Agilent 1200 HPLC -- A genral procedure is described for HPLC analysis to obtains separation, such as shown below. The following is a general description of the instrument. It gives spectral information from 190 to 950 nm with dual-lamp design with the Agilent 1200 Series diode array detectors ensure outstanding sensitivity. Photodiode arrays with 1024 elements, programmable slit width (1, 2, 4, 8, 16 nm) and the selection of nine different flow cells enable optimization of resolution, linearity and sensitivity. High resolution chromatography reveals more small particle size and long separation columns are key for higher efficiency and resolution. The Agilent 1200 Series Rapid Resolution system uses the the ZORBAX Rapid Resolution HT columns with 1.8 μm particles, providing resolution of complex samples.

Agilent 1200 Series quaternary pump -- The quaternary pump provides highest flexibility in solvent mixing and is recommended for a wide range of research and routine applications, and in method development.
• Speeds up method development, preparation of mobile phases and flushing the HPLC system by offering convenient access of up to four solvents for isocratic or gradient analysis
• Keeps maintenance to a minimum for lower operation costs by using robust materials such as stainless steel, titanium, gold, ruby, sapphire, ceramics, PEEK and PTFE that withstand most demanding applications, ensuring long lifetime
• The optional active seal wash option is recommended for high salt mobile phases to prevent corrosion
• A wide flow range of up to 10 mL/min for broad HPLC work, including semi-preparative separations
• Incorporated vacuum degasser, which is mandatory for low pressure gradient mixing, offers high degassing efficiency, trouble-free operation and highest performance

Software -- ChemStation is the software for Agilent instruments for UV/VIS, GC, LC, LC/MSD, CE, CE/MSD, and A/D system. It contols Instruments, data acquisition, and data evaluation systems. The software is designed to run on IBM-compatible personal computers under Microsoft Windows XP Professional operating environment. The ChemStation user interface is designed into Views that group software functionality according to typical analytical tasks. The following three standard views are present in all software configurations:
• the Method and Run Control view for controlling and acquiring data from the instrument,
• the Data Analysis view for reviewing and reevaluating data that has been acquired,
• the Report Designer view for designing specific report layouts.

As well as configurations that support instrument diagnostics and verification procedures. A Navigation Pane is available on the left side of all ChemStation views. The Navigation Pane contains the Navigation Button, to allow rapid switching between ChemStation views, and the tree-based ChemStation Explorer. The contents of the ChemStation Explorer are view-dependent, giving access to different ChemStation elements. Each view consists of a set of standard user elements including menus and toolbars. The standard toolbar provides rapid access to the common system specification information such as methods and sequences. The Method and Run Control view additionally incorporates a system status bar, a sample information area, that may be configured for single runs or automated runs, and a schematic instrument interface diagram for GC, CE and LC configurations. The schematic instrument interface diagram uses hot spots to allow rapid access to instrument parameters and an animated graphical overview of the status of each analysis as it proceeds. The schematic instrument diagram may be turned off if it is not required, to save memory and other Windows resources. The Data Analysis view extends the standard toolbar to specific data analysis modes including integration, calibration, reporting, annotation, signal comparison and additional specialized modes if the modules are installed. Each of these separate data analysis modes are supported with a mode-specific toolset. The Report Designer view allows the user to graphically define the layout of a specific report style in a graphical object orientated fashion. It also uses a set of toolbars specific to this task.

Data Acquisition -- The status of the instrument is continually monitored and updated on the display, along with the elapsed run time of the analysis both when the software is a visible window and when it is iconized. The transactions that occur during the analysis, including any errors and the instrument conditions at the start and the end of the analysis, are recorded in the system’s logbook, an extract of which is stored with every data file. The instrument conditions, such as flow, temperature, pressure and solvent composition for liquid chromatographs may be recorded and stored with each data file. These instrument parameters can be displayed and plotted to testify to the quality of each analysis. The exact nature of the parameters recorded depends both on the technique and the capabilities of the configured instrument. One or more display windows may be used to monitor the data being acquired by the instrument in real time. The data are displayed in real measurement units such as mAU, Volts, degrees, or pressure in bar. The windows may each show multiple overlaid chromatographic/electropherographic signals or instrument parameters, such as pressure. The display default settings may be adjusted and are remembered by the system so users can set their own preferred settings as the instrument default. The window has zoom capability and the cursor may be used to display a specific signal’s response at any point in time. During an analysis, the complete functionality of the ChemStation can be used through the offline copy. While acquisition is running, the Data Analysis part of the online session of an instrument is not accessible, and data review has to be performed in the offline copy. A snapshot function is available for users who wish to start processing data before the analysis is completed. The snapshot has to be taken in the offline copy of the instrument sessions and is immediately present for review. The layout of the signal and status information windows, including the
components of the schematic instrument interface diagram is saved automatically.

Data Analysis—Display
The Data Analysis view extends the standard tool bar with task-grouped data analysis functions including integration, calibration, reporting, annotation, and signal comparison toolsets. The following key graphical operations are possible:
• single or multisignal displays selectable when loading the chromatogram/electropherogram,
• overlays of chromatogram/electropherogram from different samples,
• subtraction of one chromatogram/ electropherogram from another,
• graphical vertical and horizontal alignment of signals to help visual comparison,
• signal inversion or mirroring to help visual comparison,
• graphical zoom and scrolling functions,
• adjustment of display attributes including selection of tick marks, baselines, axes, retention/migration times and compound names (the user can also select the font for the RT and compound labels, adjust the size and orientation of the display, select the display as overlaid or separated and select scaling factors),
• the chromatogram/electropherogram display may include graphical overlays of instrument parameters depending on the capability of the configured instrument,
• user-defined annotations may be interactively added to the display, with the selection of font, size, text rotation and color (once defined, the annotations may be graphically moved, edited or deleted),

• copy the display to the Windows clipboard in both metafile and bitmap format,
• a pick mode function to display the values of individual data points in detector units, and
• export of time/intensity digitized points to the Microsoft Windows clipboard.

Data Analysis—Quantification
The ChemStation’s calibration mode of the data analysis view allows simultaneous display of:
• the signal or signals being calibrated with an indication of the current compound’s retention/migration time window,
• the calibration table whose display may be configured from a comprehensive selection of calibration parameters, and
• the calibration curve for the compound being analyzed.
All the calibration mode windows are linked so that changes in one are automatically reflected in all the others. This mode allows graphical selection and modifications of the calibration data. Quantification is based on %, Normalized %, External standard, External standard %, Internal standard and Internal standard % calculations calculated on either peak area or height. Calibrations may be multilevel and include
multiple internal standard definitions. Calibration histories are automatically saved and can be used to weight the recalibration calculations.

What Happens When a Method is Run?
The run-time checklist dialog box specifies the parts of the method to execute when a run is started. There are eight parts to the run-time checklist:
• pre-run command or macro,
• data acquisition,
• standard data analysis,
• analysis method for second signal (GC only),
• customized data analysis,
• save GLP data,
• post-run command or macro, and
• save copy of method with data (RUN.M).
When running a method the specified parts of the method defined in the Run Time Checklist dialog box are executed. Method Operation -- The figure above shows an overview of the ChemStation status during the method operation, where all parts of the Run Time Checklist are selected. The following list shows the flow of the method operation when all parts of the Run Time Checklist are selected.
1. Prerun Command Macro -- Does a task before the analysis is started.
2. Data Acquisition - Does injector program. Injects sample. Acquires raw data. Stores data.
3. Save Copy of Method with Data (RUN.M) - optional by Run Time Checklist
4. Save Copy of Method with Data (ACQ.M) - ChemStation default
5. Data Analysis (Process Data) Loads data file. Integrates data file. Identifies and quantifies peak. Searches spectral library if available. Checks peak purity if available. Prints report.
6. Customized Data Analysis Executes your macros.
7. Save GLP Data Saves binary register GLPSave.Reg
8. Postrun Command Macro Does a task after completion of the analysis, for example, generates a customized report.
9. Save Copy of Method with Data (DA.M) - ChemStation default.

Naming Conventions
The following rules enable the ChemStation to create and process valid names for files and directories:
The following characters are not allowed as part of a file or directory name:
• < > : " / \ | @ % * ? blank (spaces), etc.
If these characters are used in the installation folder, the reprocessing copy does not start, and if the character % is used in the installation folder, some 'Agilent Chemstation' shortcuts do not work properly.
The following rules apply in addition:

The following reserved device names cannot be used as the name of a file:
• CON, PRN, AUX, NUL
• COMx (where x is a number from 1 to 9)

• LPT1x (where x is a number from 1 to 9)
Also avoid these names followed by an extension (e.g. Nul.txt).