GE HEALTHCARE HiPrep 16/10 CM FF User manual

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HiPrep 16/10 CM FF
HiPrep 16/10 DEAE FF
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Instructions 28-4026-56 AB
GE Healthcare
HiPre
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Instructions
HiPrep™16/10 CM FF and HiPrep 16/10 DEAE FF are prepacked, ready to use columns
for ion exchange chromatography. They provide fast, preparative separations of
proteins and other biomolecules. See below for column characteristics.
Column data
Matrix 6% highly cross-linked spherical agarose
Mean particle size 90 µm
Bed volume 20 ml
Bed height 100 mm
i.d. 16 mm
Column composition Polypropylene
Recommended flow rate12–10 ml/min (60–300 cm/h)
Maximum flow rate110 ml/min (300 cm/h)
Maximum pressure over the 0.15 MPa, 1.5 bar, 22 psi
packed bed during operation, ∆p3
HiPrep column hardware 0.5 MPa, 5 bar, 73 psi
pressure limit3
Storage +4 ºC to +30 ºC in 20 % ethanol
CM DEAE
Type of exchanger weak cation weak anion
Charged group -0-CH2COO- -N+(C2H5)2H
pH stability
short term 2–14 1–14
working 6–10 2–9
long term 4–13 2–13
Total ionic capacity 0.09–0.13 0.11–0.16
mmol H+/ml medium mmol Cl-/ml medium
Dynamic binding capacity
(mg/ml medium)2
HSA (Mr68 000) N.D 110
Ribonuclease A (Mr13 700) 50 N.D
1 Water at room temperature. Flow rate is determined by v • η ≤ 10 ml/min where v=flow rate and
η=viscosity.
2 Determination of dynamic binding capacity: Samples were applied at 75 cm/h until 50 %
breakthrough. Columns: 0.5 x 5 cm. Buffers: 0.05 M Tris, (+2 M NaCl in the elution buffer), pH 7.5
(DEAE), 0.1 M acetate, (+2 M NaCl in the elution buffer), pH 5.0 (CM).
3 Many chromatography systems are equipped with pressure gauges to measure the pressure at
a particular point in the system, usually just after the pumps. The pressure measured here is the
sum of the pre-column pressure, the pressure drop over the medium bed, and the post-column
pressure. It is always higher than the pressure drop over the bed alone. We recommend keeping
the pressure drop over the bed below 1.5 bar. Setting the upper limit of your pressure gauge to
1.5 bar will ensure the pump shuts down before the medium is overpressured.
If necessary, post-column pressure of up to 3.5 bar can be added to the limit without exceeding
the column hardware limit. To determine post-column pressure, proceed as follows:
To avoid breaking the column, the post-column pressure must never exceed 3.5 bar.
1. Connect a piece of tubing in place of the column.
2. Run the pump at the maximum flow you intend to use for chromatography. Use a
buffer with the same viscosity as you intend to use for chromatography. Note the
backpressure as total pressure.
3. Disconnect the tubing and run at the same flow rate used in step 2.
Note this backpressure as pre-column pressure.
4. Calculate the post-column pressure as total pressure minus pre-column pressure.
If the post-column pressure is higher than 3.5 bar, take steps to reduce it (shorten
tubing, clear clogged tubing, or change flow restrictors) and perform steps 1–4 again
until the post-column pressure is below 3.5 bar. When the post-column pressure
is satisfactory, add the post-column pressure to 1.5 bar and set this as the upper
pressure limit on the chromatography system.
First time use
Ensure an appropriate pressure limit has been set. Equilibrate the column for first time
use or after long-term storage by running:
1. 100 ml start buffer (low ionic strength) at 5 ml/min at room temperature (see
section “Choice of buffer” for buffer recommendations).
2. 100 ml elution buffer at 5 ml/min at room temperature.
3. 100 ml start buffer at 5 ml/min at room temperature.
These HiPrep columns can be used directly in ÄKTAdesign™systems without the need
for any extra connectors.
Try these conditions rst
Flow rate: 5 ml/min at room temperature
Start buffer: See section “Choice of buffer”
Elution buffer: Start buffer + 1 M NaCl
Gradient: 0–100% elution buffer in 200 ml (10 CV)
Equilibration before a new run
Proceed according to steps 2 and 3 in the section “First time use”. Extended
equilibration may be needed if detergents were included in the eluent.
Please read the back of these instructions for information onoptimizing a separation.
Buers and solvent resistance
De-gas and filter all solutions through a 0.45 µm filter toincrease column life-time.
Daily use
All commonly used aqueous buffers (see “Column data” for recommended pH)
Guanidine hydrochloride, up to 6 M
Urea, up to 8 M
Cleaning
Sodium hydroxide, up to 1 M
Ethanol, up to 70 %
Acetic acid, up to 1 M
Isopropanol, up to 30 %
Avoid
Oxidizing agents
Cationic detergents and buffers (CM)
Anionic detergents and buffers (DEAE)
Phenol
Sample recommendations
Net charge of protein: Positive (CM), negative (DEAE)
Recommended Not more than 10–20 % of the dynamic capacity
sample load: (see section“Column data”).
Preparation: Dissolve the sample in start buffer, filter
through 0.45 µm or centrifuge at
10 000 × g for 10 min
Delivery/storage
The column is supplied in 20% ethanol. If the
column is to be stored for more than two days
after use, clean the column according to the
procedure described under “Cleaning-in-place
(CIP)”. Then equilibrate with at least 100 ml of
20 % ethanol at a flow rate of 5 ml/min at room
temperature.
DO NOT OPEN THE COLUMN!
Instructions
HiPrep 16/10 CM FF or
HiPrep 16/10 DEAE FF
Connectors
2 × Union M6 female/
1/16" male
Bed length 100 mm, i.d. 16 mm
Choice of buer
To avoid local disturbances in pH caused by buffering ions participating 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 the isoelectric point for anion
exchangersor at least 1 pH unit below the isoelectric point for cation exchangers.
Figure 1 and Figure 2 list a selection of standard aqueous buffers.
Table 1 lists suggested volatile buffers used in cases where the purified substance has
to be freeze-dried.

imagination at work
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GE Healthcare Europe GmbH
Munzinger Strasse 5, D-79111 Freiburg, Germany
GE Healthcare UK Ltd
Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK
GE Healthcare Bio-Sciences Corp
800 Centennial Avenue, P.O. Box 1327, Piscataway, NJ 08855-1327, USA
GE Healthcare Bio-Sciences KK
Sanken Bldg, 3-25-1, Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan
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HiPrep, HiTrap, ÄKTAdesign, FPLC, and Drop Design are trademarks of GE Healthcare Ltd.
GE tagline and GE Monogram are trademarks of General Electric Company.
All goods and services are sold subject to the terms and conditions of sale of the company within
GE Healthcare which supplies them. General Electric Company reserves the right, subject to any
regulatory and contractual approval, if required, to make changes in specications and features shown
herein, or discontinue the product described at any time without notice or obligation.
© 2006 General Electric Company – All rights reserved.
GE Healthcare Bio-Sciences AB, a General Electric Company.
www.gehealthcare.com/protein-purication
www.gehealthcare.com
GE Healthcare Bio-Sciences AB
Björkgatan 30
751 84 Uppsala
Sweden
28-4026-56 AB 08/2006
Elanders Östervåla 2006 12345
Elanders Östervåla 2006 12345
Elanders Östervåla 2006
Elanders Östervåla 2006
Elanders Östervåla 2006 12345
Elanders Östervåla 2006 12345
Elanders Östervåla 2006
Elanders Östervåla 2006
Table 1. Volatile buffer systems.
pH Substances
2.3–3.5 Pyridine/formic acid
3.0–5.0 Trimethylamine/formic acid
4.0–6.0 Trimethylamine/acetic acid
6.8–8.8 Trimethylamine/HCl
7.0–8.5 Ammonia/formic acid
8.5–10.0 Ammonia/acetic acid
7.0–12.0 Trimethylamine/CO2
8.0–9.5 Ammonium carbonate/ammonia
8.5–10.5 Ethanolamine/HCl
Optimization
Perform your first run according to ”Try these conditions first”. If the obtained results
are unsatisfactory, consider the following:
Action Eect
Change pH/buffer salt Selectivity change,weaker/stronger binding
(see Figs.1 and 2 for buffers)
Change salt, counter ions, Selectivity change
and/or co-ions
Smaller sample loading Improved resolution
Lower flow rate Improved resolution
Shallower gradient Improved resolution, but broader peaks and decreased
concentrationin fractions
For more information, please check www.gehealthcare.com/protein-purification
or refer tot he handbook “Ion Exchange Chromatography and Chromatofocusing,
Principles and Methods”, see ordering information.
Cleaning-in-Place (CIP)
Regular cleaning
Wash the column with 40 ml of 2 M NaCl at a flow rate of 5 ml/min at room
temperature after each run to elute material still bound to the column.
If detergents have been used, rinse the column with 100 ml distilled water followed by
40 ml of 2 M NaCl at a flow rate of 5 ml/min at room temperature.
Re-equilibrate the column with at least 100 ml start buffer at a flow rate of 5 ml/min
at room temperature, until the UV baseline and pH/conductivity values are stable.
More rigorous cleaning
Reverse the flow direction and run the following sequence of solutions at a flow rate
of 5 ml/min at room temperature.
1. 80 ml of a 2 M NaCl solution (removes ionically bound proteins from the column)
followed by 50 ml distilled water.
2. 80 ml of a 1 M NaOH solution (removes precipitated proteins, hydrophobically
bound proteins, and lipoproteins from the column) followed by 50 ml distilled
water.
3. 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
or
30 ml 0.5 % non-ionic detergent in acidic solution (e.g. 0.1 M acetic acid) followed
by 100 ml 70% ethanol (to remove the detergent) and 60 ml distilled water.
After cleaning, equilibrate the column before use withapproximately 100 ml start
buffer at a flow rate of 5 ml/minat room temperature in the normal flow direction.
DO NOT OPEN THE COLUMN!
Trouble shooting
Symptom Remedy
Increased backpressure Reverse the flow direction and pump 100 ml elution buffer at a flow
over the column rate of 5 ml/min through the column. Return to normal flow
direction and run 100 ml buffer at a flow rate of 5 ml/min through
the column. If back-pressure is not decreased reverse the flow
direction again and follow the rigorous cleaning instructions.
Loss of resolution and/or Clean the column according to the procedure described in the
decreased sample recovery section “More rigorous cleaning”.
Air in the column Reverse the flow direction and pump 100 ml of well de-gassed start
buffer through the column at a flow rate of 5 ml/min.
Ordering information
Product No. per pack Code No.
HiPrep 16/10 CM FF 1 (20 ml) 17-5091-01
HiPrep 16/10 DEAE FF 1 (20 ml) 17-5090-01
Companion products No. per pack Code No.
HiPrep 26/10 Desalting 1 (53 ml) 17-5087-01
HiPrep 26/10 Desalting 4 (53 ml) 17-5087-02
HiTrap™IEX Selection Kit* 7 × 1 ml 17-6002-33
HiTrap DEAE FF 5 × 1 ml 17-5055-01
HiTrap DEAE FF 5 × 5 ml 17-5154-01
HiTrap CM FF 5 × 1 ml 17-5056-01
HiTrap CM FF 5 × 5 ml 17-5155-01
* Contains 7 different ion exchange media
Accessories No. per pack Code No.
HiTrap/HiPrep 1/16" male connector to ÄKTAdesign 8 28-4010-81
To connect columns with 1/16" connections to FPLC™System:
Union M6 female/1/16" male* 5 18-3858-01
Literature No. per pack Code No.
Handbook, Ion Exchange Chromatography and
Chromatofocusing, Principles & Methods 1 11-0004-21
Ion Exchange Chromatography,
Media and Column Guide 1 18-1127-31
* Two units (in red polypropylene) are included in the HiPrep package.
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Figure 1. Recommended buffer substances for anion exchange chromatography.
Figure 2. Recommended buffer substances for cation exchange chromatography.
Column performance control
We recommend checking the column performance at regular intervals. Figure 3
describes how to check the function of HiPrep 16/10 CM FF and HiPrep 16/10 DEAE FF.
Sample:
1. Ribonuclease A 1.5 mg/ml
2. Cytochrome C 0.4 mg/ml
3. Lysozyme 0.4 mg/ml
Sample volume: 100 µl
Gradient: 0–100% elution
buffer in 400 ml
(20 CV)
Start buffer: 20 mM Sodium
phosphate, pH 6.8
Elution buffer: 20 mM Sodium
phosphate,
0.5 M NaCl, pH 6.8
Flow rate: 4.0 ml/min, room
temperature
a) HiPrep 16/10 CM FF
Sample:
1. α-amylase 2.0 mg/ml
2. α-Lactalbumin 1.0 mg/ml
3. Trypsin inhibitor 3.0 mg/ml
Sample volume: 100 µl
Gradient: 0–100% elution
buffer in 400 ml
(20 CV)
Start buffer: 20 mM Tris,
pH 7.5
Elution buffer: 20 mM Tris,
1.0 M NaCl,
pH 7.5
Flow rate: 4.0 ml/min, room
temperature
b) HiPrep 16/10 DEAE FF
Figure 3. Typical chromatogram from a function test of (a) HiPrep 16/10 CM FF and
(b) HiPrep 16/10 DEAE FF.
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