HPLC Cation Exchange

 aLP purification

Buffer A is 10 mM NaHPO₄ pH 7.2

Buffer B is 10 mM NaHPO₄, 0.25 M NaOAc pH 7.2

 

SGPB purification

Buffer A is 5 mM NaOAc, 2 mM CaCl₂ pH 4.8

Buffer B is 5 mM NaOAc, 2 mM CaCl₂, 0.25 M NaCl pH 4.8

   

1.     Degas all solutions (~10 minutes).  (Degas SDS wash solution using the vacuum apparatus in the DeRisi/Walter lab.)

2.     Flip column right side up (flow arrow pointing towards detector.  Always attach column drop-to-drop :  pump ddH₂O through Pump A at 0.05 ml/min.  Once a drop is visible in the line, attach that line to the column.  Once a drop is visible coming out of the column, attach the output line to the column. 

3.     Turn on detector UV lamp.  If it gets stuck in the wrong mode hit “Edit,” hit “Prev.” or “Next” keys to find “single wavelength mode” and press “enter” key.  Connect UV detector output tubing to the waste tube (not fraction collector).

4.     Disconnect tubing leading directly to column and plug column.  Prime pumps A and B with water.  (**If you can’t prime the pumps, its probably because there is too much air in the line.  To combat this evil crime of HPLC, you will have to prime manually.  Take a syringe that corresponds to the blocked pump from the HPLC equipment and attach it to the line for that pump.  Rotate the stopcock so that you are blocking off the line to the pump itself [this means that the stopcock should be facing towards the line heading to the pump.]  Pull the plunger back on the syringe so that you suck back a decent amount of water from the reservoir.  Rotate the stopcock so that you block off the line heading to the reservoir [i.e. the stopcock knob is facing towards the line heading to the reservoir.]  Then push water thru the line until it shoots out of the open tube.  Remove the syringe and rotate the stopcock to the original position blocking the syringe entrance and prime with the “prime” button on the pump to ensure proper priming.**)  Reconnect the tubing to the column.  Manually ramp up pump A flow rate (Increase by 0.01 ml/min for first 0.1 ml/min, then increase by 0.1 ml/min until reach desired flow rate) to 4.00 ml/min.  Wash column with ~10 ml water.  Ramp pump back down to 0.00 ml/min.

5.     Change solutions so that pump A is in Buffer A and pump B is in Buffer B.  Manually ramp up pump A flow rate to 4.00 ml/min.  Wash column with small amount of Buffer A.  Zero UV detector when flow rate is at 4.00 ml/min.

6.     Perform dummy run to be sure nothing extraneous is going to elute during real run.

Open Dynamax > DA (DA alias is on desktop)

File > Open Method > HR16/10 0-250

Control > Select Pumps

System > System Status (check that correct pumps are selected)^*

System > Device Status (make sure 12 reads an interface, if not turn chart recorder on and off)

Control > Run Control > Set Data Name (add D for dummy run, select destination)

Control > Run Control > Ramp to Start (ramp time 0:30, hold time 0:00)

Control > Run Control > Start Run (when ramp is over)

Method > Show Graph (to see gradient)

Data > Show Data Trace (to see chart recorder)

Note pressure during run __________psi

**when run is over**

Control > Stop Pumps

Close out the “Run Control” window

Control > Deselect Pumps

 

7.      Load sample.  Be sure sample was diluted 4-fold to desalt or dialyzed.  Sterile filter sample solution, but do not degas.  Transfer to a 50 ml conical tube for loading.  Disconnect tubing leading directly to column and plug column.  Prime pump C with water.  Reconnect the tubing to the column (drop-to-drop).  Place pump C in sample solution.  Select Control > Manual Control.  Set acquisition time to about 40 minutes longer than you think the load will take and start acquisition.  Manually ramp up pump C flow rate to 4.00 ml/min.  Be sure that solution level does not fall lower than pump—do not pump air into column.  Save load flow-through and monitor UV absorbance (Data > Show Data Trace).

8.     Leave computer settings the same but ramp pump C flow rate to 0.00 ml/min and ramp pump A flow rate up to 4.00 ml/min.  Wash column with Buffer A for 10 minutes.  Save wash flow-through and monitor UV absorbance (Data > Show Data Trace).  Ramp pump A flow rate to 0.00 ml/min and Stop acquisition (Control > Manual Control).  Turn off power to pump C.

9.     Perform elution run.  Change UV detector outlet tubing connection to fraction collector.  Place rack (code 1) with 80 tubes in fraction collector.

File > Open Method > HR16/10 0-250

Control > Select Pumps

System > System Status (check that correct pumps are selected)^*

System > Device Status (make sure 12 reads an interface)

Control > Run Control > Set Data Name (select destination)

Control > Run Control > Ramp to Start

Control > Run Control > Start Run (when ramp is over)

Start fraction collector simultaneously with run!!

Method > Show Graph (to see gradient)

Data > Show Data Trace (to see chart recorder)

Note pressure during run __________psi

**when run is over**

Control > Stop Pumps

Close out the “Run Control” window

Control > Deselect Pumps

 

10.  Methanol wash.  Flip column upside down.  Place pump A in ddH₂O + 0.02 % NaN₃ and pump B in Methanol. 

File > Open Method > HR16/10Wash

Control > Select Pumps

System > System Status (check that correct pumps are selected)^*

System > Device Status (make sure 12 reads an interface)

Control > Run Control > Set Data Name (select destination)

Control > Run Control > Ramp to Start

Control > Run Control > Start Run (when ramp is over)

Method > Show Graph (to see gradient)

Data > Show Data Trace (to see chart recorder)

**when run is over**

Control > Stop Pumps

Close out the “Run Control” window

Control > Deselect Pumps

 

11.  With the column still upside down, place pump A in ddH₂O + 0.02 % NaN₃ and pump B in 0.25 M NaOH + 0.25% SDS. 

File > Open Method > HR16/10NaOH/SDS

Control > Select Pumps

System > System Status (check that correct pumps are selected)^*

System > Device Status (make sure 12 reads an interface)

Control > Run Control > Set Data Name (select destination)

Control > Run Control > Ramp to Start

Control > Run Control > Start Run (when ramp is over)

Method > Show Graph (to see gradient)

Data > Show Data Trace (to see chart recorder)

**when run is over**

Control > Stop Pumps

Close out the “Run Control” window

Control > Deselect Pumps

 

12.  Repeat methanol wash (step 9).

13.  Disconnect line leading from pump C to the column.  Run 6 M GndHCl through loop and let sit overnight.  Rinse pump C thoroughly with ddH₂O + 0.02 % NaN₃.  Reconnect pump C line to column.

^*If pumps don’t show up (“off-line”), try turning off the integrator (the black box on top of the UV detector), then turn it back on.