i 71-7070-00
3602Edition AE
Q Sepharo Fast Flow
Q Sepharo™ Fast Flow is a strong anion
exchanger with excellent flow properties and high
capacity for proteins of all pI values. The ion
exchange group is a quaternary amine which
remains charged and maintains consistently high
capacities over the entire working range, pH 2-12.
Q Sepharo Fast Flow belongs to the
BioProcess™ Media family. BioProcess Media are
paration media developed, made and supported
for industrial scale - especially the manufacture
drum error
of healthcare products. With their high physical
and chemical stability, very high batch-to-batch
reproducibility, and Regulatory Support File
back-up, BioProcess Media are ideal for all stages
of an operation - from process development
through scale-up and into production.
Large quantities can be delivered at short notice.
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Table 1. Medium characteristics.
Type of ion exchanger:
Strong anion Total ionic capacity:
0.18-0.25 mmole/ml gel Available capacity*:Thyroglobulin (M r 669 000) 3 mg/ml HSA (M r 68 000)120 mg/ml α-lactalbumin (M r 14 300)110 mg/ml Bead structure:6% highly cross-linked agaro
Bead size range:45-165 µm
橡皮英语Mean particle size:90 µm
Linear flow rate:400-700 cm/h at 25 °C, 0.1 MPa (1 bar, 14 psi), XK 50/30
column, 15 cm bed height, mobile pha 0.1 M NaCl
Max. operating pressure:0.3 MPa (3 bar, 42 psi)
pH stability working range:2-12
long term:2-12
short term:1-14
Chemical stability:All commonly ud aqueous buffers,1.0 M NaOH, 8 M urea,
8 M guanidine hydrochloride, 24% ethanol (tested at 40 °C
for 7 days).
Physical stability:Negligible volume variation due to changes in pH or ionic strength.
Autoclavable:
In 0.1 M NaCl at 121 °C for 30 min.*
The available capacity was de te rmine d in a 0.5 x 5 cm column at a line ar flow rate of 300 cm/h. Starting buffe r:0.05 M Tris, pH 8.3. Elution buffer: 0.05 M Tris, 2 M NaCl, pH 8.3.**The range s give n are e stimate s ba d on our knowle dge and e xpe rie nce . Ple a note the following:
pH stability, long te rm re fe rs to the pH inte rval whe re the ge l is stable ove r a long pe riod of ti
me without adve r e ffe cts on its sub que nt chromatographic pe rformance .
pH stability, short te rm re fe rs to the pH inte rval for re ge ne ration, cle aning-in-place and sanitization proce dure s.Preparation of the medium
Q Sepharo Fast Flow is supplied pre-swollen in 20% ethanol.Prepare a slurry by decanting the 20% ethanol solution and replace it with starting buffer in a ratio of 75% ttled gel to 25% buffer.The starting buffer should not contain agents which significantly increa the viscosity. The column may be equilibrated with viscous buffers at reduced flow rates after packing is completed.
Packing Se pharo Fast Flow
1.Equilibrate all material to the temperature at which the
chromatography will be performed.
2.De-gas the gel slurry.
lfesteem3.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. Clo 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.
6.Open the bottom outlet of the column and t the pump to
run at the desired flow rate. Ideally, Fast Flow gels are晴朗
packed at a constant pressure not exceeding 3 bar (0.3 MPa) in XK columns. If the packing equipment does not include a pressure gauge, u a packing flow rate of at least 400 cm/h
(15 cm bed height, 25 °C, low viscosity buffer).
If the recommended pressure or flow rate cannot be obtained, u the maximum flow rate the pump can deliver. This should also give a reasonably well-packed gel.
Note:Do not exceed 75% of the packing flow rate in
subquent chromatographic procedures.
7.Maintain the packing flow rate for 3 bed volumes after a
constant bed height is reached.
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U of an adaptor
Adaptors should be fitted as follows:
1.After the medium has been packed as described above, clo
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 at the top.
2.Inrt the adaptor at an angle into the column, ensuring that
耻辱的意思
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 tubings is displaced by buffer.
Valves on the inlet side of the column should be turned in all directions during this procedure to ensure that air is removed.
5.Lock the adaptor in position, open the column outlet and
start the buffer flow. Pass buffer through the column at the
packing flow rate until the medium bed is stable. Re-position the adaptor on the medium surface as necessary. Equilibration
Before starting a run, make sure that the ion-exchange bed has reached equilibrium. This is done by pumping starting buffer through the column until the conductivity and/or pH of the effluent is the same as for that of the in-going solution.
The column is now equilibrated and ready for u.
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Binding
•The most common procedure is to let the molecules of interest bind to the ion exchanger and allow other molecules to pass through.
However, in some cas it may be more uful to bind
“contaminants” and let the molecules of interest flow through.•For adsorption, it is critical to choo a buffer with an appropriate pH. Plea refer to Table 2. The ionic strength of the buffer should be kept low so as not to interfere with
sample binding. Recommended operating pH is within 0.5
pH units of the buffer’s pK
财务总监培训a and at least one pH unit above
the isoelectric point (pI) of the molecule of interest.
Table 2. Suggested buffers for u with Q Sepharo Fast Flow
Buffer Counter ion Concentration pKa (25°C) N-methylpiperazine Cl-20 mM 4.8 piperazine Cl-20 mM 5.7
orgyHCOO-
L-histidine Cl-20 mM 6.2
bis-Tris Cl-20 mM 6.5
bis-Tris propane Cl-20 mM 6.8 triethanolamine Cl-20 mM7.8
CH
3COO-
introducedTris Cl-20 mM8.2
N-methyldiethanolamine SO
42-50 mM8.5
Cl-
CH
3COO-
purple什么意思diethanolamine Cl-
1,3-diaminopropane Cl-20 mM
ethanolamine Cl-20 mM
piperazine Cl-20 mM
1,3-diaminopropane Cl-20 mM
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