I have packed a column with ~50 mL (height=28.5 cm) sephadex G-50 superfine, as required for the high resolution chromatography of a ~9.5 kDa peptide, in a BIORAD econo-column (d=1.5 cm, h=30 cm, max. backpressure 0.1 MPa). I randomly selected a flow rate of 0.8 mL/min which doesn't create a backpressure of more than 0.1 MPa on Akta Prime Plus system for packing. I wanted to know how to calculate the maximum flow rate that can be applied on the column using Darcy's Law
U = K x delta P x L-1
L know the value of, L= bed height= 28.5 cm
I dont know what to put or how to get the value of delta P, which is pressure drop over the packed bed expressed in cm water.
and K which is constant for the proportionality depending on the properties of the bed material and the buffer, which is given as 36 for sephadex G-50, fine but not for the superfine. Which I can assume reduces by a factor of 3.33 as that of sephadex G-25, so 10.8.
Hi,
I think you need not theoretical values. You want to purify some protein. The right value shoud be around 0.5 ml/min for your column. Simply work at pressure just below max. recommended for Sephadex. I would recommend to calculate efficency by injection of 200 ul of 2% acetone at different flow rates 0.2-0.8 ml/min to find optimal value for this column (select those flow rate which gives peak with minimal width at half height) Note that maximal sample volume should not be more than 500 ul, 200-250 ul is optimal.
The value of P and other useful information is given here:
http://www.gelifesciences.com/webapp/wcs/stores/servlet/ProductDisplay?categoryId=11520&catalogId=10101&productId=21009&storeId=11787&langId=-1
pressure drop cm H2O/bed height=15
I will say that same follow the reference that Adam gave you. GE recommends a minimum of 60 cm/hr. linear flow. To determine the actual ml/minute flow rate with the known cross section of 1.5 cm, the flow rate will be 1.8 ml/min. at a minimum.
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