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Superflo® Column Applications

*General Column Information: Benefits | Applications | FAQ | Column Packing

Fast Ion Exchange Ion exchange fractionation of ascites fluid 
Fast Affinity Affinity purification of an anti-melanoma antibody
High Throughput Hydrophobic Interaction Hydrophobic interaction chromatography using axial and radial flow columns
Fast Desalting Fast desalting of a protein solution 
Low Cost Reverse Phase Reverse phase of nucleotides
Direct Purification Direct purification of crude cell lysates (when speed counts)
Tandem Radial Flow Tandem radial flow column chromatography of a refolded recombinant protein sample

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
Yeild: 
     2600 mg estimated recovery
     3126 mg actual recovery
Purity: 
     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 

Sample:
     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 

Column: Superflo® 250 ml Stainless Steel
Packing: Nu-Gel® C-18, 40-60 microns
Sample: 5mg CMP, 5mg UMP, 5mg ATP
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.



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