Starting the system:

1. Open Valve on Helium tank to supply pressure,

2. Turn on switches for reagents to be used on the Reagent Delivery Module.

The reagent 1 switch currently controls reservoirs 1 and 4.

3. Turn on Eluent Degas Module by turning system switch to the on position. Then, turn on the reservoirs that are going to be used (Usually 1 and 4 for Anions, but is possible to run up to four different reservoirs).

4. Enter log in name: DUNBAR

5. Enter project name: C

6. Select "k" to start Windows 3.1

7. Enter Password: SEMAS

8. When in Windows, double click on the Main Menu Icon in the Dionex AL-450 window. This will start the program with which the entire HPLC system will be run.

Rinsing the Column:

Checking the Standard:

1. Double click on the Run icon in the analysis box. This will bring up the AL-450 model 2-Run window. You may already be there from the rinse step.

2. To load a method, click on Load on the menu bar. From this, select Method.

3. For an Anion column standard, select ANIONSJ2.MET from the file window. Then click on OK. This will close the load window and ...Loading the GPM event steps will be displayed the Dionex Window. Watch the panel on the Gradient Pump Display to insure communication between the computer and HPLC system.

4. Inject a sample into the sample port using the appropriate syringe. Push enough into the port such that drops are noticed falling into the waste (not to be confused with eluent drops).

5. Click on Run from the menu bar. This will bring down Start, End, and Abort. Click on Start.

6. The start method window should appear. Enter a sample description (up to 30 characters). Data file name will automatically be generated. In the box labeled start, make sure Automatically is selected.

7. Click on OK. This will start the run. A real time plot of the chromatogram will be displayed. After 0.2 minutes, the piston valve for the sample chamber should activate, and should close after 0.7 minutes.

Running a Sample:

1. Running a sample follows the same procedure as running the standard. However, it may be necessary to use a different method depending on the analysis that is to be performed. A report will automatically be generated at the end of each run.

Shutting Down the System:

1. To shut down the system, open the Rinse2.met method file as was done above. Click on run to begin flushing the column. At the end of this run, the pump will automatically shut off.

2. Turn off the Helium

3. Turn off the Reagents

4. Turn off the Eluent Degas Module as well as the Individual Reservoirs.

5. Log off the computer by exiting windows and pressing F5 at the prompt.

Developing a Method:

1. Double click on Method in the main menu. This brings up the AL-450 Method window.

2. Click on System button to choose the system to be monitored. Currently, Dionex is the only system. Click on OK to return to the method window.

3. Select the detector(s) that are going to be used.

4. To set up the detector, click on Detector 1. This will bring up the Select Detector 1 window.

5. Click on Configured Dionex Detector.

6. Click on the arrow pointing down to select the detector that is to be used.

For Anions, select CDM II Conductivity Detector

For Metals, select Variable Wavelength Detector

7. Click on OK to return to the method window.

8. After selecting detectors, the computer will automatically generate recommended values for the plot minimum and maximum. These usually work very well, but can be adjusted in post-data processing.

9. Select the appropriate run time by entering it into the box.

10. Select a sampling rate. The is the speed at which the interface talks to the computer. 5 Hz is suitable for most applications.

11. In the Instrument Control Box, click on Gradient to setup the eluent to be used and injection time for the sample. This will bring up 2 windows, the Gradient Editor and the Gradient Pump Setup (this one will be active).

12. To label the eluents, click on boxes next to the appropriate reservoir (E1, E2, E3, or E4).

13. Change the labels on switch V5 by clicking on the boxes next to the switch position (1 is on, 0 is off). Label position 1 as Inject and position 0 as Load.

14. For switch V6, label position 1 as Metals and position 0 as Anions.

15. In the File Description Box, it is possible to include a brief discussion of what this gradient method is to be used for.

16. Click on OK to go to the Gradient Editor window.

17. At time 0.0, set flow rate at the appropriate level.

18. The comment window will allow you to each step of the gradient elution.

19. The Eluents column will allow you to set the percentage of each reservoir passing through the column. Set the initial eluent mixture.

20. Adjust time for next step in the gradient elution. To inject a sample, select inject on switch V5. Allow enough time for the eluent to pass through the injection loop, then select load on the same switch. Continue to adjust the gradient elution levels for the rest of the run, if necessary.

21. The graph will continually show the mixture of each reservoir over the period of the run.

22. Click on File from the menu line.

23. Click on Save Gradient File as...

24. Enter a name for the file and click on OK.

25. Double click on the box in the upper left corner to close this window. This will return you to the AL-450 Method Window.

26. Next to the Gradient button, the gradient file name should be displayed.

27. Click on the Timed Events button to bring up the Timed Events editor.

28. At time Init, the settings for this window should be:

                            Time: Init

AGP-I: Start

                            CDM-2: Cell

                            Temp. Comp.: 1.7

29. At time 0.0, need to reset gradient program and turn on the offset for the detector to set the baseline at 0. The settings should be:

                            Time: 0.0

                            AGP-I: Start

                            Run

                            Reset

                            CDM-2: Offset

                            Cell

Temp. Comp. and Range need not be changed.

30. When sample is loaded, the program must be told to begin sampling data and the reset for the gradient pump program must be turned off. The settings should look like:

                            AGP-I: Start

                            Run

                            CDM-2: Offset

                            Cell

                            ACI: Begin Sampling

31. After the run is complete, stop sampling the data and the gradient pump. The settings should look like:

                            AGP-I: Start

                            CDM-2: Offset

                            Cell

32. 0.2 minutes after the end of the run, turn off the offset for the detector.

33. Save the file by clicking on File and then Save As...

34. Double click upper left corner of window to close.

35. Click on the box next to Save Data As to save all data collected to disk.

36. To save to a specific place, click on Directory... This will bring up the Save As window which will allow you to edit the name of the file as well as location of the files. Filenames are limited to 5 characters, because the system uses the rest to identify system used and prevent file overlap. Double click the upper left corner of this window to exit.

37. Click on Detector 1 to begin setting up the detector. This will bring up the Data Processing Parameters Window.

38. Click on Integrate to modify the integration parameters:

Peak width: This is the minimum width a peak must have to be recognized by the system. 5 to 10 seconds is usually satisfactory.

Peak Threshold: This is the minimum slope a peak must have to be recognized.

Area Reject: sets the minimum area for a peak before discarding the data as noise

39. Click on OK to return to the Data Processing Parameters Window.

40. Click on Data Events to set up how the processing software deals with the data. This will allow you to manipulate the data at different times throughout the chromatograph. Some of the more important events are:

Start and Stop Peak Detection: tells software when to start and stop recognizing peaks

Double Peak Threshold: doubles the minimum slop a peak must have to be recognized.

For further information, please refer to the manual, pages 6-28 and 6-29.

41. To add an event, select the event and the time for which it is to occur and then click on Add Event. To remove one, select the event and click on Delete Event.

42. Click on Exit to return to the Data Processing Parameters window.

43. Click on Calibration Parameters to set-up the parameters with which the software will use to calculate concentration.

44. Levels is the number of data points used to create the line for the calibration curve.

45. Fit type allows you to specify the type of equation used to create the calibration curve (i.e. linear, quadratic, etc.)

46. Force zero forces the calibration plot to intersect the origin.

47. Calibration update allows you to select whether new calibration data will replace the old data or be averaged with it.

48. Standardization selects whether internal or external standards are to be used.

49. Response for unknowns allows you to select whether the area under the curve or the height of the curve is reported for unknown peaks.

50. Defaults are the initial values with which the software is set-up at the start of a run.

51. Sample volume is a factor that relates sample sizes. If doubling the sample size during a run, double this number to maintain results.

52. Dilution factor allows you to set-up method to automatically account for the dilution of a sample and report as the concentration of the original sample.

53. Unknown Response Factor should be left at zero.

54. Calibration Standard Volume serves the same purpose as the sample volume but for the calibrating species.

55. Internal standard amount in samples is the amount of internal standard auto injected into the sample. Not used with external standards (Leave at 1)

56. Amount Units: the units desired to express concentration.

57. Click on OK to return to the data processing parameters window.

58. Click on Components. This will bring up the component table window and Add Components window (active).

59. Component name: Name of component to be detected and printed on report. Maximum of 16 characters.

60. Retention Time: The time the analyte is expected to remain in the column in minutes. If not known, enter an approximate time. The exact value can be determined from the first run.

61. Retention Window: This is the amount by which the peak may shift from the expected time and still be recognized. Can be expressed in minutes or percent of expected retention time. A 3% window is usually satisfactory.

62. Calibrate by allows you to chose whether the area under the curve of the peak height is used to determine the calibration curve.

63. Amount Limits (high and low): These are the limits of concentration that you wish to detect. For the low amount, the lower detection limit of the analyte is used.

64. Reference Component #: Entering a value here will automatically shift the retention windows for the other components based on the shift of this one.

65. Click on OK to return to the Data Processing Parameters. Click on OK to return to the Editor window. Save the file by clicking on File and then Save As...

Creating and/or modifying the calibration curve:

1. Prepare solutions with known concentration of the analytes. These should be the same as the concentrations input in the levels of the components.

2. Run the samples using the method that the calibration plots are to be used for.

3. To setup the plot, return to the main menu and double click on Optimize.

4. Click on File and open the data file for the run with the concentrations for level 1.

5. Click on Operations, then select Calibrate...

6. This will bring up the component update window. Select Level 1, choose the type of options. Can print the calibration update and Confirm changes.

7. Click on OK to automatically input the data for the concentration level. Repeat this for each of the levels.

Hints For Data Storage:

1) For samples that are fairly regular, establish a method specifically for this analysis. This is beneficial because it is then possible to name the data files specifically for these analyses and/or store them in specific places.

2) If the data needs to be separated by dates, two different things are possible:

1) Instead of allowing the datafile to be stored as a name, edit the filename in the method editor such that it contains the date: For example, make the file name SeptXXXX.XXX or 111295XX.XX. It will then be easy to see when each test was run.