Determination of Polyaromatic Hydrocarbon Mixtures.

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IF THE INSTRUMENT IS ALREADY OPERATIONAL, GOT TO SECTION 3.

1. Instrument Start-up:

1.1. Before starting the experiment, the reservoir bottle of mobile phase (70:30 acetonitrile:water) should be at least half-full.

1.2. Turn on the power for the detector and the pump control units. The detector takes 30 minutes to warm-up for the best performance.

1.3. If the red reset button on the pump is lit, press to reset the pump.

1.4 For the first run of the day, turn on the computer system, bringing up the LOGIN window on the computer. (If in the Program Manager Window, double-click on the Millenium Express ICON to bring up the LOGIN window.) Enter the User Name: DEMO; TAB to the Password block; and enter the user password.

2. Priming the pump (Waters 510)

2.1. Place a beaker under the plastic tubing bypass outlet of the pump. Rotate the large toggle switch on the pump to "Bypass"; Rotate counter-clockwise (open) the round "Bypass Valve".

2.2. Use the 5.0 mL syringe to withdraw a few mL of solvent. Air bubbles will come out with solvent at first.

2.3. Continue withdrawing solvent until no more air bubbles are observed; close (clockwise) the bypass valve. Reset the large toggle switch on the pump to "Flow to Column". Discard solvents to the waste bottle.

3. Preparation for Sample Injection: (Procedure used for all subsequent injections)

3.1. 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.

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3.2. 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 ~50 mL of solution in the syringe

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

4.1. Flow Calibration

4.1.1. Unscrew the plastic waste tubing from the detector.

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

4.1.3. Place a waste beaker at the outlet of the detector.

4.1.4. Proceed through Section 5 as though running a sample, but inject only solvent.

4.1.5. At Section 5.8 adjust the pump flow rate to 1.0, 2.0, and 3.0 ml/min for each of three calibration runs. This adjustment is made by double-clicking on the pump (PCM) icon in the "Quick Set: HPLC_996" window; then changing the flow rate selection to the appropriate value for each run. After the three calibration runs, return the flow rate to 1.0 ml/min.

4.1.6. During each flow calibration run, observe the flowrate at the outlet tubing of the detector which drains to the waste bottle. Once the flow stabilizes, place a dry 5 mL volumetric flask at the detector outlet tubing, and measure the time, to the nearest second, required to fill the flask to the mark. Repeat the timed 5 mL collection at the 2.00 ml/min rate; and again at the 3.00 ml/min. rate. Calculate the flowrates. Discard beaker and flask contents to waste bottle.

4.2. Column Calibration

Following the directions in Addendum B, calculate the value of theoretical plates (N) obtained for each analyte done by the group.

4.3. Record the results for all three flow rates and the theoretical plates values in your laboratory notebook. Also record results in the HPLC Instrument Calibration Record Book.

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5. HPLC (Waters Millenium System) Operation