Introduction to the HPLC ChemStation and Acquisition - Agilent

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 Views�Method and Run Control

�Data Analysis

�Report Layout

�Verification (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, includingintegration 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 through

instrument parameters required to acquire

data for one analysis or access the

parameter windows by selection.

Note: Edit Entire Method does not

access 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 µl standard� Up to 1500 µl with multi-draw

kit.

�Up to 900 µl in a single drawusing expanded injectionupgrade 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) delayvolume of 6.2 ul

� INJECTOR PROGRAMfor programming custom injection steps

Widest dynamic injection range:

0.1 µl-1.5 ml

From pump

To 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 to

narrowest

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 Excel

using a custom macro.

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Saving the Method

Save a method by selecting Save Method or Save Method As from the

Method 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 minutes

prior to your first analysis for warm-up

by 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 % A

until all air bubbles have cleared.

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Prepare the Instrument – Prime the Pump

4. Pump 5 mL/min at 100%B until all

air bubbles have cleared.

5. Pump 5 mL/min at 100 % for each

remaining channel.

6. Change the composition to that

of your next run and continue.

7. Change the flow rate to that of your

next run.

8. Close the purge valve.

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Prepare the Instrument –Instrument Actuals

Allows you to review

your instrument

parameters, check

pump pressure

and 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

DEFAULT.D

ACQRES.REG DAD1.UV DAD1A.CH GLPSAVE.REG LCDIAG.REG RUN.LOG SAMPLE.MAC

005-0101.D

DEMO

DATA METHODS SEQUENCE TEMP VERIFY

1 BACKUP Core REPSTYLE SPECLIBS

Chem32

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.