Neutral •

C6H9N3O2 155.2 C6H7N3O 137.2 Basic (+ve) •

C6H13NO2 131.2 C6H11NO 113.2 Neutral •

C6H13NO2 131.2 C6H11NO 113.2 Neutral •

C6H14N2O2 146.2 C6H12N2O 128.2 Basic (+ve) •

C5H11NO2S 149.2 C5H9NOS 131.2 Neutral •

C9H11NO2 165.2 C9H9NO 147.2 Neutral •

C5H9NO2 115.1 C5H7NO 97.1 Neutral •

C3H7NO3 105.1 C3H5NO2 87.1 Neutral •

C4H9NO3 119.1 C4H7NO2 101.1 Neutral •

C11H12N2O2 204.2 C11H10N2O 186.2 Neutral •

C9H11NO3 181.2 C9H9NO2 163.2 Neutral •

C5H11NO2 117.1 C5H9NO 99.1 Neutral •

18-1022-18 AK 107
Appendix 8
Analysis and characterization
Analytical assays are essential to follow the progress of purification. They are used to assess the
effectiveness of the purification in terms of yield, biological activity, recovery and degree of
purification. The importance of a reliable assay for the target molecule cannot be overemphasized.

Protein detection and quantification

Detection and quantification of the target protein are needed when optimizing purification
protocols. For over-expressed proteins, the high concentration in itself can be used for
detection of the target protein fraction in a chromatogram, but in such a case verification
of the identity of the protein in the final preparation is needed. Specific detection of tagged
proteins can often be accomplished by analyzing the presence of the tag by activity or
immunoassay, or simply by the spectral properties of the tag. Specific detection of the target
protein can be obtained by functional assays, immunodetection, and mass spectrometry.
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is the key method for checking purity of
proteins. The target protein band can often be identified using the apparent Mr obtained by
including standard molecular weight markers in the analysis. Subsequent verification of protein
identity should always be obtained. Optimizing purification protocols may require functional
assays to assess the intactness of the target protein. In general:
• The relative yield of tagged protein can often be determined by measuring the absorbance
at 280 nm because the purity after a single purification step is high, that is, most of the
eluted material may be considered to be the target protein. The extinction coefficient of the
target protein will be needed. A good estimation may be obtained by theoretical calculation
from the amino acid composition of the protein.
• The yield of protein may also be determined by standard chromogenic methods (e.g., Lowry,
BCA™ protein assay, Bradford, etc.).
• Immunoassays (Western blot, ELISA, immunoprecipitation, etc) can be used for quantitation
if a suitable standard curve can be produced. In this case, it is not necessary to purify the
tagged protein so long as a purified standard is available. Therefore, these techniques may
be used for quantitation during protocol development. The immunoassay technique is also
particularly suitable for screening large numbers of samples when a simple yes/no answer
is required (e.g., when testing fractions from a chromatographic run).

Purity check and protein characterization

Purity
Purity of the target protein is most often estimated by SDS-PAGE, capillary electrophoresis,
RPC or mass spectrometry. The absorbance at 280 nm gives a rough but fast and non-
destructive estimate of total protein content in crude samples. Lowry or Bradford assays are
also frequently used to determine the total protein. The Bradford assay is particularly suited
to samples where there is a high lipid content which may interfere with the Lowry assay.
Absorbance data can be calibrated to high accuracy for pure proteins.
Obtaining a single band in electrophoresis is indicative of a pure protein, although potentially
other proteins may co-migrate. At this stage, additional methods based on orthogonal
separation principles, such as chromatography in analytical scale should be used to confirm
homogeneity/purity.
108 18-1022-18 AK
Characterization
Characterization of purified components can be divided into physical, chemical and biological/
functional characterization.
Physical parameters
Physical parameters important for identification, further experimental work, and
documentation include mass, isoelectric point and UV/Vis spectral properties.
Mass is conveniently determined with mass spectrometry (MS) also for proteins, with accuracy
better than 0.1%, providing a good fingerprint. Differences in single amino acids or attached
monosaccharides are clearly revealed.
Isoelectric point is determined by analytical isoelectric focusing (IEF) with accuracy better than
0.1 pH units and may thus sometimes even reveal variations among non-charged amino acids
in the vicinity of charged ones.
The UV/Vis spectrum can sometimes be unique for a specific protein, in particular if light-
absorbing cofactors (flavins, heme groups, other metal ions etc) are present. The specific
extinction coefficient can also be calibrated, allowing very accurate concentration
determination once the protein is free from contaminants.
Chemical characterization
Chemical characterization includes total amino acid composition, complete or partial amino
acid sequence, MS fragment pattern (peptide maps) and determination of posttranslational
modifications including glycosylation. The total amino acid composition is still carried out in
specialized laboratories, while most other data can be obtained by different applications of MS.
Functional characterization
Biological/functional characterization comprises enzymatic specificity and kinetic parameters
including the action of inhibitors. Examples of important non-enzymatic properties are binding
to signal substances, toxins, lectins, and antibodies. The interaction of a component with
detecting antibodies may also be found in this category.
Enzymatic properties are determined by a multitude of specific protocols. Readily reversible
protein-ligand interactions are typically determined by electrophoretic or chromatographic
methods, whereas slow (strong) protein-ligand interactions are determine by filter binding
assays. Protein-protein interactions and many protein-ligand interactions conveniently studied
with Biacore™ assays that also allow the study of binding kinetics.

Sensitivity . Quantitative Living . Linear dynamic

limit cells range
Coomassie blue staining 20 ng +++ no 7
Negative staining 15 ng + no 3
Silver staining 200 pg ++ no 3
Fluorescent staining 400 pg ++++ no 104
Fluorescent labeling a few pg ++++++ no 104

Radioactive labelling:
X-ray film 1 pg +++ yes 20
Phosphor-imager plates 0.2 pg ++++ yes 105
Stable isotope labelling < 1 pg ++++ (with MS) yes ?

18-1022-18 AK 109
Appendix 9
Storage of biological samples
The advice given here is of a general nature and cannot be applied to every biological sample.
Always consider the properties of the specific sample and its intended use before following any
of these recommendations.

General recommendations
Add stabilizing agents when necessary. Stabilizing agents are often required for storage
of purified proteins.
Serum, culture supernatants, and ascites should be kept frozen at -20°C or -70°C, in small
aliquots.
Avoid repeated freeze/thawing or freeze drying/redissolving that may reduce biological
activity.
Avoid conditions close to stability limits, in terms of for example pH or salt
concentrations, reducing or chelating agents.
Keep refrigerated at 4°C in a closed vessel to minimize bacterial growth and protease
activity. For prolonged storage at 4°C (more than 24 h), add a preserving agent (e.g.,
merthiolate 0.01%).
Sodium azide can interfere with coupling methods, and some biological assays, and can
be a health hazard. It can be removed by using a desalting column (see Chapter 5).

Common storage conditions for purified proteins

Store as a precipitate in a high concentration of ammonium sulfate, for example 4.0 M.
Freeze in 50% glycerol, especially suitable for enzymes.
Avoid using preserving agents if the product is to be used for a biological assay.
Preserving agents should not be added if in vivo experiments are to be performed. Store
samples in small aliquots and keep frozen.
Sterile filter to prolong storage time.
Add stabilizing agents, for example, glycerol (5% to 20%), serum albumin (10 mg/ml),
ligand (concentration is selected based on concentration of active protein) to maintain
biological activity. Remember that any additive will reduce the purity of the protein and
may need to be removed at a later stage.
Avoid repeated freeze/thawing or freeze drying/redissolving that may reduce biological
activity.
Certain proteins, including some mouse antibodies of the IgG3 subclass, should not
be stored at 4°C as they precipitate at this temperature (cryoproteins). Store at room
temperature in the presence of a preserving agent

110 18-1022-18 AK
Appendix 10
Molecular weight estimation and molecular
weight distribution analysis
Unlike electrophoretic techniques, gel filtration provides a means of determining the molecular
weight or size (Stokes radius) of native or denatured proteins under a wide variety of conditions
of pH, ionic strength and temperature, free from the constraints imposed by the charge state of
the molecules. In order to understand and follow the procedures outlined, it is important to have
read Chapter 7, Gel filtration in theory.
For molecular weight determination, several theoretical models have been proposed to describe
the behavior of solutes during gel filtration. Most models assume that the partition of solute molecules
between the particles and surrounding liquid is an entirely steric effect. However, in practice a
homologous series of compounds demonstrate a sigmodial relationship between their various
elution volume parameters and the logarithm of their molecular weights. Thus molecular weight
determination by gel filtration can be made by comparing an elution volume parameter, such as
Kav of the substance of interest, with the values obtained for several known calibration standards.
A calibration curve is prepared by measuring the elution volumes of several standards, calculating
their corresponding Kav values (or similar parameter), and plotting their Kav values versus the
logarithm of their molecular weight. The molecular weight of an unknown substance can be
determined from the calibration curve once its Kav value is calculated from its measured elution
volume. Various elution parameters, such as Ve, Ve/Vo, Kd, and Kav have been used in the literature
for the preparation of calibration curves but the use of Kav is recommended since: 1) it is less
sensitive to errors which may be introduced as a result of variations in column preparation and
column dimensions, 2) it does not require the unreliable determination of the internal volume (Vi)
as is required with Kd.
For good estimation of molecular weight, the calibration standards must have the same relationship
between molecular weight and molecular size as the substance of interest. Calibration Kits from
GE Healthcare provide well-characterized, globular protein standards for protein molecular weight
estimation. The Low Molecular Weight (LMW) Gel Filtration Calibration Kit contains five individually
lyophilized proteins with molecular weights in the range 6500 to 75 000 and Blue Dextran 2000
(see Table A10.1). The High Molecular Weight (HMW) Gel Filtration Calibration Kit contains five
individually lyophilized proteins with molecular weights in the range 43 000 to 669 000 and Blue
Dextran 2000 (see Table A10.2). Blue Dextran 2000 determines the void fraction in the column.
These well-defined protein standards show excellent behavior in gel filtration and enable simple,
reliable calibration of gel filtration columns.

Table A10.1. Characteristics of Gel Filtration Calibration Kit LMW

Protein (weight per vial) Molecular weight (Mr) Source

Aprotinin (10 mg) 6500 Bovine lung
Ribonuclease A (50 mg) 13 700 Bovine pancreas
Carbonic anhydrase (15 mg) 29 000 Bovine erythrocytes
Ovalbumin (50 mg) 43 000 Hen egg
Conalbumin (50 mg) 75 000 Chicken egg white
Blue dextran 2000 (50 mg) 2 000 000

18-1022-18 AK 111
Table A10.2. Characteristics of Gel Filtration Calibration HMW

Protein (weight per vial) Molecular weight (Mr) Source

Ovalbumin (50 mg) 43 000 Hen egg
Conalbumin (50 mg) 75 000 Chicken egg white
Aldolase (50 mg)
1
158 000 Rabbit muscle
Ferritin1 (15 mg) 440 000 Horse spleen
Thyroglobulin (50 mg) 669 000 Bovine thyroid
Blue dextran 2000 (50 mg) 2 000 000
1
These proteins are supplied mixed with sucrose or mannitol to maintain stability and aid their solubility.

Typical calibration results from chromatographic runs and calculated calibration curves using
prepacked Superdex columns are shown in Figures A10.1 and A10.2.

The method used for Figures A10.1 and A10.2:

Sample: Proteins from Gel Filtration Calibration Kits LMW and HMW:
aprotinin (Apr), RNase A (R), carbonic anhydrase (CA), ovalbumin (O), conalbumin (C),
aldolase (Ald), ferritin (F) and thyroglobulin (T)
Sample vol.: Figures A10.1 and A10.2 100 μl
Buffer: 50 mM phosphate buffer, 150 mM NaCl, pH 7.2
Flow rate: Figure A10.1 0.5 ml/min
Figure A10.2 0.6 ml/min
Detection: 280 nm

mAU Kav
1.00

0.90
O CA
400 R 0.80

Ald C 0.70
Aprotinin

RNase A
0.60
300 F Carb. anh
0.50
Apr Ovalbumin
0.40
Conalbumin
200 0.30
Aldolase

0.20
Ferritin
0.10
100
0.00
3 4 5 6
10 10 10 10
Mr logarithmic scale

0
0 5 10 15 20 25 ml

Fig A10.1. Chromatographic separation and calibration curve for the standard proteins on Superdex 200 10/300 GL column.

Kav
mAU 1.00

0.90
O 0.80
400
CA 0.70
C R
0.60
300
0.50 Aprotinin
Apr
0.40 RNase A

200 0.30 Carb. anh

0.20 Ovalbumin
Conalbumin
0.10
100
0.00
3 4 5 6
10 10 10 10
Mr logarithmic scale

0
0 5 10 15 20 25 ml
Fig A10.2. Chromatographic separation and calibration curve for the standard proteins on Superdex 75 10/300 GL column.

112 18-1022-18 AK
Many of the parameters important for a successful molecular weight determination are the
same as for any high resolution fractionation:
Use a medium with the correct fractionation range for the molecules of interest. The
expected molecular weight values should fall in the linear part of the selectivity curve
(see gel filtration media fractionation guide Chapter 1 page 16).
Use a prepacked column whenever possible. Homemade columns must be packed very
carefully (see Appendix 1).
Use freshly prepared calibration standards, selected so that the expected molecular
weight values are covered by the entire calibration range. Always filter Blue Dextran
before use. Apply samples in a volume less than 2% of the total column volume.
Use the same buffer for the separation of calibrants and sample, for example 50
mM sodium phosphate, 0.15 M NaCl at pH 7. Use the recommended flow rate for the
prepacked column or medium selected.
If the molecular weight is unknown, use a medium with a wide fractionation range such
as Sephacryl HR. This is also recommended for molecular weight distribution analysis
and for polymeric materials such as dextrans and polyethylene glycols.
Performing a molecular weight determination in the presence of urea, guanidine
hydrochloride or SDS transforms polypeptides and proteins to a random coil
configuration and so reduces structural differences. Differences will be seen in the
resulting molecular weight values when compared to values acquired under non-
denaturing conditions.
Deviation from a Kav:log Mr calibration curve may occur if the molecule of interest does not have
the same molecular shape as the standards.

Performing a molecular weight determination

1. If using a self-packed column, prepare a fresh, filtered solution of Blue Dextran 2000 (1.0 mg/ml)
in the running buffer. Apply Blue Dextran to the column, using a volume <2% of the total
column volume (Vt) to determine the void volume (Vo), and to check the column packing.
2. Dissolve the selected calibration references in the running buffer (at concentrations
recommended by the manufacturer). Allow a few minutes for dissolution, stirring gently.
Do not heat or mix vigorously. If necessary, filter the calibration solution.
3. Apply the calibration solution to the column, in a volume <2% of the total column volume (Vt).
4. Determine the elution volumes (Ve) for the standards by measuring the volume of the
eluent from the point of application to the centre of the elution peak.
5. Calculate the Kav values for the standards and prepare a calibration curve of Kav versus
the logarithm of their molecular weights, as follows:
Ve – Vo
Kav =
Vt – Vo

where Ve = elution volume for the protein

Vo = column void volume = elution volume for Blue Dextran 2000
Vt = total bed volume
Use a computer to plot the Kav value for each protein standard against the corresponding
logarithmic molecular weight and use Excel to calculate the regression line.

18-1022-18 AK 113
 6. Apply the sample in a volume <2% of the total column volume (Vt) and determine the
elution volume (Ve) of the molecule of interest.
7. Calculate the corresponding Kav for the component of interest and determine its
molecular weight from the calibration curve.

A calibrated column can be used for extended periods as long as the column is kept in
good condition and not allowed to dry out, eliminating the need to set up a separate
experiment for each determination.

114 18-1022-18 AK
Related literature
Purification Code no.
Antibody Purification Handbook 18-1037-46
Strategies for Protein Purification 28-9833-31
Recombinant Protein Handbook, Principles and Methods 18-1142-75
Purifying Challenging Proteins, Principles and Methods 28-9095-31
Affinity Chromatography Handbook: Principles and Methods 18-1022-29
Ion Exchange Chromatography Handbook: Principles and Methods 18-1114-21
Hydrophobic Interaction and Reversed Phase Chromatography 11-0012-69
Handbook, Principles and Methods
Sample preparation for analysis of proteins, peptides and carbohydrates, Selection guide 18-1128-62
Gel Filtration columns and media, Selection guide and product profile 18-1124-19
Solutions for antibody purification, Selection Guide 28-9351-97
Ion Exchange columns and media, Selection Guide 18-1127-31
Affinity chromatography columns and media, Selection Guide 18-1121-86
HiTrap-convenient protein purification, Column Guide 18-1129-81
ÄKTA Protein purification by design 28-4026-97
Prepacked chromatography columns for ÄKTA design systems, Selection guide 28-9317-78
Column Packing - The Movie, CD 18-1165-33
Pure simplicity for tagged proteins, Brochure 28-9353-64
Years of experience in every column, Brochure 28-9090-94

Protein Analysis
www.gelifesciences.com/proteinanalysis_techsupport

18-1022-18 AK 115
Ordering information
High-resolution chromatography

Column Quantity Code no.

High-resolution fractionation
Superdex
Superdex Peptide PC 3.2/30 1 × 2.4 ml column 17-1458-01
Superdex 75 PC 3.2/30 1 × 2.4 ml column 17-0771-01
Superdex 200 PC 3.2/30 1 × 2.4 ml column 17-1089-01
Superdex Peptide 10/300 GL 1 × 24 ml column 17-5176-01
Superdex 75 10/300 GL 1 × 24 ml column 17-5174-01
Superdex 200 10/300 GL 1 × 24 ml column 17-5175-01
Superdex 200 5/150 GL 1 × 3 ml column 28-9065-61
Superdex 75 5/150 GL 1 × 3 ml column 28-9205-04
HiLoad 16/60 Superdex 30 prep grade 1 × 120 ml column 17-1139-01
HiLoad 26/60 Superdex 30 prep grade 1 × 320 ml column 17-1140-01
HiLoad 16/60 Superdex 75 prep grade 1 × 120 ml column 17-1068-01
HiLoad 26/60 Superdex 75 prep grade 1 × 320 ml column 17-1070-01
HiLoad 16/60 Superdex 200 prep grade 1 × 120 ml column 17-1069-01
HiLoad 26/60 Superdex 200 prep grade 1 × 320 ml column 17-1071-01
Superdex 30 prep grade 150 ml 17-0905-01
Superdex 75 prep grade 150 ml 17-1044-01
Superdex 200 prep grade 150 ml 17-1043-01

Superose
Superose 6 PC 3.2/30 1 × 2.4 ml column 17-0673-01
Superose 12 PC 3.2/30 1 × 2.4 ml column 17-0674-01
Superose 6 10/300 GL 1 × 24 ml column 17-5172-01
Superose 12 10/300 GL 1 × 24 ml column 17-5173-01
Superose 6 prep grade 125 ml 17-0489-01
Superose 12 prep grade 125 ml 17-0536-01

Sephacryl
HiPrep 16/60 Sephacryl S-100 HR 1 × 120 ml column 17-1165-01
HiPrep 26/60 Sephacryl S-100 HR 1 × 320 ml column 17-1194-01
HiPrep 16/60 Sephacryl S-200 HR 1 × 120 ml column 17-1166-01
HiPrep 26/60 Sephacryl S-200 HR 1 × 320 ml column 17-1195-01
HiPrep 16/60 Sephacryl S-300 HR 1 × 120 ml column 17-1167-01
HiPrep 26/60 Sephacryl S-300 HR 1 × 320 ml column 17-1196-01
HiPrep 16/60 Sephacryl S-400 HR 1 × 120 ml column 28-9356-04
HiPrep 26/60 Sephacryl S-400 HR 1 × 320 ml column 28-9356-05
HiPrep 16/60 Sephacryl S-500 HR 1 × 120 ml column 28-9356-06
HiPrep 26/60 Sephacryl S-500 HR 1 × 320 ml column 28-9356-07
Sephacryl S-100 HR 150 ml 17-0612-10
Sephacryl S-100 HR 750 ml 17-0612-01
Sephacryl S-200 HR 150 ml 17-0584-10

116 18-1022-18 AK
Column Quantity Code no.
Sephacryl S-200 HR 750 ml 17-0584-01
Sephacryl S-300 HR 150 ml 17-0599-10
Sephacryl S-300 HR 750 ml 17-0599-01
Sephacryl S-400 HR 150 ml 17-0609-10
Sephacryl S-400 HR 750 ml 17-0609-01
Sephacryl S-500 HR 150 ml 17-0613-10
Sephacryl S-500 HR 750 ml 17-0613-01
Sephacryl S-1000 SF 750 ml 17-0476-01

Desalting and group separations

HiTrap Desalting 5 × 5 ml columns 17-1408-01
HiPrep 26/10 Desalting 1 × 53 ml column 17-5087-01
HiPrep 26/10 Desalting 4 × 53 ml column 17-5087-02
PD-10 Desalting Column 30 gravity-fed columns 17-0851-01
Empty PD-10 Desalting Column 50 gravity-fed empty columns 17-0435-01
PD Spin Trap G-25 50 columns 28-9180-04
PD MultiTrap G-25 4 × 96-well plates 28-9180-06
PD MiniTrap G-25 50 columns 28-9180-07
PD MidiTrap G-25 50 columns 28-9180-08
PD MiniTrap G-10 50 columns 28-9180-10
MiniSpin Adapter 10 28-9232-43
MidiSpin Adapter 10 28-9232-44
PD-10 Spin Adapter 10 28-9232-45
Collection plate 500 µl V-bottom 5 × 96-well plates 28-4039-43
LabMate PD-10 Buffer Reservoir 10 18-3216-03
Sephadex G-10 100 g 17-0010-01
Sephadex G-10 500 g 17-0010-02
Sephadex G-25 Coarse 100 g 17-0034-01
Sephadex G-25 Coarse 500 g 17-0034-02
Sephadex G-25 Fine 100 g 17-0032-01
Sephadex G-25 Fine 500 g 17-0032-02
Sephadex G-25 Medium 100 g 17-0033-01
Sephadex G-25 Medium 500 g 17-0033-02
Sephadex G-25 Superfine 100 g 17-0031-01
Sephadex G-50 Fine 100 g 17-0042-01
Sephadex G-50 Fine 500 g 17-0042-02

Separation in organic solvents

Sephadex LH-20 25 g 17-0090-10
Sephadex LH-20 100 g 17-0090-01
Sephadex LH-20 500 g 17-0090-02

Calibration Kits
Gel Filtration Calibration Kit LMW 1 28-4038-41
Gel Filtration Calibration Kit HMW 1 28-4038-42

18-1022-18 AK 117
Column Quantity Code no.

Empty columns
XK columns
XK 16/20 column 1 18-8773-01
XK 16/40 column 1 18-8774-01
XK 16/70 column 1 18-8775-01
XK 16/100 column 1 18-8776-01
XK 26/20 column 1 18-1000-72
XK 26/40 column 1 18-8768-01
XK 26/70 column 1 18-8769-01
XK 26/100 column 1 18-8770-01
XK 50/20 column 1 18-1000-71
XK 50/30 column 1 18-8751-01
XK 50/60 column 1 18-8752-01
XK 50/100 column 1 18-8753-01

Tricorn columns
Tricorn 10/100 1 28-4065-15
Tricorn 10/150 1 28-4064-16
Tricorn 10/200 1 28-4064-17
Tricorn 10/300 1 28-4064-18
Tricorn 10/600 1 28-4064-19

HiScale columns
HiScale 16/20 1 28-9644-41
HiScale 16/40 1 28-9644-24
HiScale 26/20 1 28-9645-14
HiScale 26/40 1 28-9645-13
HiScale 50/20 1 28-9644-45
HiScale 50/40 1 28-9644-44

Solvent resistant columns

SR 25/45 column 1 19-0879-01
SR 25/100 column 1 19-0880-01

Accessories and spare parts

Packing Connector XK 16 1 18-1153-44
Packing Connector XK 26 1 18-1153-45
Packing equipment 10/100 (Tricorn) 1 18-1153-25
Packing Connector 10-10 1 18-1153-23

More details and products can be found on www.gelifesciences.com/protein-purification

118 18-1022-18 AK
18-1022-18 AK 119
120 18-1022-18 AK
 GE Healthcare

Gel filtration – Principles and Methods

GE, imagination at work, and GE monogram are trademarks of General Electric Company.
ÄKTA, ÄKTAexplorer, ÄKTAmicro, ÄKTAprime, ÄKTApurifier, ÄKTAxpress, Biacore, BioProcess,
HiLoad, HiPrep, HiScale, HiTrap, MabSelect, MidiTrap, MiniTrap, MultiTrap, Sephacryl,
Sephadex, Sepharose, SpinTrap, Superdex, Superose, and Tricorn are trademarks of
GE Healthcare companies.
US patent numbers 5,284,933 and 5,310,663, and equivalent patents and patent
applications in other countries (assignee: Hoffman La Roche, Inc) relate to the purification
and preparation of fusion proteins and affinity peptides comprising at least two adjacent
histidine residues (commonly known as the histidine-tag technology).
Any customer that wishes to use Chelating Sepharose Fast Flow, Ni Sepharose 6 Fast Flow
or IMAC Sepharose 6 Fast Flow for non-research/commercial applications under these
patents is requested to contact Hoffman-La Roche AG, Corporate licensing, attention
Dr Andreas Maurer, CH-4070 Basel, Switzerland, telephone +41 61 687 2548, fax +41 61
687 2113, for the purpose of obtaining a license.
All third party trademarks are the property of their respective owners.
© 2010 General Electric Company—All rights reserved.
First published Dec. 2000.

Gel filtration
All goods and services are sold subject to the terms and conditions of sale of the company
within GE Healthcare which supplies them. A copy of these terms and conditions is available
on request. Contact your local GE Healthcare representative for the most current information.
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please visit www.gelifesciences.com/contact UK
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