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Large scale columns
• BPGTM variable bed, gla ss columns. Inner d iameters from 100 –450 mm,
bed volumes from 2.4–131 litres; b ed height max 83 cm.
• CHROMAFLOWTM variable be d columns. Inner di ameters from
400–600 mm.
3.2 Packing lab-scale columns1. Assemble t he column (and pack ing reservoir i f necessary) .
2. Remove air from t he column dead space s by flushing the e nd-piece
and adaptor with packi ng buffer. Make sure no ai r has been trapped
under the column bed sup port. Close t he column outlet le aving the bed
support covered wit h packing buffe r.
3. Re-suspend m edium stored in it s container by shak ing (avoid stir ring the
sedimented medium) . Mix the packing b uffer with the me dium to form
50–70% slurry (sedime nted bed volume/s lurry volume = 0.5 –0.7).
4. Pour the slurr y into the column in a si ngle continuous m otion. Pouring
the slurry down a glass r od held against t he column wall will mi nimize
the introduction of air bubbles.
5. If using a packi ng reservoir, immed iately fill the re mainder of the
column and reservoir w ith packing buf fer. Mount the adaptor o r lid of
the packing reservo ir and connect the c olumn to a pump. Avoid
trapping air bubbles u nder the adaptor or i n the inlet tubing .
6. Open the bot tom outlet of the col umn and set the pump to r un at the
desired flow rate. Ide ally, Sepharose 4 Fa st Flow based med ia are
packed at a constant pres sure of approximat ely 1 bar (0.1 MPa). If t he
packing equipment do es not include a press ure gauge, use a packi ng
flow velocity of approx imately 400 cm/ h (15 cm bed height, 25 °C, l ow
viscosity buffer).
If the reco mmended pressu re or flow rate cannot b e obtained, use
the maximum flow rate t he pump can deliver. This s hould also give a
reasonably well-packed bed.
Note: Do not exceed 75% of the packi ng flow velocity i n subsequent
chromatographic pro cedures using the sa me pump.