JtC(t)dt
3.2 Materials and methods.
The systems under study consisted o f a 0.05m diameter STREAMLINE™ (ST-50) expanded bed and a 0.05m diameter XK50/40 packed bed. RTD characteristics were performed on several different diameter packed beds used in this study, however only the results generated for the XK50 packed bed are considered alongside the ST-50 expanded bed in this chapter. RTD calculations for the different sized packed columns were primarily conducted to evaluate the efficiency o f colunm packing rather than the determination o f dispersion coefficients and other mixing parameters.
3.2.1 Experimental apparatus.
The experimental set-up is shown schematically in Fig. 3.1.
3.2.2 Column packing-ST-50 STREAMLINE™ expanded Bed
A peristaltic pump (Model 505DU, Watson Marlow, Falmouth, Cornwall, UK) was used to pump water into the bottom o f the STREAMLINE™ 50 column until a liquid level of approximately 0.4m was present in the open column. A second 505DU pump was connected to a Im length o f rigid plastic tubing and used to remove any trapped air from underneath the bottom distributor plate and mesh. Suction was applied until no further air bubbles could be observed rising up the transparent plastic tubing and a residual height o f 0.1m o f water remained in the column. Approximately 300mL of settled prototype STREAMLINE™ Phenyl (low sub) matrix was re-suspended in its storage buffer o f 20%(v/v) ethanol. Once the gel was suspended the column was tilted off the vertical and the gel was carefully poured down the sides o f the glass column to prevent any air bubbles becoming trapped under the matrix. After addition o f the matrix, the column was filled to the brim with Dl-water and the gel was left to settle. The upper movable adapter was then attached to the top o f the column. Care was taken to prevent air becoming trapped under the top adapter.
3.2.3 Column packing-XK50/40 packed Bed.
The correct packing of packed beds of chromatographic matrices is more critical to the overall performance o f the adsorption step than in expanded beds. For the RTD
Key to symbols:
EB: 5cm Expanded Bed PB: 5cm Packed Bed
Bubble Trap
cPOj 4 port-4way Jacobo Valve
Hydraulic Pump
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20%EtOH Hydraulic Resevoir %-E ^ V 5 ^ EB iMy-8 Valve #1 PB IMV-8 Valve #2^ Feed Pump — # —4 port-2way Jacobo Valve
Y
V 4 ►Waste pH UV280 mS/cm V 3 IF J 3- v i t Buffer A Buffer B SuperFrac j-» fra i L*-yyai Factions ste Data loging Computer liSii )Fig.3 .1 Schem atic diagram o f the ST-50 STR EA M LIN E ™ expanded bed and X K 50/40 packed bed colum ns, along with the ancillary pum ps and detection equipm ent.
studies, a final packed bed height o f approximately 0.15m of Phenyl Sepharose'^^F (low sub) was required. To this end, 820mL of settled matrix in a final volume o f IL 20%(v/v) ethanol was re-suspended to a give a 0.82%(v/v) slurry which was degassed in a buckner vacuum flask using helium for 0.15h. The XK 50 column was connected to the ancillary pipework and air removed from the lower adapter in a similar fashion to the expanded bed. Leaving 0.05m o f water in the bottom o f the column, 365mL of the slurry was poured down a glass rod held against the side o f the XK column. The column was immediately filled to the top with DI water and the upper adapter fixed to the top o f the column. Water was pumped in a downward direction at 0.30m/h until the matrix had packed down to approximately 0.15-0.2m bed height. The upper adapter was lowered to the surface of this bed and the flowrate increased by 0.3m/h and the bed further compressed. This packing process continued until the flowrate reached 4m/h at which the adapter was set and fixed at a final bed height o f 0.125m.
3.2.4 Bed expansion characteristics.
The expansion characteristics o f a batch of prototype matrix STREAMLINE™ Phenyl (low sub) was investigated in a STREAMLINE™ 50 column with a variety of process liquids.
3.2.4.1 Expansion tests.
Before the first bed expansion test was conducted, the bed was initially expanded using DI water, starting at a low superficial liquid velocity and increasing the velocity in 0.50/h steps, every 0.5h, up to a final liquid velocity o f 4m/h. The matrix in the bed was allowed to reach a steady state with the particles o f differing sizes stratifying within the column to occupy their equilibrium positions within the bed. After approximately 0.5h at this flowrate, flow was stopped and the bed allowed to settle. Using DI water at 20°C as the process liquid, the flowrate was initially increased in steps until the surface o f the bed became unstable and diffuse. At each flowrate the bed was allowed to reach a steady state, i.e. a constant bed height, before the expanded bed height at that flowrate was measured. After changing the flowrate, 0.3h was required for bed height to stabilise.
The expansion o f the hydrophobic matrix was also performed in the presence o f the equilibration buffer; 0.78M ammonium sulphate in phosphate buffer (0.02M, pH7), again at 20°C. The bed was first expanded to a flowrate of 2m/h in DI water until a steady bed height was achieved. The upper movable adapter was positioned 10cm higher than the position of the upper surface o f the bed, and the Ouidising liquid changed to the ammonium sulphate buffer. Once both the expanded bed height and the on-line conductivity readings has stabilised the bed was allowed to settle prior to measuring the expansion o f the bed against flowrate for this new buffer system.
To simulate the expansion characteristics o f the prototype matrix in diluted yeast homogenate, both 20%(v/v) and 30%(v/v) glycerol solutions were employed as fluidising solutions since their physical properties lie close to that o f the diluted yeast homogenate used in this study (Chapter 2). Diluted yeast homogenate could not be used directly as its turbidity prevented the visualisation o f the top o f bed.