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Large scale columns
• BPG
TM
variable bed, glass columns. Inner diameters from 100–450 mm,
bed volumes from 2.4–131 litres; bed height max 83 cm.
• CHROMAFLOW
TM
variable bed columns. Inner diameters from
400–600 mm.
3.2 Packing lab-scale columns
1. Assemble the column (and packing reservoir if necessary).
2. Remove air from the column dead spaces by flushing the end-piece
and adaptor with packing buffer. Make sure no air has been trapped
under the column bed support. Close the column outlet leaving the bed
support covered with packing buffer.
3. Re-suspend medium stored in its container by shaking (avoid stirring the
sedimented medium). Mix the packing buffer with the medium to form
50–70% slurry (sedimented bed volume/slurry volume = 0.5–0.7).
4. Pour the slurry into the column in a single continuous motion. Pouring
the slurry down a glass rod held against the column wall will minimize
the introduction of air bubbles.
5. If using a packing reservoir, immediately fill the remainder of the
column and reservoir with packing buffer. Mount the adaptor or lid of
the packing reservoir and connect the column to a pump. Avoid
trapping air bubbles under the adaptor or in the inlet tubing.
6. Open the bottom outlet of the column and set the pump to run at the
desired flow rate. Ideally, Sepharose 4 Fast Flow based media are
packed at a constant pressure of approximately 1 bar (0.1 MPa). If the
packing equipment does not include a pressure gauge, use a packing
flow velocity of approximately 400 cm/h (15 cm bed height, 25 °C, low
viscosity buffer).
If the recommended pressure or flow rate cannot be obtained, use
the maximum flow rate the pump can deliver. This should also give a
reasonably well-packed bed.
Note: Do not exceed 75% of the packing flow velocity in subsequent
chromatographic procedures using the same pump.