Editing Tosoh TSK-Gel Gel Filtration

== Important Guidelines<br>  ==

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.<br> 

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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.<br><br> 

'''3. Backflushing '''<br> 

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<br>about one time out of three cases.<br> 

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*Shut off the pump and let the column bed "relax" for about 15 minutes.<br> 
*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).<br> 
*Return the column to original operating conditions.<br><br>

'''4. Column washing'''<br> 

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).<br>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,<br>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).<br>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<br>guanidine-HCI.<br>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.<br> 

'''New protocol for column washing:<br>''' 

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

'''5. Column Storage'''<br> 

If the column will not be used for several days, it should be stored as follows:<br>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.<br> 

<br>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.<br>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<br>column overnight as long as the buffer is not corrosive. It is highly recommended to maintain a low flow (&lt; 0.5 ml/min) of solvent through the column to prevent precipitation of salts.<br> 

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

MW range: 10,000-300,000<br>Particle Size: 13<br> 

Capacity: 10-100 mg<br> 

pH Range: 2-7<br><br><br><br><br><br> 

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[[Category:Protocols]]