Editing LCC 500-501 Operation (section)

== General Use  ==

Standard settings: 

{|
|-
| conc. B 
| 50.0 (therefore 50% of the fluid pushed through the system comes from pump A and 50% from B)
|-
| ml/min 
| 0.5
|-
| cm/mL 
| 1.0
|-
| PT 
| 0.0.0.0.0.0.0.0.0
|-
| valve 
| 1.1 directly to column
|-
| <br> 
| 1.2 through loading loop and then to column- ONLY WITH SYRINGE IN
|-
| <br> 
| 1.3 skips column and goes to waste- for wash
|-
| flow c 
| 0.0
|-
| monitor 
| 0
|-
| level 
| 0.0
|-
| ml/mark 
| 0.0
|-
| wash A.B 
| 1.0 to wash reservoir of A
|-
| <br> 
| 0.1 to wash resevoir of B
|-
| <br> 
| 1.1 to wash both reservoirs
|-
| <br> 
| 0.0 no washing
|-
| integrate 
| 0
|-
| feedtube
|-
| sync A.B 
| 0.0
|}

===== Chart recorder:  =====

*set to: ext 
*run at: mm/sec 
*chart speed: 20 
*set rec to: on 
*Have the pens “zero” down to set pens and up for the run 
*Put pens down for writing. 
*red-&gt; conductivity 
*blue-&gt; UV- 280nm 
*set for “cal”, not “var”

===== To wash pumps:  =====

Use filtered degassed buffer only with the FPLC. Air bubbles on the column can ruin the column resolution. 

Put the tube to pump A in bottle A and the tube to pump B in bottle B, or put both tubes in one bottle if you only have one buffer. Be sure they are always under the liquid level. 

Set the valve to 1.3 and the flow rate to 5 ml/min. 

Let this run until both pumps have fully equilibrated in the new solutions. Then stop the run. 

===== To wash the column:  =====

Connect a column to port 1 of the switching valves. Be sure that there is liquid in the tube directly connecting to the column so that no air enters the column. If the tube is dry then switch the pumps on to run to the column before connecting the column to get liquid in the tube. Let fluid run into any connecting nozzles to remove any air. 

Connect the bottom of the column to the UV monitor tubing, again assuring not to force any air into the detector's flow cell. If you do end up with air in the flow cell, this may present as the detector signal jumping all over the place and the plotter flying up and down wildly. If this happens, use a syringe to force fluid through the flow cell to try and get the bubble out. 

Set the ml/min to something low, like 0.5 ml/min and the chart recorder to 1 or 0.5 cm/ml 

Watch the UV and conductivity meters and allow them to equilibrate, meaning that the conductivity monitor and detector signals stabilize. 

Watch that the pressure does not go above 2 MPa with any size exclusion column, nor above 0.5 MPa with any ion exchange column. 

You can increase the ml/min if the pressure remains low. 

===== Trouble with columns:  =====

If the back pressure on the column is high, there can be several problems. First, when ethanol is present in the column, the pressure will be higher than usual. This is fine, just use a low flow rate until the ethanol is flushed out. If the pressure remains high, the first place to check is the pre-column filter. Replace the filter with an extra from the FPLC drawer. If this does not help, then the flow cell my be clogged or the column may be dirty. Check column or UVis information sheets to determine cleaning solutions. 

If the alarm is going off on the column for any reason, the machine will usually pause itself. Push the alarm reset button on the main computer to reset, then push continue (if you have figured out what caused the alarm and corrected it). 

===== Loading a sample:  =====

Connect a sample loop of similar size to the volume you wish to load on the column to ports 2 and 6 on the switching valve. Use a syringe with water in it to wash the sample loop by inserting the syringe into the syringe port on the switching valve. Push the syringe in all the way, but do not push it too hard. Inject the water through the sample loop, it will come out of a tube which should be held above the level of the loading syring so that the water does not drain out when done. Again, this is to keep air bubbles from forming in the sample loop which then might be injected onto a column. 

Then load sample in syringe into sample loop. The sample volume should ideally be larger than the sample loop so that no air bubles will be present in the loop after loading. If the sample volume is smaller than the loop be careful that the water injected for cleaning is straight against the sample volume (no air bubble). 

Leave the syringe in place after loading the sample loop. (leave syring in place during all times that the valve is switched to 1.2) 

When the valve is switched to 1.2, the sample loop will be part of the flow to the column and the sample will inject onto the column. Once enough volume has gone through the loop to clear it out, switch the valve back to 1.1. Now you can safely remove the syringe and clean the loop (or reload the sample loop). 

===== Running a method:  =====

The gel filtration column is run with a constant buffer percentage and does not require a formal method to run. This can be run directly off the manual control and the fraction collector can be run manually as well (see info. about fraction collector below). However if you wish to write a method for it you may. 

The ion exchange columns require a salt gradient for elution. This requires a method file. Method files are stored in 5 different method banks (1-5) and then in 10 different methods in each bank (0-9). 

To set up a method: 

There are some standard methods that can be loaded into the method bank 5 with the command Clear Memory 501. See inside the back cover of the controller manual for the standard methods. 

To edit a method, choose “method file” from the front panel. Then type 5 and enter for the 5th bank. Then use the forward button to get to “program method” and enter the method you wish to edit (example 0). Push “forward” to step through the commands of the method. An example with a few notes is below. The commands such as ml/mark and integrate have to do with the printing out of a small version of the run on the little onboard printer as the method runs. 

Use the delete key to remove a command. Use the insert key to insert a command; once you have pushed the insert key you can scroll through the commands with the forward button until you reach the one you wish to insert, then hit enter. At this point you can enter the time you want the command for. Then use the change key to change the value of the newly inserted command. Use the change key to change the time or value of a specific command. 

When you have the method the way you want it, choose exit from the front panel. 

Now you can run your method by typing the method number and hitting enter (it will run the method of the method bank you were last in) 

example of method: 

{|
|-
| 0.00 
| conc&nbsp;%B 
| 0.0
|-
| 0.00 
| ml/min 
| 1.00
|-
| 5.00 
| valve.pos 
| 1.2 
| this is to '''Inject''' sample loop volume
|-
| 5.00 
| port.set 
| 6.1 
| this turns the '''fraction collector''' to run
|-
| 5.00 
| clear data
|-
| 5.00 
| monitor 
| 1
|-
| 5.00 
| level&nbsp;% 
| 5.0
|-
| 5.00 
| min/mark 
| 2.0
|-
| 5.00 
| integrate 
| 1
|-
| 10.0 
| conc&nbsp;%B 
| 0.0
|-
| 10.0 
| valve.pos 
| 1.1 
| '''Load'''; this stops going through th sample loop
|-
| 30.00 
| conc&nbsp;%B 
| 35.0
|-
| 34.00 
| conc&nbsp;%B 
| 100
|-
| 38.00 
| conc&nbsp;%B 
| 100
|-
| 38.00 
| port.set 
| 6.0 
| this stops the fraction collector
|-
| 38.00 
| integrate 
| 0
|-
| 38.00 
| prt pk 
| 1.05
|-
| 38.00 
| conc&nbsp;%B 
| 0.0
|-
| 43.00 
| conc&nbsp;%B 
| 0.0
|}

To switch between the values being given in min or in ml, use the calibration key and in the first choice use 0 for minutes, 1 for ml, and 2 for dl. Then use the exit key to leave the calibration mode. 

===== Trouble with a program:  =====

As our Pharmacia system has aged, it has become prone to complete power and memory failures (frustrating I know). This will present as an error reading Check Code 190. This will require you to first reset the calibration on the pumps and the recorder by pressing the calibrate button on the controller and entering 110 for Pump AB and 200 for Rec. You will now be able to run manual methods and to reprogram old methods that have been cleared from the memory. 

===== Fraction Collector:  =====

The fraction collector can be told to run or stop from the onboard computer but can not be set for the size fractions to collect this way. The fraction collector is set by using its own key pad and I always use the simple time-based collection. 

To set the fraction size: 

#Press 1,9, then store return 
#Press 7 
#Press number for fraction size in fractions of a minute (based on your flow rate). example: 0.5 for half a minute 
#Press store return 
#Press run if you want to start it yourself (if not using a method) otherwise do not push this and leave it after step 4- the method file will start it when it reaches the port-set 6.1 command.

Put tubes in the fraction collector- use glass test tubes if collecting large fractions or put small eppendorf type tube in the glass test tubes if collecting small fractions (using the test tubes as holders for the small tubes). Set the exit stream tube over the first tube. The fraction collector will automatically move from tube to tube and will also mark the chart recorder for each tube moved. 

===== When you are finished running the FPLC:  =====

The column and system should be stored in filtered degassed 24% ethanol/H2O when not in use. Put this solution through the column at 0.5 ml/min until it is completely rinsed through. The pressure will rise when the ethanol is present in the column- this is normal. Disconnect the column from the FPLC and store capped (room temp is alright for many columns). Reconnect where the column was to keep the system from drying out. 

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