Voser and Walliser'74! viewed scaleup as a three step process involving selection of a process strategy, evaluation of the maximum and optimal bed height and finally column design. The selection of a process strategy involves choosing the direction of flow, frequency of backwashing, the operation with positive head pressure or only hydrostatic pressure, and the use of a single column or a series of columns in batch or semi-continuous operation. The maximum feasible bed height is determined by keeping the optimal laboratory-scale specific volume velocity (bed volume/hour) constant. The limiting factors will be either pressure drop or unfavorable adsorption/desorption kinetics since the linear velocity also increases with increasing bed height. Bed heights from 15 cm to as high as 12 m have been reported. The column diameter is then selected to give the required bed volume. The column design combines these column dimensions with the practical considerations of available space, needed flexibility, construction difficulty and flow distribution and dilution for the columns.

The scaleup considerations of column chromatography for protein isolation has been described by Charm and Matteo.I75] When several hundred liters of a protein feedstream must be treated, the resin may be suspended in the solution, removed after equilibration by filtration and loaded into a column from which the desired proteins may be eluted. Adsorption onto a previously packed column was not recommended by them since they feared suspended particles would clog the interstices of the column, causing reduced flow rates and increased pressure drops across the column. The reduced flow rate may lead to loss of enzyme activity because of the increased time the protein is adsorbed on the resin.

It is important that all of the resin slurry be added to the column in one operation to obtain uniform packing and to avoid the formation of air pockets in the column. An acceptable alternative, described by Whatman,[76] allows the addition of the adsorbent slurry in increments. When the resin has settled to a packed bed of approximately 5 cm, the outlet is opened. The next increment of the slurry is added after the liquid level in the column has dropped. It is important that the suspended adsorbent particles do not completely settle between each addition.

Stacey and coworkers1771 have used the relationships shown in Table 15 to scaleup the purification from an 8 mg protein sample to a 400 mg sample. The adsorbent used in both columns was a Delta-Pak wide-pore C-18 material. When eluting the protein, the flow rate should change so that the linear velocity of the solvent through the column stays the same. The flow rate is proportional to the cross-sectional area of the column. The gradient duration must be adjusted so that the total number of column volumes delivered during the gradient remain the same. As with size exclusion chromatography, the mass load on the preparative column is proportional to the ratio of the column volumes. Figure 24 shows that the chromatograms from the 8 mg separation is very similar to that obtained for the 400 mg sample.

Table 15 Scale-Up Calculations^77'

Small Scale Preparative Scale

Column Dimensions 0.39 x 30 cm 3 x 25 cm

Flow Rate Scale Factor

Sample Load Scale Factor (3.0)2 x 25

Gradient Duration Calculation 40 min 33 min

__L5_x_40__ _ jg 7 Column 90 x (GradientDuration) _ ? (0.195)2 *x 30 ~ ' volume (1.5)2**25

Grad. Duration = 33 min.

Figure 24. Laboratory and preparative scale separation of cytochrome C digest.'77]

Ladisch178' has worked with a variety of column sizes ranging from 2 to 16 mm in diameter and 10 to 600 cm in length. His experience is that published semi-empirical scaleup correlations are useful in obtaining a first estimate on large scale column performance.

When scaling-up a chromatographic process, it may be necessary to change the order of certain steps from that used in the laboratory. Gel filtration, though a frequent first step at the laboratory scale, is not suitable for handling large scale feedstream volumes.1795 When gel filtration is used to separate molecules of similar molecular weights, sample sizes may range from 1% to 5% of the total gel volume. Thus, a 100 liter feedstream would require a gel filtration column of 2,000 to 10,000 liters. When one is separating a large molecule from small molecules, as in desalting operations, the applied volume may be up to 30% of the gel volume.

On the other hand, ion exchange chromatography is a very good first step because its capacity is approximately 30 mg of protein per ml of resin. This capacity is relatively independent of feed volume. For the same 100 liter feedstream, only a 20 liter ion exchange column would be required.

5.6 Pressure Drop

The pressure drop across an ion exchange bed has been represented by an equation180' which depends on the average particle diameter, the void fraction in the bed, an exponent and a friction factor dependent on the Reynolds number, a shape factor, the density of the fluid, the viscosity of the fluid and the flow rate.

While that equation has internally consistent units (English system), the variables are not normally measured in those units. Another disadvantage is that one must check graphs of the exponent and friction factor versus the Reynolds number to use the equation.

For laminar flow with spherical particles, the equation can be simplified to:

AP 0.0738// (cp) V0 (/ / min) (l-g)» Eq. (28) ~(bar/cm) =---—

For most ion exchange resins, the void volume is about 0.38, so that (1- s3)/ £3 = 4.34 and:

AP 0.32^ (cp) V0 (/ / min) Eq. (29) T / cm) =-—--

Table 16 shows the agreement between results from experiments and those calculated with this equation for several ion exchange resins.

Table 16. Pressure Drop for Commercial Ion Exchange Resins

Flow Rate Mean Bead Pressure Drop (bar/cm) Resin (L/min) Diameter (mm) Calculated Measured

Dowex SBR-P

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