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Superflo® Column Applications

Fast Ion Exchange

Ion exchange fractionation of ascites fluid
Sample: 150 mL ascites fluid
Column: 1500mL Superflo® Column
Packing: DEAE Cellulose
Flow Rate: 150 mL/min
Start Buffer: 10mM phosphate, pH 8.5
Step Gradient: 60 mM NaCl in start buffer
Radial flow columns are routinely used in initial capture steps after fermentation and cell disruption. The radial flow design of Superflo® columns helps overcome typical problems encountered with most ion exchange media. These packings, especially cellulose, are inexpensive and offer high capacities. However, on axial columns they characteristically yield low flow rates, high backpressures, and swell and shrink with buffer changes. With the radial flow design, fast flow rates are easily achieved with only a peristaltic pump. You save time and can use inexpensive packings and equipment.

Saxena, V. and Well, A.E., Biochromatography, Vol 2(2), p.90, 1987.

Fast Affinity

Affinity purification of an anti-melanoma antibody on Protein A Sepharose®* in a 1500mL Superflo® column
Flow Rate:
   104 mL/min loading
   107 mL/min wash
   92 mL/min elution
   2600 mg estimated recovery
   3126 mg actual recovery
   95% (HPLC gel filtration)
   IgG2A (double immunodiffusion)
Dilute cell culture fluids can be purified without prior concentration using affinity chromatography and Superflo® columns. High purification and recovery levels are possible with columns due to the high flow rates and therefore reduced residence times in the column. There is lower nonspecific binding and less proteolysis than in conventional columns.

Saxena, V., et al, American Laboratory News, October, 1987, p.112.

High Throughput Hydrophobic Interaction

Hydrophobic interaction chromatography using axial and radial flow columns

Axial Radial
Bed Volume 80 mL 100 mL
Packing Phenyl Sepharose® Phenyl Sepharose®
Load 40 mL, 80 mg protein 50 mL, 100 mg protein
Flow Rate 3 mL/min 60 mL/min
Total Time 6 hours 20 minutes

The comparison chart above shows the results of hydrophobic interaction chromatography using axial and radial flow column. Additionally, resolution was superior with the Superflo® column.

Fast Desalting

Fast desalting of a protein solution
   40g BSA in 20L
   1M NaCl
Flow Rate:
   1.8 L/min loading at 3 psi
   5.0 L/min run at 16 psi
Elution Buffer:
   Tap water
The low bed of Superflo® columns makes them ideal for fast desalting applications. Using dextran, polyacrylamide, or agarose packings, desalting can be performed at several times the speed previously possible. In this example, a 20 L sample was fully desalted in 45 minutes in a 100 L Superflo® Column.

Low Cost Reverse Phase

Reverse phase of nucleotides
Sample: 5mg CMP, 5mg UMP, 5mg ATP
Column: Superflo® 250 ml Stainless Steel
Packing: Nu-Gel® C-18, 40-60 microns
Flow Rate: 30 ml/min
Solvent A: 100% methanol
Solvent B: 0.05M tetrabutyl ammonium phosphate, 0.02M KH2PO4,5% methanol
Linear Gradient: 0 to 100% B over 60 minutes
Reverse phase separations are generally associated with high pressures, high equipment costs, and difficult packing procedures. However, with radial flow, preparative HPLC separations can be achieved at low pressures (10-20 psi). Capital equipment costs are low and the separations can easily be scaled up for large process applications.

Direct Purification

Direct purification from crude cell lysates (when speed counts)
The Superflo® column system provides a rapid, low-pressure method for directly processing total crude cell lysates. Crude lysate is pumped directly onto a column packed with fast flow ion exchanger. The large porosity of the Superflo® column bed support and the interstitial space of the agarose beads permits fast, low-pressure flow of the lysate without clogging by cell debris.

Tandem Radial Flow

Tandem radial flow column chromatography of a refolded recombinant protein sample
Peak I: Q-Sepharose®* flow-through
Peak II: Q-Sepharose®* and S-Sepharose®* flow-through
Elution Buffer: 10mM PO4, 1nM EDTA, 500mM NACl
Peak III: S-Sepharose®* peak
In the example above, tandem radial flow anion and cation exchange columns were used to partially purify and concentrate a dilute recombinant protein. The sample was first passed through a column packed with Q-Sepharose®* to remove contaminating proteins and endotoxins. A column packed with S-Sepharose®* was connected to the outlet of the first column to further purify the product. The tandem arrangement enabled the rapid processing of multiple preparations.

Data courtesy of Creative Biomolecules, Inc.