GE HEALTHCARE HiPrep 16/10 CM FF Instructions

GE Healthcare
Instructions 28-4026-56 AB
HiPrep 16/10 CM FF
HiPrep 16/10 DEAE FF
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
1
Recommended flow rate 2–10 ml/min (60–300 cm/h)
1
Maximum flow rate 10 ml/min (300 cm/h)
Maximum pressure over the 0.15 MPa, 1.5 bar, 22 psi
3
packed bed during operation, ∆p
HiPrep column hardware 0.5 MPa, 5 bar, 73 psi
3
pressure limit
Storage +4 ºC to +30 ºC in 20 % ethanol
CM
Type of exchanger weak cation
-
Charged group -0-CH COO
2
pH stability
short term 2–14
working 6–10
long term 4–13
Total ionic capacity 0.09–0.13
+
mmol H /ml medium
Dynamic binding capacity
2
(mg/ml medium)
HSA (M 68 000) N.D
r
Ribonuclease A (M 13 700) 50
r
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
for any extra connectors.
Try these conditions first
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.
Instructions
DEAE
weak anion
+
-N (C H ) H
2 5 2
1–14
2–9
2–13
▼
0.11–0.16
-
mmol Cl /ml medium
110
N.D
™
systems without the need
Connectors
2 × Union M6 female/
HiPrep 16/10 CM FF or
1/16" male
HiPrep 16/10 DEAE FF
Buffers 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!
Choice of buffer
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.
▼
HiPrep