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Life Sciences

Instructions 28-9413-23 AC Prepacked columns

HiPrep™ CM FF 16/10
HiPrep DEAE FF 16/10
HiPrep Q FF 16/10
HiPrep SP FF 16/10
HiPrep Q HP 16/10
HiPrep SP HP 16/10
HiPrep CM FF 16/10, HiPrep DEAE FF 16/10, HiPrep Q FF 16/10, HiPrep SP
FF 16/10, HiPrep Q HP 16/10, and HiPrep SP HP 16/10 are prepacked,
ready to use columns for ion exchange chromatography. They provide
fast preparative separations of proteins and other biomolecules. The
columns are used in an optimal way with liquid chromatography systems
such as ÄKTA™.
Table of Contents
1 Product description .................................................................................. 3
2 Optimization ................................................................................................ 8
3 Operation ...................................................................................................... 9
4 Cleaning-in-place ..................................................................................... 12
5 Adjusting pressure limits in chromatography system
software ........................................................................................................ 13
6 Storage .......................................................................................................... 15
7 Troubleshooting ......................................................................................... 16
8 Ordering information .............................................................................. 17

Please read these instructions carefully before using the HiPrep

columns.

Intended use
HiPrep columns are intended for research use only, and shall not
be used in any clinical or in vitro procedures for diagnostic
purposes.

Safety
For use and handling of the product in a safe way, please refer to
the Safety Data Sheet.

2 28-9413-23 AC
1 Product description
HiPrep column characteristics
HiPrep columns are made of biocompatible polypropylene that does
not interact with biomolecules. The columns are delivered with
stoppers at the inlet and outlet. The arrow on the column label
indicates the column orientation and the recommended flow
direction, see Figure 1.

Fig 1. HiPrep 16/10 column

Note: HiPrep columns cannot be opened or refilled

Note: Make sure that the connector is tight to prevent leakage.
Table 1. Characteristics of HiPrep 16/10 column

Column volume (CV) 20 ml

Column dimensions 16 × 100 mm
Column hardware pressure limit 1 5 bar (0.5 MPa)
1 The pressure over the packed bed varies depending on a range of parameters such as
the characteristics of the chromatography medium and the column tubing used.

28-9413-23 AC 3
Properties of IEX chromatography media
Q Sepharose Fast Flow, SP Sepharose Fast Flow, DEAE Sepharose
Fast Flow, and CM Sepharose Fast Flow are based on 6% highly
cross-linked agarose with an average bead size of 90 µm. The media
have good flow properties and high loading capacities.
Q Sepharose High Performance, and SP Sepharose High Performance
are based on rigid, highly cross-linked, 6% agarose with an average
bead size of 34 µm. The smaller bead size will result in higher resolu-
tion and sharper peaks.
The functional groups are coupled to the matrix via chemically stable
ether linkages and remain charged over the entire pH working range,
as well as maintain high capacity.
Type of Ion exchanger type Functional group
media
Q Strong anion exchanger Quaternary amine group
SP Strong cation exchanger Sulfoethyl group
DEAE Weak anion exchanger Diethylaminoethyl group
CM Weak cation exchanger Carboxymethyl group

The characteristics of the different media are listed in Tables 2, 3

and 4.

4 28-9413-23 AC
 Table 2. Characteristics of Q Sepharose FF and SP Sepharose FF
Q Sepharose FF SP Sepharose FF
Matrix Highly cross-linked 6% agarose
Average particle size 90 µm 90 µm
(d50v) 1
Ion exchange type Strong anion Strong cation
Charged group -N+(CH3)3 -CH2CH2CH2SO3-
Total ionic 0.18 to 0.25 mmol Cl-/ml 0.18 to 0.25 mmol H+/ml
capacity medium medium
Dynamic binding 120 mg HSA/ml medium 70 mg Ribonuclease A/ml
capacity 2 medium
Recommended flow 150 cm/h 150 cm/h
velocity 3
Maximum flow 300 cm/h 300 cm/h
velocity 3
pH stability 4
Working range 2 to 12 4 to 13
Cleaning-in-place 1 to 14 3 to 14
Chemical stability All commonly used aqueous buffers, 1 M NaOH,
8 M urea, 6 M guanidine hydrochloride, 70% ethanol
Avoid Oxidizing agents, and Oxidizing agents, and
anionic detergents/ cationic detergents/
buffers buffers
Storage 4°C to 30°C in 20% 4°C to 30°C in
ethanol 0.2 M sodium acetate in
20% ethanol
1 d50v is the average particle size of the cumulative volume distribution.
2 Running conditions:
Q Sepharose FF and DEAE Sepharose FF: 0.05 M Tris-HCl, pH 7.5 at 75 cm/h.
SP Sepharose FF: 0.1 M sodium acetate, pH 5.0 at 75 cm/h.
3 Water at room temperature.
For viscous buffers and samples the flow velocity must be optimized. Starting with a low
flow velocity is recommended.
4 Working range: pH interval where the medium can be handled without significant change
in function.
Cleaning-in-place: pH interval where the medium can be subjected to cleaning-in-place
without significant change in function.

28-9413-23 AC 5
 Table 3. Characteristics of DEAE Sepharose FF and CM Sepharose FF
DEAE Sepharose FF CM Sepharose FF
Matrix Highly cross-linked 6% agarose
Average particle size 90 µm 90 µm
(d50v) 1
Ion exchange type Weak anion Weak cation
Charged group -N+(C2H5)2H -O-CH2COO-
Total ionic 0.11 to 0.16 mmol Cl-/ml 0.09 to 0.13 mmol H+/ml
capacity medium medium
Dynamic binding 110 mg HSA/ml medium 50 mg Ribonuclease A/ml
capacity 2 medium
Recommended flow 150 cm/h 150 cm/h
velocity 3
Maximum flow 300 cm/h 300 cm/h
velocity 3
pH stability 4
Working range 2 to 12 4 to 13
Cleaning-in-place 1 to 14 2 to 14
Chemical stability All commonly used aqueous buffers, 1 M NaOH,
8 M urea, 6 M guanidine hydrochloride, 70% ethanol
Avoid Oxidizing agents, and Oxidizing agents, and
anionic detergents/ cationic detergents/
buffers buffers
Storage 4°C to 30°C in 20% 4°C to 30°C in 20%
ethanol ethanol
1 d50v is the average particle size of the cumulative volume distribution.
2 Running conditions:
Q Sepharose FF and DEAE Sepharose FF: 0.05 M Tris-HCl, pH 7.5 at 75 cm/h.
SP Sepharose FF: 0.1 M sodium acetate, pH 5.0 at 75 cm/h.
3 Water at room temperature.
For viscous buffers and samples the flow velocity must be optimized. Starting with a low
flow velocity is recommended.
4 Working range: pH interval where the medium can be handled without significant change
in function.
Cleaning-in-place: pH interval where the medium can be subjected to cleaning-in-place
without significant change in function.

6 28-9413-23 AC
 Table 4. Characteristics of Q Sepharose HP and SP Sepharose HP
Q Sepharose HP SP Sepharose HP
Matrix Highly cross-linked 6% agarose
Average particle size 34 µm 34 µm
(d50v) 1
Ion exchange type Strong anion Strong cation
Charged group -N+(CH3)3 -CH2CH2CH2SO3-
Total ionic 0.14 to 0.20 mmol Cl-/ml 0.15 to 0.20 mmol H+/ml
capacity medium medium
Dynamic binding 70 mg BSA/ml medium 55 mg Ribonuclease A/ml
capacity 2 medium
Recommended flow 90 cm/h 90 cm/h
velocity 3
Maximum flow 150 cm/h 150 cm/h
velocity 3
pH stability 4
Working range 2 to 12 4 to 13
Cleaning-in-place 1 to 14 3 to 14
Chemical stability All commonly used aqueous buffers, 1 M NaOH,
8 M urea, 6 M guanidine hydrochloride, 70% ethanol
Avoid Oxidizing agents, and Oxidizing agents, and
anionic detergents/ cationic detergents/
buffers buffers
Storage 4°C to 30°C in 20% 4°C to 30°C in
ethanol 0.2 M sodium acetate in
20% ethanol
1 d50v is the average particle size of the cumulative volume distribution.
2 Running conditions:
Q Sepharose HP: 10.0 mg/ml BSA in 0.02 M Tris-HCl, pH 8.2 at 150 cm/h.
SP Sepharose HP: 5 mg/ml Ribonuclease in 0.1 M sodium acetate, pH 6.0 at 150 cm/h.
3 Water at room temperature.
For viscous buffers and samples the flow velocity must be optimized. Starting with a low
flow velocity is recommended.
4 Working range: pH interval where the medium can be handled without significant change
in function.
Cleaning-in-place: pH interval where the medium can be subjected to cleaning-in-place
without significant change in function.

28-9413-23 AC 7
2 Optimization
Optimizing the process
The aim of designing and optimizing an ion exchange separation
process is to identify conditions that promote binding of the highest
amount of target molecule in the shortest possible time with highest
possible product recovery. To reduce time, sample and buffer
consumption during optimization the method should be designed
in laboratory scale.

Optimizing binding conditions

Screen for optimal binding conditions by testing a range of pH values
within which the target protein is known to be stable. If the isoelectric
point (pI) of the target protein is known, then begin with a narrower
pH range, for example, 0.5 to 1 pH unit away from pI. In some cases
the sample conductivity is equally important as the pH when
screening for optimal binding conditions.
Screening for buffer concentration at the temperature where the
process is intended to be run will give the optimal dynamic binding
capacity.

Optimizing elution conditions

Linear ionic strength gradients should always be used for method
development or when starting with an unknown sample. Linear
ionic strength gradients are easy to prepare and very reproducible
when generated by a suitable chromatography system. The results
obtained can then serve as a basis from which to optimize the
separation.
Step-wise elution allows the target protein to be eluted in a more
concentrated form, thus decreasing buffer consumption and
shortening cycle times. Due to the high concentrations of protein in
the eluted pool it might in rare cases be necessary to decrease the
flow rate and thereby avoid exceeding the maximum back pressure
for the column.

8 28-9413-23 AC
Automated buffer preparation
Users of ÄKTA chromatography systems with BufferPrep or BufferPro
functionality can select from a range of buffer recipes to conveniently
screen media over a range of pH values and elution conditions.

3 Operation
Prepare buffers
To avoid local disturbances in pH caused by buffering ions partici-
pating in the ion exchange process, select a buffer with buffering
ions of the same charge as the substituent groups on the ion
exchanger.
The start buffer pH should be chosen so that substances to be bound
to the ion exchanger are charged, that is, at least 1 pH unit above
pI for anion exchangers or at least 1 pH unit below pI for cation
exchangers.
The elution buffer is usually of the same composition and pH as the
start buffer, but it contains additional salt, most often sodium
chloride. The pH of the start buffer should be at least 0.5 to 1 pH unit
above pI of the target molecule when using an anion exchanger and
at least 0.5 to 1 pH unit below pI when using a cation exchanger.
The buffer species and buffer concentration are important for
reproducible and robust methods. The buffer concentration depends
partly on the buffer capacity at a given pH and should be at least
10 mM (only rarely above 100 mM). Where the conductivity of the
buffers needs to be considered, it can be increased by increasing
the buffer concentration or adding sodium chloride.
Try the following buffers for samples with unknown charge properties.
Anion exchange
Start buffer: 20 mM Tris-HCl, pH 8.0
Elution buffer: 20 mM Tris-HCl, 1 M NaCl, pH 8.0

28-9413-23 AC 9
 Cation exchange
Start buffer: 50 mM sodium acetate, pH 5.0
Elution buffer: 50 mM sodium acetate, 1 M NaCl, pH 5.0
or
Start buffer: 50 mM MES, pH 6.0
Elution buffer: 50 mM MES, 1 M NaCl, pH 6.0
Note: Water and chemicals used for buffer preparation should be
of high purity. Filter buffers through a 0.22 µm or a 0.45 µm
filter before use.

Prepare the sample

Step Action
1 Adjust the sample to the composition of the start buffer,
using one of these methods:
• Dilute the sample with start buffer.
• Exchange buffer using a HiPrep 26/10 Desalting, HiTrap™
Desalting or PD-10 Desalting column.
2 Filter the sample through a 0.45 µm filter or centrifuge at
10 000 × g for 10 min immediately before loading it to the
column. This prevents clogging and increases the life time
of the column when loading large sample volumes.

Recommended flow rates

The table below outlines recommended flow rates for the different
media types under different conditions. For viscous buffers and
samples the flow rate must be optimized. Starting with a low flow
rate is recommended.
Table 5. Recommended flow rates for HiPrep IEX columns.
Media type First time use or Experimental Cleaning-in-place
after long time condition (CIP)
storage in 20%
EtOH
High performance 0.8 ml/min 3 ml/min 3 ml/min
Fast flow 2.0 ml/min 5 ml/min 5 ml/min

10 28-9413-23 AC
Purification
Collect fractions throughout the separation.
Flow rate: See Table 5.
Column tubing: Choose the optimal tubing kit for the column and
the application you intend to run. (i.d.: 0.25, 0.50 or 0.75 [mm]). A
tubing with wider inner diameter gives broader peaks whereas a
tubing with a smaller inner diameter gives higher back pressure.

Step Action
1 Remove the stoppers and connect the column to the system.
Avoid introducing air into the column.
Note:
To prevent leakage, ensure that the connectors are tight. Use
fingertight 1/16" connector (28-4010-81).
2 Wash with 1 column volume (CV) distilled water. This step
removes the ethanol and avoids the precipitation of buffer
salts upon exposure to ethanol. The step can be omitted if
precipitation is not likely to be a problem.
3 Equilibrate the column with at least 5 CV start buffer or until
the UV baseline, eluent pH and conductivity are stable.
4 Adjust the sample to the chosen starting pH and conductivity
and load on the column.
5 Wash with 5 to 10 CV start buffer or until the UV trace of the
effluent returns to near baseline.
6 Elute, either by linear gradient elution or a step elution, see
below. If required, the collected eluted fractions can be buffer
exchanged or desalted.
• Linear gradient elution
Elute with 0% to 100% elution buffer (up to 1 M NaCl) in
10 to 20 CV.
• Step elution
Elute with 5 CV elution buffer including NaCl at chosen
concentration. Repeat at higher NaCl concentrations until
the target protein has been eluted.

28-9413-23 AC 11
 Step Action
7 Wash with 5 CV of 1 M NaCl (100% elution buffer) to elute
any remaining ionically bound material.
8 If required, perform a CIP to clean the column.
9 Re-equilibrate with 5 to 10 CV start buffer or until the UV
baseline, eluent pH, and conductivity reach the required
values.
To save time, higher flow rates during regeneration and
re-equilibration steps can be used.
Note: Do not exceed the maximum recommended flow and back
pressure for the column.

4 Cleaning-in-place
Regular cleaning
Wash the column with 40 ml of 2 M NaCl at room temperature after
each run to elute material still bound to the column. See Table 5 for
recommended flow rates.
If detergents have been used, wash the column with 100 ml distilled
water followed by 40 ml of 2 M NaCl at room temperature.
Re-equilibrate the column with at least 100 ml start buffer at room
temperature, until the UV baseline and pH/conductivity values are
stable.

12 28-9413-23 AC
Rigorous cleaning
Reverse the flow direction and run the following sequence of solutions
at room temperature:
• 80 ml of a 2 M NaCl solution (removes ionically bound proteins
from the column) followed by 50 ml distilled water.
• 80 ml of a 1 M NaOH solution (removes precipitated proteins,
hydrophobically bound proteins, and lipoproteins from the col-
umn) followed by 80 ml distilled water.
• 80 ml of 70% ethanol or 30% isopropanol (removes proteins,
lipoproteins, and lipids that are strongly hydrophobically bound
to the column) followed by 60 ml distilled water.
After cleaning, equilibrate the column with approximately 100 ml
start buffer at room temperature before use.

5 Adjusting pressure limits in

chromatography system software
Pressure generated by the flow through a column affects the packed
bed and the column hardware, see Figure below. Increased pressure
is generated when running/using one or a combination of the
following conditions:
• High flow rates
• Buffers or sample with high viscosity
• Low temperature
• A flow restrictor
Note: Exceeding the flow limit (see Table 2, 3 and 4) may damage
the column.

28-9413-23 AC 13
 pre-column pressure

pressure over the

packed bed

post-column pressure

Fig 2. Pre-column and post-column measurements.

ÄKTA avant
The system will automatically monitor the pressures (pre-column
pressure and pressure over the packed bed, Δp). The pre-column
pressure limit is the column hardware pressure limit (see Table 1).
The maximum pressure the packed bed can withstand depends on
media characteristics and sample/liquid viscosity. The measured
value also depends on the tubing used to connect the column to the
instrument.

ÄKTAexplorer™, ÄKTApurifier™, ÄKTAFPLC™ and other

systems with pressure sensor in the pump
To obtain optimal functionality, the pressure limit in the software
may be adjusted according to the following procedure:

Step Action
1 Replace the column with a piece of tubing. Run the pump
at the maximum intended flow rate. Note the pressure as
total system pressure, P1.
2 Disconnect the tubing and run the pump at the same flow
rate used in step 1. Note that there will be a drip from the
column valve. Note this pressure as P2.

14 28-9413-23 AC
 Step Action
3 Calculate the new pressure limit as a sum of P2 and the
column hardware pressure limit (see Table 1). Replace the
pressure limit in the software with the calculated value.
The actual pressure over the packed bed (Δp) will during run
be equal to actual measured pressure - total system pressure
(P1).
Note: Repeat the procedure each time the parameters are changed.

6 Storage
If the column is to be stored for more than two days after use, clean
the column according to the procedure described in section Cleaning-
in-Place (CIP). Then equilibrate as follows:
HiPrep CM FF 16/10, HiPrep DEAE FF 16/10, HiPrep Q FF 16/16 and
HiPrep Q HP 16/10: equilibrate with at least 100 ml of 20% ethanol.
HiPrep SP FF 16/10 and HiPrep SP HP 16/10: equilibrate with at least
100 ml of 0.2 M sodium acetate in 20% ethanol.
Store at 4°C to 30°C. Do not freeze.
Make sure that the column is tightly sealed to avoid drying out.

28-9413-23 AC 15
7 Troubleshooting
Problem Possible cause/corrective action
High back pressure The column is clogged.
during the run Reverse the flow direction and try to pump 100 ml
elution buffer through the column. Return to normal
flow direction and run 100 ml buffer through the
column at low flow rate. If back pressure is not de-
creased, reverse the flow direction again and follow
the rigorous cleaning protocol in Section Cleaning-
in-place (CIP).
High viscosity of solutions.
Use lower flow rate.
Loss of resolution Insufficient elution and CIP.
and/or decreased Follow the rigorous cleaning protocol in Section
sample recovery Cleaning-in-place (CIP). Optimize the elution condi-
tions, the CIP protocol and/or perform CIP more
frequently.
Unstable pressure Air in the column.
curve Reverse the flow direction and pump 100 ml of well
de-gassed start buffer through the column at room
temperature.

Note: See Table 5 for recommended flow rates.

16 28-9413-23 AC
8 Ordering information
Product Quantity Code No.
HiPrep CM FF 16/10 1 × 20 ml 28-9365-42
HiPrep DEAE FF 16/10 1 × 20 ml 28-9365-41
HiPrep Q FF 16/10 1 × 20 ml 28-9365-43
HiPrep SP FF 16/10 1 × 20 ml 28-9365-44
HiPrep Q HP 16/10 1 × 20 ml 29-0181-82
HiPrep SP HP 16/10 1 × 20 ml 29-0181-83

Related products Quantity Code No

HiTrap IEX Selection Kit, 7 different IEX media 7 × 1 ml 17-6002-33
HiTrap Q FF 5 × 1 ml 17-5053-01
5 × 5 ml 17-5156-01
HiTrap SP FF 5 × 1 ml 17-5054-01
5 × 5 ml 17-5157-01
HiTrap DEAE FF 5 × 1 ml 17-5055-01
5 × 5 ml 17-5154-01
HiTrap CM FF 5 × 1 ml 17-5056-01
5 × 5 ml 17-5155-01
HiTrap SP HP 5 × 1 ml 17-1151-01
5 × 5 ml 17-1152-01
HiTrap Q HP 5 × 1 ml 17-1153-01
5 × 5 ml 17-1154-01
HiPrep 26/10 Desalting 1 × 53 ml 17-5078-01
4 × 53 ml 17-5087-02

28-9413-23 AC 17
 Accessories Quantity Code No
HiTrap/HiPrep, 1/16" male connector for ÄKTA 8 28-4010-81
(For connection of columns with 1/16" fittings
to ÄKTA)

Related literature Code No.

Ion Exchange Chromatography and Chromatofocusing 11-0004-21
Handbook, Principles and Methods
Ion Exchange Columns and Media, Selection Guide 18-1127-31
Prepacked chromatography columns for ÄKTA systems, 28-9317-78
Selection Guide

18 28-9413-23 AC
For local office contact information, visit
www.gelifesciences.com/contact
GE Healthcare Bio-Sciences AB
Björkgatan 30
751 84 Uppsala
Sweden
www.gelifesciences.com/protein-purification

GE, imagination at work and GE monogram are trademarks of General Electric Company.
ÄKTA, ÄKTAexplorer, ÄKTAFPLC, ÄKTApurifier, HiPrep, HiTrap, and Sepharose are trademarks of
GE Healthcare companies.
© 2006-2012 General Electric Company – All rights reserved.
First published Jun. 2006
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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imagination at work
28-9413-23 AC 11/2012