1. Verifying That Everything Is Turned On

HPLC Procedure

1. Verifying that everything is turned on:

Make sure that the power strip is on.

Check the Pump flip the switch to on.

The two detectors have buttons that will light up if they are on. Press these if the lights are off. If the detectors need to be turned on wait 15 – 20 minutes before running the samples.

The column is on when it is plugged in. It has the black cord, plug it into the wall.

2. Flushing the system with the mobile phase:

First flush the reference side. On the pump there is a metal switch that moves from left to right, it should be in the left position, this runs the mobile phase through the detectors with the sample. After turning the pump on, move this switch to the right to verify that the reference side has clean mobile phase liquid in it. After verifying that the mobile phase is coming out of the reference side, move switch back to the left.

3. Preparing the test sample:

Rinse the syringe with the sample to test. Fill the syringe with 40 micro-liters of sample. Verify that the top switch reads load, if this is not the case when the plug is released it will come shooting out with 2000 psi behind it, very dangerous. Unlock the plug for the fill reservoir and place it in the hole on the top switch. Inject the sample. Replace the plug into for the fill reservoir and lock it.

4. Starting the software:

Double click on the diamir icon. You will be prompted for a password - it is written in pen on the computer case upper left hand corner - it is 5503. Now that you have loaded the software:

Choose File: Open: Method

Select 2signals.meth

On the left hand side of the screen on the bottom of the screen select the System Tab. Make sure that the Box next to Waters HPLC is checked.

5. Preparing the software for acquisition:

Select Acquisition: Quick Start

Select the method from the drop down box. This needs to be the same as the method selected at the beginning, i.e. 2signals.meth.

Click OK

Enter your file name in the file prefix box; this is not old format so it can be longer than 8 characters.

A description may also be entered if you want, in the description box.

6. Acquiring the data:

This step requires two people, as the switch does not yet work for automatic logging. Have one person stand at the computer and another by the pump. They will need to move the switch from load to inject and click the start button at the same time. Now you should see the graphs beginning. After 30 seconds move the switch back to load. The next sample can be injected to start when this sample is done.

Run the sample until it has cleared the column, 10 – 12 minutes. Once the sample has cleared the column select the stop button, the red one with a white X. Select OK.

7. Printing your chromatograph.

Select:

File: Open: Chromatograph

Select your file name. To print it, press the printer icon, or select File: Print.

This printout will have the start and stop times of all the peaks as well as the areas, heights and percent areas of each of the peaks.