DEAE Sephadex A-25

DEAE Sephadex A-50

QAE Sephadex A-25
QAE Sephadex A-50

CM Sephadex C-25
CM Sephadex C-50
SP Sephadex C-25
SP Sephadex C-50
Ion exchange resins

Instructions for Use

cytiva.com 71710400 AG
Table of Contents
1 Introduction ............................................................................................. 3
2 BioProcess resins ................................................................................... 3
3 Resin Characteristics ........................................................................... 4
4 Operation .................................................................................................. 7
5 Maintenance ............................................................................................ 13
6 Ordering information ........................................................................... 16

Read these instructions carefully before using the products.

Safety
For use and handling of the products in a safe way, refer to the
Safety Data Sheets.

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1 Introduction
DEAE Sephadex™ A-25 and DEAE Sephadex A-50 are weak
anion exchangers. The ion exchange group is
diethylaminoethyl which remains charged and maintains
consistently high capacity below pH 9.
QAE Sephadex A-25, and QAE Sephadex A-50 are strong anion
exchangers. The ion exchange group is diethyl-(2-hydroxy-
propy)aminoethyl which remains charged and maintains
consistently high capacity over the entire pH range.
CM Sephadex C-25 and CM Sephadex C-50 are weak cation
exchangers. The ion exchange group is a carboxy methyl
group which remains charged and maintains consistently high
capacity above pH 6.
SP Sephadex C-25 and SP Sephadex C-50 are strong cation
exchangers. The ion exchange group is a sulphopropyl group
which remains charged and maintains consistently high
capacity over the entire pH range.

2 BioProcess resins
BioProcess™ chromatography resins are developed and
supported for production-scale chromatography. BioProcess
resins are produced with validated methods and are tested to
meet manufacturing requirements. Secure ordering and
delivery routines give a reliable supply of resins for
production-scale. Regulatory Support Files (RSF) are available
to assist process validation and submissions to regulatory
authorities. BioProcess resins cover all purification steps from
capture to polishing.

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3 Resin Characteristics
Table 1. Anion exchangers resin characteristics
DEAE DEAE QAE QAE
Sephadex Sephadex Sephadex Sephadex
A-25 A-50 A-25 A-50
Matrix Cross-linked dextran, spherical
Type of ion exchanger Weak Weak Strong Strong
anion anion anion anion
Ionic capacity (mmol/g dry 3.0-4.0 3.0-4.0 2.6-3.4 2.6-3.4
resin)
Available capacity1
Thyroglobulin (Mr 669 000) 1 2 1.5 1.2
HAS (Mr 68 000) 30 110 10 80
α-lactalbumin (Mr 14 300) 140 50 110 30
Particle size distribution, 40-100 40-100 40-100 40-100
dry (μm)2
Recommended operating ≥ 120 cm/h ≥ 60 cm/h ≥ 100 cm/h ≥ 60 cm/h
flow velocity3
pH stability, operational4 2-13 2-12 2-13 2-12
pH stability, CIP5 2-13 2-12 2-13 2-12
pH ligand fully charged6 Below 9 Below 9 Entire pH Entire pH
range range
Chemical stability Stable to commonly used aqueous buffers
Physical stability Negligible Volume Negligible Volume
volume changes volume changes
variation due to variation due to
due to changes in due to changes in
changed in pH or ionic changes in pH or ionic
pH or ionic strength. pH or ionic strength
strength. strength.
Autoclavability 30 min at 121°C in 0.1 M sodium chloride
1 The available binding capacity was estimated in 0.05 M Tris-HCl, pH 8.3.
2 ≥ 80% volume share within given range.
3 5 cm diameter, 10 cm bed height, at room temperature using 0.02 M Sodium chloride.

4 71710400 AG
 4 pH range where resin can be operated without significant change in function.
5 pH range where resin can be subjected to cleaning- or sanitization-in-place without
significant change in function.
6 pH range where ligand is fully charged; although the ligand is fully charged throughout the
range stated, only use the resin within the stated stability ranges.

Table 2. Cation exchangers resin characteristics

CM CM SP SP
Sephadex Sephadex Sephadex Sephadex
C-25 C-50 C-25 C-50
Matrix Cross-linked dextran, spherical
Type of ion exchanger Weak Weak Strong Strong
cation caton cation cation
Ionic capacity (mmol/g dry 4.0-5.0 4.0-5.0 2.0-2.6 2.0-2.6
resin)
Available capacity1
IgG (Mr 160 000) 1.6 7 1.1 8
Bovine COHb (Mr 69 000) 70 140 70 110
Ribonuclease (Mr 13 700) 190 120 230 100
Particle size distribution, 40-100 40-100 40-100 40-100
dry (μm)2
Recommended operating ≥ 120 cm/h ≥ 100 cm/h ≥ 100 cm/h ≥ 100 cm/h
flow velocity3
pH stability, operational4 2-13 2-12 2-13 2-12
pH stability, CIP5 2-13 2-12 2-13 2-12
pH ligand fully charged6 Above 6 Above 6 Entire pH Entire pH
range range
Chemical stability Stable to commonly used aqueous buffers
Physical stability Negligible Volume Negligible Volume
volume changes volume changes
variation due to variation due to
due to changes in due to changes in
changed in pH or ionic changes in pH or ionic
pH or ionic strength. pH or ionic strength
strength. strength.

71710400 AG 5
 CM CM SP SP
Sephadex Sephadex Sephadex Sephadex
C-25 C-50 C-25 C-50
Autoclavability 30 min at 121°C in 0.1 M sodium chloride
1 The available binding capacity was estimated in 0.1 M Acetate buffer, pH 5.0.
2 ≥ 80% volume share within given range.
3 5 cm diameter, 10 cm bed height, at room temperature using 0.02 M Sodium chloride.
4 pH range where resin can be operated without significant change in function.
5 pH range where resin can be subjected to cleaning- or sanitization-in-place without
significant change in function.
6 pH range where ligand is fully charged; although the ligand is fully charged throughout the
range stated, only use the resin within the stated stability ranges.

Sephadex is particular suitable as a basis for an ion exchange

matrix, since it is hydrophilic and shows very low non-specific
adsorption. Proteins, nucleic acids and other labile biological
molecules are not adsorbed to or denatured by the resin.
DEAE Sephadex A-50, QAE Sephadex A-50 , CM Sephadex
C-50 and
SP Sephadex C-50 are prepared from Sephadex G-50 and have
a greater porosity and higher available capacity for larger
molecules (Mr > 30 000) than DEAE Sephadex A-25, QAE
Sephadex A-25, CM Sephadex C-25 and SP Sephadex C-25,
which are prepared from Sephadex G-25.
DEAE Sephadex A-25, QAE Sephadex A-25, CM Sephadex
C-25 and SP Sephadex C-25 can have a higher available
capacity for molecules with molecular weights over 100 000
since such molecules only bind on the surface of the resin
bead. Here the higher charge density of the A-25 resins can be
advantageous.

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4 Operation
Preparing the resin
DEAE Sephadex A-25, DEAE Sephadex A-50, QAE Sephadex
A-25, QAE Sephadex A-50, CM Sephadex C-25, CM Sephadex
C-50, SP Sephadex C-25 and SP Sephadex C-50 are supplied
as dry powders.
Weigh out the required amount of dry powder and suspend it
in the binding buffer. Note that the swelling factor is
dependent on the buffer used. For a general guideline, see the
table below.
Table 3. Swelling volumes for Sephadex resins
Resin Approximate volume per 1 g dry powder in saline
buffer
DEAE Sephadex A-25 ~ 7 mL
QAE Sephadex A-25 ~ 7 mL
DEAE Sephadex A-50 ~ 20 mL
QAE Sephadex A-50 ~ 25 mL
CM Sephadex C-25 ~ 7 mL
SP Sephadex C-25 ~ 7 mL
CM Sephadex C-50 ~ 25 mL
SP Sephadex C-25 ~ 25 mL

Sephadex ion exchangers must be swollen at the pH to be

used in the experiment. Note that in ultra pure water, the
bands will swell too much, too quickly, which can cause
breakage. Complete swelling takes 1 to 2 days at room
temperature. Swelling at high temperature also serves to
degas the resin. Vigorous stirring, for example with a magnetic
stirrer, must be avoided in order not to damage the particles.
Stir the required amount of ion exchanger into an excess of
binding buffer.

71710400 AG 7
 The binding buffer must contain the same ion as that originally
present in the ion exchanger.
After the initial swelling, remove the supernatant and wash
the ion exchanger extensively on a Büchner funnel with
binding buffer.
Prepare a slurry with binding buffer in a ratio of 75% settled
resin to 25% buffer. The binding buffer must not contain
agents which significantly increase the viscosity. The column
can be equilibrated with viscous buffers at reduced flow rates
after packing is completed.
Packing the Sephadex resins

Step Action
1 Equilibrate all material to the temperature at which
the chromatography will be performed.

2 Degas the resin slurry.

3 Eliminate air from the column dead spaces by flushing

the end pieces with buffer. Make sure no air has been
trapped under the column net. Close the column outlet
with a few centimeters of buffer remaining in the
column.

4 Pour the slurry into the column in one continuous

motion. Pouring the slurry down a glass rod held
against the wall of the column will minimize the
introduction of air bubbles.

5 Immediately fill the remainder of the column with

buffer, mount the column top piece onto the column
and connect the column to a pump.

8 71710400 AG
 Step Action
6 Open the bottom outlet of the column and set the
pump to run at the desired flow rate. This should be at
least 133% of the flow rate to be used during
subsequent chromatographic procedures. However,
the maximum flow rate, see Table 1, on page 4 and
Table 2, on page 5, is typically employed during
packing.
Note:
If you have packed at the maximum linear flow rate, do
not exceed 75% of this in subsequent chromatographic
procedure.

7 Open the bottom outlet of the column and set the

pump to run at the desired flow rate. This should be at
least 133% of the flow rate to be used during
subsequent chromatographic procedures. However,
the maximum flow rate, see Table 1, on page 4 and
Table 2, on page 5, is typically employed during
packing.

8 After packing columns with DEAE Sephadex A-50, QAE

Sephadex A-50, CM Sephadex C-50 or SP Sephadex
C-50 we recommend layering about 0.5 cm Sephadex
G-25 Coarse, swollen in the same buffer as the ion
exchanger, onto the top of the bed to act as a bed
surface protectant. This must not be done for a column
packed with DEAE Sephadex A-25, QAE Sephadex
A-25, CM Sephadex C-25 or SP Sephadex C-25 where
an adapter will be fitted.

71710400 AG 9
Using an adapter
An adapter is less suitable for columns packed with DEAE
Sephadex A-50, QAE Sephadex A-50, CM Sephadex C-50 or SP
Sephadex C-50 because of bed volume variations, due to
changes in pH or ionic strength, during elution.
Adapters must be fitted as follows:

Step Action
1 After the resin has been packed as described above,
close the column outlet and remove the top piece from
the column. Carefully fill the rest of the column with
buffer to form an upward meniscus.

2 Insert the adapter at an angle into the column,

ensuring that no air is trapped under the net.

3 Make all tubing connections at this stage. There must

be a bubble-free liquid connection between the
column and the pump.

4 Slide the plunger slowly down the column so that the

air above the net and in the capillary tubings is
displaced by eluent. (Valves on the inlet side of the
column must be turned in all directions during this
procedure to make sure that air is removed).

5 Lock the adapter in position on the resin surface. Open

the column outlet and start the eluent flow. Pass
eluent through the column at the packing flow rate
until the resin bed is stable. Re-position the adapter on
the resin surface as necessary.

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Equilibration
Before starting a run, make sure that the resin has reached
equilibrium. This is done by pumping start buffer through the
column until the conductivity and/or pH of the effluent is the
same as that of the in-going start buffer.
The column is now equilibrated and ready for use.
Binding
The most common procedure is to let the molecules of
interest bind to the ion exchanger and allow the others to pass
through. However, in some cases it can be more useful to bind
“contaminants” and let the molecules of interest remain in the
flow through.
For adsorption, it is critical to choose a buffer with an
appropriate pH. See the tables below. The ionic strength of the
buffer must be kept low, so as not to interfere with sample
binding. The recommended operating pH is within 0.5 pH units
of the buffer’s pKa and at least one pH unit above the
isoelectric point (pI) of the molecule of interest.
Table 4. Buffers for cation exchange chromatography
pH interval Substance Conc. (mM) Counter-ion pKa (25°C)1
1.4-2.4 Malic acid 20 Na+ 1.92
2.6-3.6 Methyl malonic acid 20 Na+ or Li+ 3.07
2.6-3.6 Citric acid 20 Na+ 3.13
3.3-4.3 Lactic acid 50 Na+ 3.86
3.3-4.3 Formic acid 50 Na+ or Li+ 3.75
3.7-4.7 Succinic acid 50 Na+ 4.21
5.1-6.1 Succinic acid 50 Na+ 5.64
4.3-5.3 Acetic acid 50 Na+ or Li+ 4.75
5.2-6.2 Methyl malonic acid 50 Na+ or Li+ 5.76

71710400 AG 11
 pH interval Substance Conc. (mM) Counter-ion pKa (25°C)1
5.6-6.6 MES 50 Na+ or Li+ 6.27
6.7-7.7 Phosphate 50 Na++ 7.20
7.0-8.0 HEPES 50 Na+ or Li+ 7.56
7.8-8.8 BICINE 50 Na+ 8.33
1 Handbook of chemistry and physics, 83rd edition, CRC, 2002–2003.

pH Substance Conc. Counter-ion pKa

interval (mM) (25°C)1
4.3-5.3 N-Methylpiperazine 20 Cl- 4.75
4.8-5.8 Piperazine 20 Cl- or HCOO- 5.33
5.5-6.5 L-Histidine 20 Cl- 6.04
6.0-7.0 Bis-Tris 20 Cl- 6.48
6.2-7.2 Bis-Tris propane 20 Cl- 6.65
8.6-9.6 Bis-Tris propane 20 Cl- 9.10
7.3-8.3 Triethanolamine 20 Cl- or 7.76
CH3COO-
7.6-8.6 Tris 20 Cl- 8.07
8.0-9.0 N-Methyldiethanolamine 20 SO4 2- 8.52
8.0-9.0 N-Methyldiethanolamine 50 Cl- or 8.52
CH3COO-
8.4-9.4 Diethanolamine 20 at pH Cl- 8.88
8.4
50 at pH
8.8
8.4-9.4 Propane 1,3-diamino 20 Cl- 8.88
9.0-10.0 Ethanoliamine 20 Cl- 9.50
9.2-10.2 Piperazine 20 Cl- 9.73
10.0-11.0 Propane 1,3-diamino 20 Cl- 10.55
10.6-11.6 Piperidine 20 Cl- 11.12
1 Handbook of chemistry and physics, 83rd edition, CRC, 2002–2003.

12 71710400 AG
Elution
For DEAE Sephadex and QAE Sephadex resins, elution is
achieved using either an increasing salt gradient (continuous
or step wise) or a decreasing pH gradient (continuous or step
wise). For CM Sephadex and SP Sephadex resins, elution is
achieved using either an increasing salt gradient (continuous
or step wise) or an increasing pH gradient (continuous or step
wise).

5 Maintenance
Regeneration
Depending of the nature of the sample, regeneration is
normally performed by washing with a high ionic strength
buffer (e.g., 1 to 2 M NaCl) and/or decreasing/increasing pH,
followed by re-equilibration in binding buffer.
In some applications, substances such as denaturated
proteins or lipids do not elute in the regeneration procedure.
These can be removed by cleaning procedures, CIP (Cleaning-
in-place).
Cleaning-In-Place (CIP)
Remove ionically bound proteins by washing the column with
0.5 to1 bed volume of a 2 M NaCl solution.
Remove precipitated proteins, hydrophobically bound
proteins and lipoproteins by washing the resin with 1 bed
volume of a 0.1 M or 0.001 M NaOH (see pH stability, CIP in
Table 1, on page 4 and Table 2, on page 5) solution followed by
binding buffer until free from alkali.

71710400 AG 13
 Strongly hydrophobically bound proteins, lipoproteins and
lipids can be removed by washing the resin with up to 70%
ethanol or 30% isopropanol.
Alternatively, wash the resin with 2 bed volumes of detergent
in a basic or acidic solution. Use for example, 0.1% to 0.5%
nonionic detergent in 0.1 M acetic acid. After treatment with
detergent always remove residual detergents by washing with
5 bed volumes of 70% ethanol.
Note: Due to the relatively large volume changes of
Sephadex based resins in organic solvents, we
recommend washing with organic solvents on a
Büchner funnel, since the resin needs to be repacked
after such treatment.
After cleaning the resin, re-equilibrate the ion exchanger
according to the recommendations above.

CAUTION
70% ethanol can require the use of
explosion-proof areas and equipment.

14 71710400 AG
Storage
Dry powders of DEAE Sephadex A-25, DEAE Sephadex A-50,
QAE Sephadex A-25, QAE Sephadex A-50, CM Sephadex C-25,
CM Sephadex C-50, SP Sephadex C-25 and SP Sephadex C-50
must be stored at 4°C to 30°C.
Store swollen resin in the presence of a suitable bacteriostat,
for example 20% ethanol or 0.01 M NaOH1 at 4°C to 30°C.
Sodium azide or thiomersal must not be used as bacteriostat.

1 In most cases, no long term stability data has been

generated by Cytiva in 0.01 M NaOH. In some cases,
accelerated studies at elevated temperature indicate
that storage in 0.01 M NaOH can be a viable option but
no guarantees can be made regarding retained function
of the product.

71710400 AG 15
6 Ordering information
Product Pack size Product code
DEAE Sephadex A-25 100 g 17017001
DEAE Sephadex A-25 500 g 17017002
DEAE Sephadex A-25 5 kg 17017003
DEAE Sephadex A-50 100 g 17018001
DEAE Sephadex A-50 500 g 17018002
DEAE Sephadex A-50 5 kg 17018003
DEAE Sephadex A-50 40 kg1 17018007
QAE Sephadex A-25 500 g 17019002
QAE Sephadex A-25 5 kg 17019003
QAE Sephadex A-50 100 g 17020001
QAE Sephadex A-50 5 kg 17020003
CM Sephadex C-25 100 g 17021001
CM Sephadex C-25 500 g 17021002
CM Sephadex C-25 5 kg 17021003
CM Sephadex C-50 100 g 17022001
CM Sephadex C-50 500 g 17022002
CM Sephadex C-50 5 kg 17022003
SP Sephadex C-25 100 g 17023001
SP Sephadex C-25 500 g 17023002
SP Sephadex C-25 5 kg 17023003
SP Sephadex C-50 100 g 17024001
SP Sephadex C-50 5 kg 17024003
1 Pack sizes available upon request

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71710400 AG V:6 07/2020