Hi Reza,
To begin with I would recommend reading the general handbook for gel filtration chromatography (GE Health Care)
http://sevierlab.vet.cornell.edu/resources/Gel_Filtration_Book.pdf
To be more specific for your purpose for high performance size exclusion chromatography (sephadex g-50) you can try reading the attached article.
Good luck!!!
Hi Reza,
It is very similar to any HPLC column, though you will want to be more careful of pressure spikes, as this media more easily crushes:
Applies To
Phenomenex Gel Filtration /Size Exclusion silica column:
BioSep-SEC-S 2000, BioSep-SEC-S 3000, and BioSep-SEC-S 4000 Columns
Yarra 3u SEC-2000, Yarra 3u SEC-3000 only.
Does not apply to Yarra SEC-4000 columns
Introduction
Each column manufactured by Phenomenex is individually prepared and tested. Every column is supplied with a Test Chromatogram and Specification Sheet, which indicates testing conditions, operating parameters, and column identity details. The Column Details, including specifications and performance test results should be entered into an appropriate information management system for easy tracking and
reference should a problem arise.
Inspection
Upon Receipt of the Column:
Verify the column you received is the one you ordered Check the column for physical damage caused by shipping Test the column IMMEDIATELY to verify performance. Record the result of your test in your Column Information Management system.
Column Test Standard
Aqueous SEC 1 P/N AL0-3042
Installation
Flush your HPLC pump and line with the filtered and degassed mobile phase advised in column test procedure. Ensure no air bubbles are trapped in the system. Set the flow rate to 0.1mL/min and connect to the column inlet, ensuring that flow is in the direction indicated by the arrow on the column. Tighten the end fitting going into the column and slowly increase flow to between 0.5 and 1.0 mL/min for 5 minutes.
Stop flow and wipe outlet end of column to remove any particulates prior to connecting to the flow cell. Connect column outlet to flow cell and pass approximately 5 column volumes through the system at 1 mL/min while observing the back pressure. A steady back pressure indicates constant flow - while fluctuations may indicate air in the system. Wide fluctuations may shock and damage the column. When a steady pressure has been attained within the prescibed limits, the column is ready for use. New columns should be tested with GFC test mix, AL0-3042 and previously used columns should be tested on a regular basis. Remember to re-equilibrate the system when changing solvents. Never change to (or from) a buffer/salt solution where the buffer/salt is not soluble in the second solvent. Again this will damage the column. Never attempt to remove the column end fittings. This will void the warranty.
Flow Rate
Adjust to maintain within pressure limits. Typical flow rates for 7.8 mm columns 0.6-1.0 mL/min for BioSep-SEC-S Columns. For Yarra SEC columns typical flow rates are from 0.6 to 1.2 mL/min. For 4.6 mm ID columns flow rates are typically between 0.2 and 0.5 mL/min for BioSep-SEC-S and Yarra SEC columns. Do not exceed 1.5 mL/min for Yarra SEC-2000 and Yarra SEC-3000 columns in 7.8 mm ID.
Pressure
For BioSep typical operating backpressures are between 400-800 PSI. Do not exceed 1500 PSI for
BioSep Columns
For Yarra SEC-2000 and Yarra SEC-3000 typical operating backpressures are between 800-1600 PSI. Do not exceed 3000 PSI for Yarra SEC-2000 and Yarra SEC-3000 Columns
Temperature
Phenomenex
HPLC
10
GFC-SEC BioSep and Yarra
Phenomenex
pH 2.5 - pH 7.5
For more information, please see attached file.
Paul
Molecular weight of my protein is 20,000 and i want to separate it's monomers from it's aggregates. Sephadex with which range of separation do you propose? ( for example, sephadex g-50 separates up to 30,000 )
Have you tried to use Yarra SEC4000 to fractionate your protein? I think this column is compatible for the high MW protein.
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