DEAE Sepharose CL-6B is weak ion exchange chromatography media with excellent flow
properties and high capacity for proteins of all pI values. The ion exchange group is a
diethylaminoethyl group which remains charged and maintains consistently high capacity
over the entire working range, pH2-13.
Gel characteristics
Type of
Weak anion
exchanger
Total ionic capacity
0.13-0.17m mol/ml gel
thyroglobulin(TG)MW669000 2mg/ml；HAS
Available capacity
MW68000 170mg/ml；α-lactalbumin MW 14300
150mg/ml
Bead structure
Bead size range
Mean particle size
6%highly cross-linked Sepharose
45-165um
90um
Max. flow rate
300cm/h
Max. operating
0.015Mpa(0.15bar，2psi)
pressure
pH stability
Long term:3-12 short term :2-14
All commonly used aqueous buffers,1.0M NaOH,
Chemical stability
8M Urea, 8M guanidine hydrochloride, ethanol,
propanol and so on.
Physical stability
Negligible volume variation due to changes in pH
or ionic strength.
autoclavable
At 0.1 M NaCl, 121℃ for 30min.
The available capacity is measured in a 0.5×5cm column in the linear flow rate of
300cm/h. The balance buffer is 0.05M Tris-HCl, pH 8.3 and the elution buffer contains 2M
NaCl.
All ranges given are estimates based on our knowledge and experience. Please note the
following aspects:
(1) pH stability, long term refers to the pH interval where the gel is stable over a long
period of time without adverse effects on its subsequent chromatographic performance.
(2) pH stability, short term refers to the pH interval for re-generation, cleaning-in-place and
sanitization procedures.
Preparing the gel:
DEAE Sepharose FF has been stored in 20% ethanol. Please remove the ethanol and
wash the Sepharose with distilled water to alcohol-free flavor. Remove the upper aqueous
phase when the gel is deposited. Then mix 75% Sepharose and 25% start buffer together.
The start buffer should not contain agents which significantly increase the viscosity.
Equilibration:
Make sure that the ion exchanger has been equilibrated before beginning the experiment.
Pump the start buffer through the column until the column is fully equilibrated when the pH
and/or conductivity of the effluent is the same as the start buffer.
1．Binding: The most common procedure in ion exchange is to bind the molecules of
interest, while allowing contaminants to pass through the column. In some cases,
however, it may be more useful to bind the contaminants and allow the molecules of
interest to pass.
2．Binding process: a suitable pH is very important in the binding process. For efficient
binding, the ionic strength of the starting buffer must be low to avoid interfering the binding
of the samples. The pH should be at least one pH unit different from the isoelectric point
(pI) of the molecules to be bound and within 0.5 pH units of the selected buffer salt's pKa.
Packing the column:
1．Equilibrate all material to room temperature and degas the slurry.
2．Pre-add 1/3 column volume buffer in the column, Pour the equilibrated 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.
3．Immediately fill the remainder of the column with buffer, mount the column top piece
onto the column and connect the column to a pump.
4．Open the bottom outlet of the column and set the pump to run at the desired flow rate.
The flow rate should under 135% of the flow rate used in the chromatography procedures
(see table 1) mainly used during the packing. (Note: if you have packed at the maximum
flow rate, do not exceed 75% of this value in subsequent chromatographic procedures）.
5．Keep the flow rate for another 3 bed volume when it reaches a height of the column
bed.
Sample application:
1. When the column has been equilibrated, close the column outlet and carefully remove
the top piece from the column to form an upward meniscus at the top.
2. Add the sample into the column, ensuring that no air is trapped under the net.
3. Connect the tube and the column, there must be a bubble-free liquid in the connect
system.
4. Add all the sample into the column and form an upward meniscus. Then begin to wash
with 5-10ml buffer.
Equilibration:
Make sure that the ion exchanger has been equilibrated before beginning the experiment.
Pump the start buffer through the column until the column is fully equilibrated when the pH
and/or conductivity of the effluent is the same as the start buffer
1．Binding: The most common procedure in ion exchange is to bind the molecules of
interest, while allowing contaminants to pass through the column. In some cases,
however, it may be more useful to bind the contaminants and allow the molecules of
interest to pass.
2．Binding process: a suitable pH is very important in the binding process. For efficient
binding, the ionic strength of the starting buffer must be low to avoid interfering the binding
of the sample. The pH should be at least one pH unit different from the isoelectric point
(pI) of the molecules to be bound and within 0.5 pH units of the selected buffer salt's pKa.
Regeneration:
Depending on the nature of the sample, regeneration is normally performed by washing
with a high ionic strength buffer (e.g. 1-2M NaCl) and/or increasing pH, followed by
re-equilibrating in binding buffer.
In some applications, substances such as denatured proteins or lipids do not elute in the
regeneration procedure. These can be removed by cleaning-in-place procedures.
CIP:
Washing the column with 0.5 bed volumes of 2 M NaCl solution after disassembling the
column to remove the bound proteins, contact time 10 to 15 minutes.
Remove hydrophilic bound proteins and lipoproteins by soaking with 2 bed volumes for
30minutes. Remove the supernatant after intermittent stirring, and then wash it to neutral
with water. Equilibrate the column with at least 3 bed volumes of start buffer.
Storage:
It is recommended to store the medium at 4℃ in 20% ethanol for long period of storage.