Paul.xu at 14:05, 27 February 2013

2013-02-27T14:05:18Z

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== Important Guidelines ==

1. Keep the salt concentration below 0.5 M in order to avoid viscosity rise and salt precipitation. Halogen salts (e.g. sodium chloride, potassium chloride, etc.) are not recommended if using a stainless steel column and/or stainless steel system and tubing. If halide ion-containing solvents are used, it is recommended to rinse the column with deionized water for long-term storage of the column. 

 

2. After use, store column in sodium azide (0.05%), 5% methanol in water, <strike>or 20% ethanol.</strike> Solvents should be passed through a 0.45 micron filter before use. 

'''3. Backflushing ''' 

Gentle backwashing can help to restore a collapsed bed to its original configuration or allow entrained air spaces to redissolve. If bed compression is severe, the column may not return to original performance. Backwashing is also a cost effective means of clearing a blocked column frit, and solves the problem about one time out of three cases. 

 

*Shut off the pump and let the column bed "relax" for about 15 minutes. 
*Reverse the flow direction and backwash the column at 0. 1 ml/min with the running solvent for at least 4 hours (if necessary, run overnight). 
*Return the column to original operating conditions. 

'''4. Column washing''' 

1. Invert the guard or column and wash with five column volumes of HPLC grade deionized water at 0.3 ml/min ( 1.5 ml/min for 21.5 mm ID columns). 2. Return the column to its normal operating orientation. At the flow rates recommended in step 1, wash with 5 column volumes of 0.3 M NaH2PO4, pH 3.5 (to remove hydrophilically adsorbed material) or run a 5 column volume 0-100% acetonitrile or methanol gradient (to remove hydrophobically adsorbed material). 3. If there is little or no improvement in column performance and/or back pressure after completing step 2, wash with five column volumes of 6 M guanidine-HCI. 4. If Step 3 proves ineffective, wash with 5 column volumes of 0.1% trypsin in 0.01 M phosphate, 0.15 M NaCl, pH 6.8, as a final resort. 

'''New protocol for column washing: '''

SW and SWxl 1. Turn the column in reverse flow direction and run at half the maximum flow rate. 2. Clean with 5 column volumes (CV) of 1M sodium chloride (pH-7.0) 3. Clean with 5CV of Milli-Q or DI water. 4. Clean with 5CV of 20% acetonitrile. 5. Clean with 5CV of Milli-Q or DI water. 6. Turn column in normal flow direction and equilibrate in mobile phase for at least 45 minutes. 

'''5. Column Storage''' 

If the column will not be used for several days, it should be stored as follows: 1. After rinsing with several column volumes of distilled water, replace the mobile phase with 0.05% sodium azide or 5% methanol in water. Use a low flow rate (0.3 ml/min for 7.5 and 7.8 mm ID columns; 1.5 ml/min for 21.5 mm ID columns) during solvent replacement. 

 2. Keep the ends of the column tightly capped. Use the protective nuts or end plugs originally furnished with the column. Do not allow the resin to dry out. The column can be safely stored for short duration (1-2 days) in HPLC grade deionized water. If the column is used daily, it is safe to leave the buffer in the column overnight as long as the buffer is not corrosive. It is highly recommended to maintain a low flow (< 0.5 ml/min) of solvent through the column to prevent precipitation of salts. 

 

155-0201 Bio-Sil SEC TSK 250 Column Dimension: 600 x 21.5 

MW range: 10,000-300,000 Particle Size: 13 

Capacity: 10-100 mg 

pH Range: 2-7 

 

 

[[Category:Protocols]]

Tosoh TSK-Gel Gel Filtration - Revision history

Paul.xu at 14:05, 27 February 2013