Running the PF2D 
 
 
 
 

Preparing the Buffers:  
 

1. Remove the Start Buffer and the Eluent Buffer from the refrigerator and let come to room temperature.  **Do not apply heat to warm them, but they can be placed in room temperature water.
2. You must pH new batches of buffers.  If they are not new, however, only pH them once a week.  **overshooting the pH can cause steep gradients in the run.
3. pH the Start Buffer to pH 8.5 (using either iminodiacetic acid 50mg/ml stock or 1M NH₄OH.
4. pH the Eluent Buffer to pH 4.0 (using either of the above listed chemicals).
5. Once they have been pHed, either put them on the machine for use or store them at 4°C.  They can be aliquoted for separate runs.  100ml of the Start Buffer and 60ml of the Eluent Buffer should be enough for 1 run.

Priming the lines and the pumps 
 

      1.  The lines and pumps must be primed before any use of the machine in order

            to introduce fluids to the lines and to remove any air bubbles. 
 

      For the 1^st dimension:

1. Open the waste valve by turning the knob 2 rotations counter clockwise.
2. On Direct Control, select position A (start buffer).  The flow can be off or on at .2mL/min w/ .5min duration.
3. Insert syringe into the priming port.
4. Turn the valve down to the “Prime Lines” position and pull out 5-7ml of fluid with the syringe.
5. Turn the valve back to the “Operate” position.
6. Remove the syringe and empty contents into waste container.
7. On Direct Control, select position B (eluent buffer).
8. Reinsert syringe, turn valve to “Prime Lines” and remove 5-7 ml of fluid.
9. Turn valve to “Operate”, remove syringe, and empty contents into waste container.
10. On Direct Control, select position C (HISS+ solvent) and prime as you did the other two lines.
11. Once you have selected the last position D (water), pull 5-7 ml of water into the syringe while the valve is in the “Prime Lines” position.  Then turn the valve to the “Operate” position. 
12. Remove the syringe and get the air bubbles out of the syringe by flicking and moving plunger up.
13. Reinsert the syringe.  Move the valve to “Prime Pump” position and push the 5-7ml of water through the pump.  Note:  if you can’t push the water through, then you probably didn’t open the waste valve.  Otherwise, it could be a clogged filter on the valve unit.
14. Turn the valve back to the “Operate” position.
15. Close the waste valve by turning clockwise.

For the 2^nd dimension: 
 

1. Open the waste valve by turning the knob 2 rotations counter clockwise.
2. Nothing needs to be selected on Direct Control, because only one position is used on pumps B and A.
3. Insert a syringe into the priming port of pump B (the top pump).
4. Turn the valve down to the “Prime Lines” position.
5. Pull out 5-7ml of liquid.
6. Turn the valve back to the “Operate” position.
7. Remove the syringe and get rid of the air in it.
8. Reinsert the syringe, turn the valve up to the “Prime Pump” position, and push the liquid back through the pump.
9. Remove the syringe and repeat the same procedure with pump A.
10. Remember that when you get rid of any waste from these pumps, be sure to put it in the acetonitrile waste container.
11. Close the waste valve by turning the knob clockwise.

Installing the columns 
 

      **Leave the pH probe off the machine until the 1^st dimension column has been

                flushed, so that all of the alcohol has been flushed out.  We do not want

                alcohol passing across the probe. 
 

      1.  The columns must be flushed every time they are put back on the machine

                  in order to remove the storing fluid from the columns.  (The 1^st dimension

                  columns are stored in 10% ethanol, and the 2^nd dimension columns are stored

                  in 100% acetonitrile.) 
 

      2.  Attach each column onto the lines by tightening the end screws firmly.  Make

                 sure you are putting each column on in the same direction each time.  The 1^st

           dimension column tells you which direction.  The 2^nd one doesn’t, but you

                 should still install it in the same direction each time. 
 

      3.  Go to Direct Control on the 32Karat PF2D software.  
 

      For the 1^st dimension column: 
 

           select the D position (Water) and turn the flow

            on to .200mL/min  over a .5 minute duration.  **Don’t leave that duration  

            time at zero or you could slam the pumps.

                  Let the water pump through the column for 45 minutes. 
 

      For the 2^nd dimension column: 
 

           select pump B (.1%TFA in Water) to be on at 100% and pumpA to be at 0%

           (*Note:  you can only change the percentage of pump B, and that in turn

               changes the percentage of pump A)

                 Turn the flow on to .75mL/min over a 0.5 minute duration.  Let it flow for 20

           minutes.  Then change pump B to 0% making pump A 100% (.08%TFA in

                 acetonitrile).  Let it flow for 20 minutes. 
 

      4.  Once the columns have been flushed, they are ready for equilibration. 
 
 

Calibrating the pH meter 
 

1. The pH meter must be calibrated before each new day’s run. (For 1^st dimension runs put back to back on the same day, you wouldn’t need to recalibrate in between.)  It can be done before equilibrating the columns or after, but it must be done before you start a run.
2. Use fresh pH buffers from Beckman.
3. If the pH meter is not connected to the 1^st dimension, remove it from the holding vial.  Otherwise, remove the pH meter from its position on the machine.
4. On Direct Control, click on “Calibrate” next to the picture of the pH meter.
5. A window will pop up showing the current calibrations.
6. Click “Calibrate”.
7. You must then enter the first value to which you are calibrating.  Enter 4.0.
8. Put the pH meter into your pH4 buffer and then click “Calibrate”.
9. It may take several minutes, but it will prompt you once it’s done.  **It is possible for an error message to pop up here (according to Amy, so I’m not sure what it says).  Just click out of it and continue.
10. Continue on with calibrating pH 7 and pH 10.01 in the same manner.  **Be sure you enter 10.01 instead of just 10.  It does make a difference.
11. Once you have finished,  put the pH meter into its position on the machine and screw in firmly.  .             **When doing this you can squirt milliQ water right into that position on the machine before screwing in the pH meter to ensure no air bubbles will be incorporated.
12. Now the pH meter is calibrated.  **It is very important to remember that later when you start a run, it will prompt you to calibrate.  Just click “Close”.  If you accidentally click “Calibrate” then you will need to calibrate the pH meter again.

Equilibrating the columns 
 

1. Each column must be equilibrated before a run.
2. Prime lines and pumps if you have not already done so (see above directions).

For the 1^st dimension: 
 

1. On Direct Control, select the A position on the pump (Start Buffer).
2. Turn the flow on to .2mL/min over a .5 minute duration.
3. Let the Start Buffer flow through the column for no less than 130 minutes (2hr 10 min).  You can go longer, but you cannot go shorter.  Do not start your timer until you are sure that the pressure is holding steady.  If it keeps dropping, then you have an air bubble somewhere.  Try priming the pump again or increase flow rate for a little bit.
4. Once the 2hr and 10 minutes has passed, the column is ready for an injection to be run.  **Be sure to have equilibrated the 2^nd column before moving on if you plan to have the dimensions run back to back, because once you start a run you can no longer equilibrate the 2^nd column.

      For the 2^nd dimension: 
 

1. On Direct Control, make sure that pump B (.1% TFA in water) is at 100% and pump A is at 0%.
2. Turn the flow on to .75mL/min with a .5 duration for 5 minutes.
3. Then switch pump B to 0% making pump A (.08% TFA in acetonitrile) at 100%.
4. Leave the flow the same and let it run for 10 minutes.

**Note: if you have just installed the 2^nd dimension column, then has really already been done, so you can skip it. 
 
 

Loading a sample and starting a 1^st dimension run 
 

      **Depending on which method you choose to run, the 2^nd dimension run can

          start automatically after the 1^st dimension.  These instructions, however, will

                deal with only running the 1^st dimension, since this is how a run will normally

                be processed. 
 

1. Make sure your lines and pump have been primed.
2. Once you have equilibrated the column for 2hrs 10min, then you are ready to run a sample.
3. Load a block onto the FC/I.
4. Turn the plate cooler on by going to Direct Control, click the FC/I tab at the top, then select “tray cooler” and check “on”.
5. Make sure that the pressure on the pump is steady.  If it keeps dropping down, then you have air in the lines.  The pressure should be steady, though, if you’ve equilibrated the column.
6. Click on the single blue arrow (which means you are starting a first dimension run).
7. A window will pop up.

Run Information:

      Sample ID:  Enter the name you want

      Method:       PF2D_Default_v2pH8_5-4_5ml_loop_2mlplate.met

                     (This method is for running the 1^st dimension on the

                      5ml loop with a pause and shutdown before running

                                  the second dimension.)

      Data Path:   Choose the folder you want data dumped into

      Data ID:      Enter the identification name you want (I enter the

                                same name as the Sample ID) 
 

leave everything else as it is, and make sure the “Vial” position says

“Start”  (meaning it’s collecting from the 1^st Dimension…otherwise if

were doing this method for the 2^nd Dimension, you would indicate the

well position from which it should inject)

      8.  Now you can click “Start”  (**there is also an option on the right side of the

      window allowing you to start the program after an entered amount of time)

      9.  A Head Pressure window will open.  At the bottom of this window there is a

           display box telling you what the machine is doing.  Once it says:  “Waiting for

           Trigger”    you can inject the sample. 
 

    10.   To inject the sample:   leave the loading port knob in the “injection” position

              which is to the right.  Pull your sample into a 10ml syringe and attach

  the CLEAN injection tip to the syringe.  Insert the injection tip into

            the loading port.  Turn the knob to the “load” position (to the left), inject

            your sample, then turn the know back to the “injection” position (to the

            right.  Now you may remove your syringe.

      (**The reason the positions are named “injection” and “load”, which seems

            backwards is because in the “load” position you load  your sample, but

            the sample is not actually injected into the lines until you switch the

            position back to “injection”.) 
 

    11.  You will notice at the bottom of the Head Pressure window, it will now register

      that the sample has been loaded.

    12.  Flush the injection port with 10 to 20 ml of MilliQ water using a 10ml syringe

      and the plastic syringe adapter.

    13.  Now clean the injection tip you used to load the sample by flushing plenty of

      alcohol and milliQ water through it. 

    14.  Let the machine run overnight.

Running the 2^nd Dimension 
 

**At this point, I usually have flushed the 1^st Dimension column for long-term storage and have removed it, since you can’t do things with the 1^st Dimension while the 2^nd Dimension is running.  See instructions below. 
 

1. Make sure the column has been installed and equilibrated.
2. Have your block on the FC/I…either left there from the previous night or pulled from the freezer, thawed, and vortexed lightly.
3. If you are going to collect the samples that are fractionated during the run, place blocks on the 2D fraction collector (see separate instructions).  If you do not plan to collect the samples, make sure the fraction collector is turned off so the fluids will go to the waste bottle.
4. On Direct control, click the FC/I tab at the top to go the FC/I controls page.
5. Click on:  Needle Wash – Okay    (**make sure there is plenty of 10% Methanol in the bottle on the FC/I.
6. Do this needle wash at least 3 times before the run to prevent or remove any crustation from the buffers.
7. Open your Sequence Data :  File – Sequence – Open

             The sequence file created from your 1^st Dimension run should be found in

             My Computer – Local disc (D) – 32Karat – Projects – Default - Sequence

8. You can edit your Sequence Data file…for example, right clicking to add a line so you can insert a shutdown step.  You should in fact do this, since you will probably only be able to collect fractions for 16 samples in the 8 blocks that can fit on the collector (if collector is set at .25 sec, 50 wells).  If you don’t plan to collect samples, you can run all of the fractionations by selecting (highlighting) all of them…otherwise, select the ones to run.  Then click on the double green arrow.
9. I leave the Sample name as it is and click “Start”.  (**there is also an option here for making the program begin after an indicated amount of time)
10. Check on the collector every now and then to make sure it’s dripping into the wells, otherwise you’re good to go.

Long Term Storage of Columns 
 

1. Each column should be flushed with the appropriate fluid (1^st D column:  10% isopropanol           2^nd D column:  100% acetonitrile)  for 45 minutes.
2. Remove the column, replace the end plugs, and store at 4°C.
3. Turn the pump flows off, and place the appropriate adapter piece between the lines that held the column  (it’s just to prevent dripping, but it doesn’t always work).