eq. (A.160)
Gel filtration is primarily used as a polishing step at the end of purification trains. It is also used for buffer exchange following ion exchange steps. In gel filtration the separation media acts as a molecular sieve, separating molecules according to differences in their molecular size and weight. The packed stationary phase consists of numerous porous beads surrounded by a mobile phase. When the sample mixture is applied at the top of the column, large molecules, unable to enter the pores of the beads, pass in between the beads and are eluted first. Smaller molecules with access to the pores are eluted after the larger molecules in order of decreasing size. For globular proteins, elution volumes are directly dependent upon the molecular weight.
In design mode of calculation, the user provides the following information:
● Sample volume per injection, BSAM (% of column volume).
● Column length (L in m) and maximum column diameter (Dmax in m) or
● Column diameter (D in m) and maximum column length (Lmax in m).
● Linear velocities (u in cm/h) for loading, elution and washing-stripping-regeneration.
● Recovery yield of component i, Yi.
● Overdesign factor, ODF.
If Q is the volume of material processed by the column per plant batch time, n is the number of cycles per batch, then the volume of material fed and processed during one cycle must be equal to one sample volume, that is:
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eq. (A.160) |
or:
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eq. (A.161) |
where V is the column volume. The value of the volume is multiplied by the overdesign factor for conservative design.
If the user decides to specify the length, L, of the column (based on experimental data), the column diameter, D, is calculated by:
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If D > Dmax, then multiple columns are assumed in parallel. Analogous calculations are done when the diameter is fixed and the length is allowed to vary.
Note that the feed streams to a chromatography column cannot be part of a recycle loop.
You can choose among four different options for specifying the loading flowrate: you can specify the feed’s linear velocity, or its volumetric flowrate, or its relative flowrate (in bed volumes) in the bed, or the empty bed contact time (the amount of time it takes for a feed volume of material to flow through an empty bed). Any variable that is not specified will be calculated by the program based on the following relations:
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The process time is calculated using the following equation:
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The feed volume in the above equation is expressed in ‘packed bed volumes’.
The recovery yield of a component is used to estimate the fraction of that component that ends up in the product stream.
The cost associated with resin replacement is estimated as for all required consumables (see Consumables Cost). For resins that last several years, the user may decide to depreciate the first resin fill-in by checking the ‘Is First Resin capitalized’ check box in the equipment consumables tab.
1. R.K. Scopes (1987). Protein Purification, Springer-Verlag.
2. H. Johansson, M. Ostling, G. Sofer, H. Wahlstrom, and D. Low (1988). Chromatographic Equipment for Large-Scale Protein and Peptide Purification. in “Advances in Biotechnological Processes”, Vol. 8, Edited by: A. Mizrahi, Published by Alan R. Liss, Inc.
3. R.W. Yost, L.S. Ettre, and R.D. Conlon (1980). Practical Liquid Chromatography - An Introduction. ThePerkin-Elmer Corporation.
The interface of this operation has the following tabs:
● Loading, see GFL Column Loading: Oper. Conds Tab
● Labor, etc, see Operations Dialog: Labor etc. Tab
● Description, see Operations Dialog: Description Tab
● Batch Sheet, see Operations Dialog: Batch Sheet Tab
● Scheduling, see Operations Dialog: Scheduling Tab
