LCC 500-501 Operation
Operation
Start-up
[edit]1. Check that all components are correctly installed
2. Turn on the pumps, monitors, recorder and fraction collector.
3. Set the flow rate on the pumps to zero and press the RUN button (E-50, P-500, P-6000).
4. Set the three chart speed selection buttons on the recorder to 10, mm/sec and "ext". Set the input range selection buttons for the channel connected to GRADIENT 1 V on the LCC-501 Plus to 1 V (10x100 mV) and the buttons for die channel connected to the monitor to the appropriate voltage. Press the input range selection button 0 on both channels to adjust both pens to zero with the zero control knob. Release both input range selection buttons 0 (REC-102).
5. Set the appropriate sensitivity with the sensitivity range selector on the UV monitor and adjust the baseline with the baseline knob or press "AUTO ZERO" on the UV-M II.
6. Turn On the LCC-501 Plus.
7. Enter the Calibration block and check the kalor for method base and calibration values for the pumps, recorder etc.
8. Program the fraction collector and set it in the start position.
9. Check that the pumps are filled with the correct buffers by activating the wash program in the Manual block.
10. To get started as fast as possible there are 9 different preprogrammed methods within the sminvare of the LCC-501 Plus. They am easily copied into Method banks in methods 0~8 with the command a CLEAR MEMORY 501.
The methods are for specific columns sizes and techniques, to optimise your methods follow the instructions included with the columns.
Note The preprogrammed methods wiil not write over existing methods.
11. If the method is to be programmed before starting the run, it is convenient to equilibrate the column during this period by setting the initial conditions through the Manual block.
12. Program the method.
13. Check the method in the Review block or by listing it on the printer.
14. Run the method.
Terminology
[edit]CALIBRATE PUMP A
[edit]Allows calibration of a pump connected to the connector PUMP A. For calibrating a peristaltic pump, connect it to the PUMP A socket and follow the procedure below. Do not forget to change the connections and re-enter PUMP A calibration value after you have finished the procedure. The
procedure is according to the following scheme:
1. Fill the pump with suitable liquid, e.g. water, (before connecting it to LCC-501 plus).
2. Connect the pump to the PUMP A connection on rear panel. Turn on the pump.
3. Set a representative flow rate on the control panel of the pump, (approx. 1 ml/min. ). Place the outlet from the pump into a graduated cylinder.
4. Press [do store]. The run LED is switched on and pump A control from the LCC-501 Plus is released so pump A can start with the flow rate set on the pump. The LCC-501 Plus is now counting pulses from the stepper motor in the pump.
5. Press [end] when a volume large enough to be measured with good accuracy has been delivered, e.g. 10 ml.
6. VOLUME.ML is displayed (or VOLUME. DL if dl mode is chosen).
Enter the collected volume and press [do store].
7. The calibration mode is terminated and the display will show PUMP CAL AB as above but with a new calibration value.
The calibration value for the Pump P-50 is 234 pulses/0.1 ml, for the P-500 is 110 pulses/0.1 ml
P-1ポンプのキャリブレーション方法
ポンプのキャリブレーション方法
1 P-1 ポンプをコミュニケーションケーブルでコントロール背面の Pump A のインターフェイスに接続
2 P-1 ポンプの流速を、5、×10 にセット
3 P-1 ポンプ出口に 10 ml 程度のメスシリンダーを置き、CALIBRATE PUMP Aで do/store ボタン
4 メスシリンダーに 5 ml 程度たまったら、end ボタン
5 ディスプレイに Volume ml と表示されるので、測定した溶液の Volume 値 をコントローラのタッチパネルで入力(検量値(1ml 送液するのに必要なパ ルス数)は自動的に PUMP CALABに入力される)
6 ポンプCとしてのみ、P-1 ポンプを使用する場合には、 PUMP CALABから 一つ進めて PUMP CAL Cに同じ値を入力し、かつ、PUMP CALABには 使用するポンプの検量値を入れ直す。
8 さらに、1でコントロール背面の Pump Aのインターフェイスに接続した ケーブルを、Pump C のインターフェイスに接続し直す
P1 tubings
Silicone tubing/P-1 i.d. 1.0 mm 2 19-4692-01
Silicone tubing/P-1 i.d. 2.1 mm 2 19-4691-01
Silicone tubing/P-1 i.d. 3.1 mm 2 19-4690-01
Fluoro-rubber tubing/P-1 i.d. 1.0 mm 2 19-7626-01
Fluoro-rubber tubing/P-1 i.d. 2.1 mm 2 19-7627-01
Fluoro-rubber tubing/P-1 i.d. 3.1 mm 2 19-7628-01
PVC tubing/P-1 i.d. 1.0 mm 2 19-7629-01
PVC tubing/P-1 i.d. 2.1 mm 2 19-7630-01
PVC tubing/P-1 i.d. 3.1 mm 2 19-7631-01
PT
[edit]
Shows the status of attached outports i.e. active or inactive e.g. .
0.0.1.0.0.0.0.0.0.0 indicates that port 2 is active. To set port 5 active enter 5.1.
Port 0 is switch valve or user-definable outport
Ports 1-4 are user-definable outports
Port 5 is fraction collector pause (1), continue (0)
Port 6 is fraction collector run (1), end (0)
Port 7 is fraction collector recycle (1), reset (0), recycle is for Frac-200.
Ports 8-9 are user-definable outports.
WASH A.B.
[edit]Activates the wash program in pumps A and B if they have the function (e.g. P-500 and P-6000). To activate the wash program in pump A enter 1.0; to activate the wash program in pump B enter 0.1; to activate the wash program in both pumps, A and B, enter 1.1.
SYNC A.B
[edit]Drives the pump pistons to an end position if the pumps are provided with the function (e.g. P-500 and P-6000). Useful for gradients of very small volumes. Enter 1.0 for driving the pump A piston to an end position, 0.1 for pump B and 1.1 for both pumps.
PORT.SET
[edit]Sets outports active or inactive e.g. 3.1 sets port number 3 active.
1 = active; 0 = inactive.
Note: Default means active is electrically 0 V on ports 0 and 3-9 and floating voltage on ports 1 and 2.
Port 0 (when not used for switch valve) rear panel no. 6
1 rear panel no. 18
2 rear panel no. 19
3 rear panel no. 2
4 rear panel no. 4
5 fraction collector, pause (1), continue (0)
6 fraction collector, run (1), end (0)
7 fraction collector, recycle (1), reset (0)
8 rear panel no. 21
9 rear panel no. 22
These instructions were originally written by Katarina Midelfort in 1999. Please get trained by current Wittrup lab personnel before using for the first time. Some things may have changed.
The fast protein liquid chromatography (FPLC) instrument is from Pharmacia. It is comprised of pumps, switching valves, sample loop, separation column, UV detector, conductivity meter, sample collector, and plotter. The instrument is run from an internal computer.
FLOW C: Pump C flow rate
VALVE 0.0: VALVE 1.3
General Use
[edit]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 |
| 1.2 through loading loop and then to column- ONLY WITH SYRINGE IN | |
| 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 |
| 0.1 to wash resevoir of B | |
| 1.1 to wash both reservoirs | |
| 0.0 no washing | |
| integrate | 0 |
| feedtube | |
| sync A.B | 0.0 |
Chart recorder:
[edit]- 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-> conductivity
- blue-> UV- 280nm
- set for “cal”, not “var”
To wash pumps:
[edit]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:
[edit]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:
[edit]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:
[edit]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:
[edit]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 %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 % | 5.0 | |
| 5.00 | min/mark | 2.0 | |
| 5.00 | integrate | 1 | |
| 10.0 | conc %B | 0.0 | |
| 10.0 | valve.pos | 1.1 | Load; this stops going through th sample loop |
| 30.00 | conc %B | 35.0 | |
| 34.00 | conc %B | 100 | |
| 38.00 | conc %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 %B | 0.0 | |
| 43.00 | conc %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:
[edit]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:
[edit]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:
[edit]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.
Methods
[edit]Old FPLC in cold Room
Method 4: Load Protein to SP FF column (valve 3.3 & 4.3), wash, then elute directly to Ni resin, wash and elute His tag protein
| Volume/ml |
Function |
Value |
Note |
| 0.00 |
VALVE.POS |
1.2 |
Inject |
| 0.00 |
VALVE.POS |
2.1 |
H2O |
| 0.00 |
VALVE.POS |
5.1 |
valve 5 by pass |
| 0.00 |
VALVE.POS |
6.1 |
valve 6 by pass |
| 0.00 |
VALVE.POS |
3.3 |
SP FF |
| 0.00 |
VALVE.POS |
4.3 |
SP FF |
| 0.00 |
FLOW C |
2.00 |
Load Sample with P1 |
| 50 |
ML/MIN |
1.00 |
sample volume |
| 40.10 |
ML/MIN |
2.0 |
start system wash (pump A, valves, tubing) |
| 40.10 |
CONC %B |
0.00 |
use water |
| 75.10 |
ML/MIN |
0.00 |
end wash |
| 75.10 |
WASH A.B |
1.1 |
|
| 75.10 | VALVE.POS | 1.1 |
Example Methods
Method # 7: Load 10 ml sample using P1 pump
| Volume/ml |
Function |
Value |
Note |
| 0.00 |
VALVE.POS |
2.2 |
Buffer A |
| 0.00 |
VALVE.POS |
4.1 |
valve 4 by pass |
| 0.00 |
VALVE.POS |
5.1 |
valve 5 by pass |
| 0.00 |
VALVE.POS |
6.1 |
valve 6 waste |
| 0.00 |
VALVE.POS |
3.1 |
Flow to position 1 valve 3 |
| 0.00 |
VALVE.POS |
1.1 |
switch MV-7 valve to Load |
| 0.10 |
FLOW C |
2.00 |
Load sample to superloop at 2 ml/min using P1 pump |
| 10.10 |
FLOW C |
0.00 |
stop pumping |
| 10.10 |
VALVE.POS |
1.2 |
switch valve to inject for sample injection |
| 10.10 |
ML/MIN |
1.0 |
inject sample |
| 10.10 |
CONC %B |
0.00 |
use Buffer A |
| 20.10 |
ML/MIN |
0.00 |
end sample injection |
Method # 0: System Wash
| Volume/ml |
Function |
Value |
Note |
| 0.00 |
VALVE.POS |
2.1 |
Wash with water |
| 0.00 |
VALVE.POS |
4.1 |
valve 4 by pass |
| 0.00 |
VALVE.POS |
5.1 |
valve 5 by pass |
| 0.00 |
VALVE.POS |
6.1 |
valve 6 waste |
| 0.00 |
VALVE.POS |
3.1 |
Flow to position 1 valve 3 |
| 0.00 |
VALVE.POS |
1.3 |
switch MV-7 valve to wash |
| 0.10 |
FLOW C |
2.00 |
Wash superloop at 2 ml/min using P1 pump |
| 40.10 |
FLOW C |
0.00 |
stop pumping |
| 40.10 |
ML/MIN |
2.0 |
start system wash (pump A, valves, tubing) |
| 40.10 |
CONC %B |
0.00 |
use water |
| 75.10 |
ML/MIN |
0.00 |
end wash |
| 75.10 |
WASH A.B |
1.1 |
|
| 75.10 | VALVE.POS | 1.1 |
Method # 1: Editable method (example)
| Volume/ml |
Function |
Value |
Note |
| 0.00 |
ML/MIN |
0.5 |
|
| 0.00 |
LOOP TMS |
8 |
Change this number according the sample volume, if it is 80 ml, loop 8 times, if it is 100 ml, loop 10 times |
| 0.00 |
CALL METH |
7 |
Load Sample using 10 ml superloop and P-1 pump |
| 0.00 |
END OF LOOP |
||
| 0.00 |
CALL METH |
8 |
Purify protein on 5 ml HisTrap HP column |
Method # 8: Purify His Tagged Protein using 5 ml HisTrap HP cartridge.
| Volume/ml |
Function |
Value |
Note |
| 0.00 |
CONC %B |
0.0 |
|
| 0.00 |
ML/MIN |
1.00 |
1 ml/min |
| 0.00 |
CLEAR DATA |
||
| 50.00 |
CONC %B |
4.0 |
20 mM (0.04X500 mM=20 mM) |
| 100.00 |
CONC %B |
8.0 |
40 mM |
| 150.00 |
PORT.SET |
6.1 |
start fraction collector |
| 150.00 |
VALVE.POS |
6.2 |
set valve 6 to position 2 |
| 150.00 |
CONC %B |
10.0 |
50 mM |
| 200.00 |
CONC %B |
60.0 |
300 mM |
| 250.00 |
CONC %B |
0 |
back to 10 mM |
| 250.00 |
PORT.SET |
6.0 |
stop collecting fractions |
| 265.00 |
ML/MIN |
0 |