Determination of Polyaromatic Hydrocarbon Mixtures.

1. Start-up and purging the solvents

1.1. Before starting the experiment, the reservoir bottles of acetonitrile and water should be at least half-full. These bottles are located on the solvent delivery tray above the pump control unit.

1.2. Open the main valve on the top of the helium tank. If necessary, rotate the regulator valve until the pointer is at the first red triangular mark.

1.3. Switch the toggle valve on the solvent delivery tray to ON. After 2 to 3 seconds, helium gas should be bubbling through the solvent in reservoirs A, B, C. (Because methanol solvent will not be used (reservoir B), the flow through B should be adjusted to zero.) A good helium flow rate is indicated when large bubbles emerge continuously from most of the holes on each sparger. Increase the helium flow through A (H₂O) and C (acetonitrile, ACN) by adjusting the knobs on the solvent delivery tray (counter-clockwise = increase). Allow helium to flow through solvents for at least ten minutes before priming the pump. Helium flow in each bottle should be left on, but at a lower rate, during the course of the experiment.

1.4. Turn on the power for the detector and the pump control units. Switches are located at the lower right and left of the units respectively. The detector takes 30 minutes to warm-up for the best performance.

2. Priming the pump

(Also see Spectra-Physics manual p. 1-15 to 1-16, Section 1.4)

2.1. Place a beaker at the outlet of the white rubber tubing, which comes out behind the column panel. The pump control panel should display %A at 100%, %B and %C at 0%, flowrate at 0 ml/min.

2.2. Press "Edit/Run Mode" on the pump control panel to change to the Run Mode (red light on the left of Run Mode should light up). Press "Purge", "Enter". Quickly open the column bypass valve. Observe the waste stream at the output of the white tubing. Air bubbles will come out with the solvents first.

2.3. Allow the flow to continue until no more air bubbles are observed, press "Stop" and close the column bypass valve. Discard solvents to the waste bottle.

2.4. Press "Edit/Run Mode" on the pump control panel. Press "%C", "100", "%A", "0" and "Enter", (100:0 ACN/H₂O solvent). Press "Edit/Run Mode", "Initialize", "1", "Enter"; you will see "F305"; then repeat 2.2 and 2.3.

2.5. Press the "Edit/Run Mode" on the pump control panel. Press "%C", "70", "%A", "30" and "Enter", (70:30 ACN/H₂O solvent). Press "Edit/Run Mode", "Initialize", "1", "Enter"; you will see "F305"; then repeat 2.2 and 2.3.

NOTE: If your are assigned to calibrate the pump, continue with section 3. Otherwise proceed to section 4. Before proceeding, check with the lab instructor to confirm that the column has been stored with the 70:30 ACN/H₂O solvent. If the column has been stored with methanol, the desired mobile phase composition must be achieved by gradually changing the solvent composition. (Substitute Section 7 for 3.2 or 4.1. below.)

3. Calibration (Do this section only if requested by the lab instructor)

3.1. Unscrew the plastic waste tubing from the detector.

3.2. Be sure that the solvent is the 70:30 ACN/water mixture.

3.3. Place a waste beaker at the outlet of the detector. Press the "Edit/Run Mode" on the pump control panel to change to Run Mode. Then press "Initialize", followed by "1" and "Enter". Observe the flowrate at the outlet of the detector. Once the flow stabilizes, place a dry 5 mL volumetric flask at the

outlet of the detector, and measure the time, to the nearest second, required to fill the flask to the mark. Repeat the timed 5 mL collection. Calculate the flowrate. Discard beaker and flask contents to waste bottle.

3.4. Record the results for all three flow rates in your laboratory notebook. Also record your results in the HPLC Instrument Calibration Record Book.

4. Stabilizing the column

4.1. On the pump control unit press "Edit/Run Mode" to return to Edit Mode. Press "Flow", "1", "Enter"; press "Edit/Run Mode", "Initialize", "1", and "Enter". The 70:30 ACN/H₂O is flowing through the column at 1.0 mL/min.

4.2. Check the highlighted messages on the detector screen. Proceed with the following detector adjustments, if "diagnosis is OK". Otherwise, check with your instructor first. Some detector commands are on the front panel, and some are displayed on the screen. The screen commands correspond to the pushbuttons (A thru E) mounted below the display screen.

4.3. To check if the column has equilibrated with the mobile phase or not, select "Monitor". A chromatogram will appear on the screen. To change the absorbance scale (y-axis) from ± 1 to ± 0.0078 AU (displayed on the screen), press the "up" cursor key. The average signal, or baseline, should be flat. About one hour is required to stabilize the column. (See Section C of the experimental writeup (p. 53) regarding sample prep during this time.)

4.4. Check pressure across the column from the pressure gauge next to the pump. With a flow of 1ml/min., the column back pressure should not be more than 67bar. If not, stop the pump and check with your instructor

When the baseline signal stabilizes (see 4.3), proceed with section 5.

5. Sample Injection

5.1. Working in the hood, place ~2 mL of 70:30 ACN/H₂O in a clean 10 mL beaker. Rinse the (100 mL) sample syringe with this solvent by filling 3 times and discarding to the waste bottle; gently wipe the syringe needle with a Kimwipe tissue. Use the syringe to withdraw sample from vial B, through the septum, filling the syringe about 3/4 full; deliver this to a waste bottle. Repeat twice. Then place the waste beaker under the two open-ended, silver capillaries at the back of the column panel.

5.2. Place the syringe needle into the solution in vial B again; withdraw about 80 mL of sample. Remove any air bubbles by slowly moving the plunger up and down while the needle tip is immersed in the solution. If bubbles remain inside the syringe, make sure they are at the top of the syringe, touching the plunger, and that there is ~80 mL of solution in the syringe.

5.3. Adjust the injector switch to "Load" position. Remove the syringe from vial B. Place the loaded syringe completely into the needle port of the injector valve. (Careful! Don't bend the needle!) Push down the plunger slowly (avoiding the injection of air bubbles) to inject sample into the sample loop, and continue until a few drops of sample may be observed coming out of the lower silver-colored capillary behind the column panel, and more than 10 mL is injected (indicating that the sample loop (10 mL) is filled). Touch a waste beaker to the capillary tip to catch any droplets. If this is the first run, quickly press "Run" on the detector unit, and the question "are you sure? 1-Yes; 0-No" may appear on the detector screen; then press "1" and pull injector valve to "inject" at the same time. For subsequent runs, press "Run", and pull injector valve to "inject" at the same time. (DISCARD waste beaker contents from the sample loop overflow.)

5.4. The programmed run-time of each injection is 12 minutes. At the end of the 12 minutes, chromatograms and results will be printed out. Wait until

"Post Analysis Menu:

1. Integrator

2. Spectrum Index

3. Chromatogram

4. Library Edit

5. Spectrum Edit"

appears on the detector screen; then go to 5.5.

 

5.5. Repeat 5.1 to 5.4 for vial C (the standard addition sample).

5.6. The next student sample runs begin at 5.1. Otherwise go to 6.

6. Shut-Down Procedure

6.1. Turn detector unit "OFF".

6.2. Press "Stop" on the pump control unit. Then turn the

power switch "OFF".

6.3. Turn off the main valve of the Helium tank (clockwise); wait until the

pointers of the gauge are at zero. Then open the regulator switch (loose

position).

6.4. Turn pump control unit toggle switch to "OFF" (up).

6.5. Sign HPLC instrument Log Sheet.

 

7. Procedure for Gradual Change in Mobile Phase Composition

From 100% Methanol to 70:30 ACN/H₂O

If instructed, use the following procedure to precede Section 4 if the column has been stored in 100% methanol.

7.1. On the pump control unit press "Edit/Run Mode" to return to Edit Mode. Press "%B", "80", "%C", "20" and "Enter". Press "Edit/Run Mode" to change to the Run Mode. Then press "Initialize", followed by "1" and "Enter". The 80:20 Methanol/ACN composition is being pumped through the column at 1.0 mL/min. Allow this to flow through the column for at least one minute.

7.2. Repeat 7.1 with 60:40 Methanol/ACN (60/40 %B/%C) for one minute.

7.3. Repeat 7.1 with 40:60 Methanol/ACN (40/60 %B/%C) for one minute.

7.4. Repeat 7.1 with 20:80 Methanol/ACN (20/80 %B/%C) for one minute.

7.5. Repeat 7.1 with 100% ACN (0/100 %B/%C) for one minute.

7.6. Press "Edit/Run Mode" to return to Edit Mode. Press "%C", "80", "%A", "20" and "Enter". Press "Edit/Run Mode" to change to the Run Mode. Then press "Initialize", followed by "1" and "Enter". The 80:20 ACN/H₂O composition is being pumped through the column. Allow this flow for at least one minute.

7.7. Repeat 7.6 with 70:30 ACN/H₂O (70/30 %C/%A). Allow the flow to continue at this composition.

NOTE: The above procedure can be modified to start with any given composition of column storage, and gradually change to any other desired mobile phase composition.