Power on the system beginning with the D-7000 interface.

Open HSM software and click the “System Status” icon on the left toolbar.  Select “Initialize.”  A connection with the instrument is set up and the program is downloaded.  This may take approximately 2 minutes to complete.  Make sure the hardware detected by the computer is correct.

Purge each solvent line for 2-3 minutes by following this procedure:

1. Hit the “Manual Set” button on the L-7100 pump
2. Open the white purge valve to the right of the pumps
3. Hit the “Pump On” button
4. Hit the “Purge” button to start and again to stop the manual purge
5. Hit the “Pump On” button to turn the pumps off
6. Close the purge valve to allow solvent flow to the column

Setting up a Method Table

***Work left-to-right on the icon list at the top of the screen***

First row icons set acquisition parameters.  Second row icons are data reprocessing functions.

Method Configuration Icon
Verify the following:

Interface Module	D-7000 I/F
Gradient Mode	LOW
Channel 1 Detector	L-7455 for DAD
Channel 2 Detector	L-7485 for FL
Column	C18
Column Oven	NONE
External Instrument Software	NONE
Autosampler	L-7200
Pump A	L-7100
Pump B	NONE
Number of Solvents	One to four

Method Information Icon
This window allows you to provide a description of the method, the name of the developer, and the solvents used.  The method name is displayed as a read-only field.

Pump Setup Icon
Setup the solvent profile for your run here.  Do not change the pressure limit values.

Autosampler Setup Icon
Use to specify syringe parameters, injection method, and needle wash.  Leave the default values for needle and injection port washing.

The L-7200 autosampler permits three injection methods:

1. Cut Injection Method – the amount of sample withdrawn by the needle is slightly larger than the amount that will be transferred from the injection valve to the column.  The middle part of the sample is injected; the leading and trailing ends are not injected.  This method provides good reproducibility, however some of the sample is wasted.
2. All Volume Injection Method – the entire sample withdrawn by the needle is transferred to into the sample loop and into the column.  This method is suitable for analysis of minute samples since no part of the sample is washed.
3. Full Loop Injection Method – the sample loop is filled with sample for measurement.  This method provides the best repeatability of sample injection volume in analysis.

Syringe capacity:  0.5 ml (standard syringe)

Sample injection volume:  0.5 ml to 400 ml  (standard loop: 100 ml)

pH range:  1 to 13

Channel 1 Detector Setup Icon
For the diode array detector (L-7455), you can specify the following:

• spectra properties such as slit width, spectral bandwidth, and spectral interval
• wavelength range and monitoring wavelength
• start and stop times
• autozero the absorbance before injection
• performance check before series start

The default spectral bandwidth is Auto and the default spectral interval is 1600 msec.

Channel 2 Detector Setup Icon

For the fluorescence detector (L-7485), you can specify the following:

• time period over which data are averaged – the default is 2.0 – a longer response time gives better noise filtering while a shorter response time gives better sensitivity
• PMT voltage (Low, Medium, or High)
• emission bandwidth – select Standard (15nm slit) or Wide (30nm slit)
• if you want to set up the Sampling Period Table

Event Signal Setup Icon
Used if an external output is connected to the D-7000 Interface to receive signals.  Do not use.

Time Summary Icon
This window displays the solvent properties and gradient over time specified in the Pump Setup section.

NOTE:  The second row of Method icons are related to data reprocessing and are briefly described below.

Calculation Method Icon
Options for data quantitation and identification can be specified here.

Component Table Icon
Used to set up, review, or modify the component table for data processing.

Integration Table Icon
Specifies how the baseline for the chromatogram is determined and how the peaks are detected.

DAD Data Processing Icon
Specifies DAD detector processing properties such as peak purity checks, setting the type of chromatogram to display, and other data options.

Chrom Display Format Icon
Specifies how the chromatogram will be displayed on the Data Acquisition and Data Processing screens as well as in a report.

DAD Display Format Icon
Use this command to display and modify the DAD display format.

Confidence Report Icon
Used to set report parameters for System Sustainability Tests, Module Performance Tests, and Data Diagnosis.

Report Format Icon
Used to select report options, including summaries, statistics, report layout and destination, as well as reported peaks.

Update Method Icon
Use this command to update all parameters of the current method to all other open windows.  This command is particularly useful if you are modifying the data processing or displaying parameters in the current method and want to update the changes to the open Data Display window.

REPORTS
Two types of reports:
Original report – raw data – this report never changes
Modified report – any changes made to an original report are saved as a modified report.  You can create any number of modified reports, however, the report is overwritten each time you save.

Starting Data Acquisition

Open a Sample Table for data acquisition and click the eyeball icon on the left tool bar.  The “Idle Monitor” window will appear.  (Make sure the system has been initialized with the proper components!)

Manually turn on the pumps.

Click on the “Start Series” button to begin data acquisition.

***Make sure to set system to SLEEP MODE if your run will continue overnight***