Introduction to the HPLC ChemStation and Acquisition

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In This Section, We Will Discuss:

• How to work in the Microsoft Windows Environment

• The structure of the ChemStation Software.

• How to set up an acquisition method.

• How to run a single sample.

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Delete temporary files on a regular basis. Use Clean Disk for Windows 2000 and XP.

Use Checkdisk to find and correct errors on the disk.

Defragment the hard drive.

Use Virus detection software.

Create an Emergency Repair disk.

Maintaining the Computer System

Accessories

Right-click drive letter

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The HPLC ChemStation Software

Add Instruments

Schedule ChemStation Tasks

Access Instrument and Software

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Method and Run Control View

Sampling Diagram

System Diagram

Online Plot

Navigation Pane

ChemStation Explorer

Navigation Buttons

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ChemStation Explorer Views

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ChemStation Views ViewsMethod and Run ControlData AnalysisReport LayoutVerification (OQ/PV)Diagnosis

Full menu or Short menu

Change Views

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View Preferences •Allows you to configure the contents of the ChemStation Explorer

•Specifies naming convention for sequence data containers

•Specifies how signals are loaded

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Parts of a Method Method information Instrument control parameters Data analysis parameters Run Time Checklist

What is a Method?

• A method comprises all the parameters necessary to perform data acquisition and data analysis, including integration and calibration parameters, for one sample. • Pre- and post-run tasks may be specified by a command or macro in the run-time checklist.

• The method is identified by a file name with a .m extension.

• Master methods are stored in Chem32\#\Methods.

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Instrument Parameters and Control: System Diagram or Menus

Click on GUI for parameters.

Instrument control via menus or GUI

Click here for instrument control.

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Create a Method

•In the Method and Run Control view, Select New Method, or double-click on DEF_LC.M.

•DEF_LC.M is loaded. This method is a template file that cannot be overwritten.

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You may use Edit Entire Method to sequentially move throughinstrument parameters required to acquire data for one analysis or access the parameter windows by selection.

Note: Edit Entire Method does notaccess all instrument parameters such as More Pump > Auxiliary, etc.

Editing a Method Using “Edit Entire Method”

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Select Portions of Method to Edit

Information about the method

Instrument parameters found in Method and Run Control view

Parameters for post-acquisition processing found in Data Analysis

Parts of the method to run

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Method Information

Fill in any information you want stored with the method.

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Time programmable composition, flow, and pressure.

Pump Parameters

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Injector Parameters - Agilent 1100/1200 Standard

Sample capacity 100 x 2 ml vials in 1 tray 40 x 2 ml vials in 1/2 tray 15 x 6 ml vials in 1/2 tray Microvials with sleeves

Injection volume 0.1 - 100 l standard Up to 1500 l with multi-draw kit.Up to 900 l in a single draw using expanded injection upgrade kit.

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NEEDLE WASHto reduce carry-over to the absolute minimum

MULTI DRAW MODEfor injection volumesgreater than 100 ul

SWITCH VALVE TO BYPASS to decrease standard loop delay volume (300ul) to a minimum (bypass) delay volume of 6.2 ul

INJECTOR PROGRAMfor programming custom injection steps

Widest dynamic injection range:

0.1 µl-1.5 ml

From pumpTo column

Metering device

To waste 4-port rotor seal

Agilent 1100/1200 Injector Special Functions

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Agilent 1200 High Throughput Samplers

Sample Capacity•2 well plates (96 and 384) plus 10 additional 2-mL vials. •108 x 2-mL vials in 2 x 54 vial plate plus 10 additional 2-mL vials. •30 x 6-mL vials in 2 x 15 vial plate plus 10 additional 2-mL vials. •54 Eppendorf tubes (0.5/1.5/2.0mL) in 2 x 27 Eppendorf tube plate. •Also compatible with the Agilent 1200 Series sample capacity extension for further expansion of the sample capacity.

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Agilent 1100/1200 DAD Parameters

5 signals (standard DAD).

8 signals (SL).

Sample signal 191 - 949 nm.

Slit programmable; 1, 2, 4, 8 and 16 nm settings.

Time programmable.

80 Hz data rate DAD (SL) for rapid resolution columns.

DAD – SL Window shown

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VWD Parameters

Time Programmable

Set peak width tonarrowest chromatographic Peak width.

VWD – G1314C

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Column Thermostat Parameters

10 degrees below ambient to 80 degrees C (Standard).

10 degrees below ambient to 100 degrees C (SL).

Two separate heated zones for two columns.

Optional valve for column switching applications.

Compartment holds up to 30 cm column.

Column identification module with injection record for GLP.

(TCC –SL shown)

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Run Time Checklist

Select items to execute during the method.

Send your report to Excelusing a custom macro.

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Saving the Method

Save a method by selecting Save Method or Save Method As from theMethod menu, or select the Save Method Tool.

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Prepare the Instrument – UV Lamp On

Turn on a UV lamp at least 20 minutesprior to your first analysis for warm-upby clicking the control button.

Balancing Ready

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Prepare the Instrument – Prime the Pump

Purge Valve

1. Make certain the vacuum degasser is on (if applicable).

2. Open the purge valve.

3. Pump 5 mL/min of 100 % Auntil all air bubbles have cleared.

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Prepare the Instrument – Prime the Pump

4. Pump 5 mL/min at 100%B until allair bubbles have cleared.

5. Pump 5 mL/min at 100 % for eachremaining channel.

6. Change the composition to thatof your next run and continue.

7. Change the flow rate to that of yournext run.

8. Close the purge valve.

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Prepare the Instrument –Instrument Actuals

Allows you to review your instrumentparameters, check pump pressureand module status at a glance.

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Prepare the Instrument - Instrument Actuals

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Edit Signal Plot

Click Change

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Run One Single Injection

To inject an individual sample, select Sample Info..., then Run Method.

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Start Method

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Follow Acquisition

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Logbook Entries

Check how the run proceeded in the Logbook.

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Directory Structure for Data Files

D EF AU L T .D

A C Q R E S .R E G D A D 1.U V D A D 1A .CH G L P S A V E .R E G L C D IA G .R EG R U N .L O G S A M P L E .M AC

005-0101 .D

D EM O

D A TA M E T H O D S S E Q U E N C E T E M P V E R IF Y

1 B A C K U P C o re R EP S T Y L E S P E C L IB S

C h em 32

C :

Instrument #

UV Spectra UV Signal Chromatograms

Logbook for Run

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Turn Off System

•Remember to flush buffers from the system.

•Do not leave 100% Acetonitrile in the system.

•Do not leave the TCC at high temperatures without column flow for extended periods.