Introduction to the HPLC

ChemStation and Acquisition

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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 Maintaining the Computer System

G Delete temporary files on a regular basis. Use Clean Disk for

Windows 2000 and XP.

G Use Checkdisk to find and correct errors on the disk.

G Defragment the hard drive.

G Use Virus detection software.

G Create an Emergency Repair disk.

Accessories

Right-click drive letter

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The HPLC ChemStation Software

Add Instruments

Access Instrument
and Software

Schedule ChemStation Tasks

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 System
Method and Run Control View Diagram
ChemStation Explorer

Sampling Online Plot

Diagram

Navigation Pane

Navigation Buttons 5
ChemStation Explorer Views

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ChemStation Views Views

Method and Run Control

Data Analysis

Report Layout

Verification (OQ/PV)

Diagnosis

Change
Views

Full menu or Short menu

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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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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.

Parts of a Method
G Method information
G Instrument control parameters

G Data analysis parameters

G Run Time Checklist

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Instrument Parameters and Control: System
Diagram or Menus
Click on GUI for parameters.

Click here for instrument control.

Instrument control via menus or GUI

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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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 Editing a Method Using “Edit Entire Method”

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.
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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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Pump Parameters

Time programmable composition, flow, and pressure.

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Injector Parameters - Agilent 1100/1200
Standard

Sample capacity
G 100 x 2 ml vials in 1 tray

G 40 x 2 ml vials in 1/2 tray

G 15 x 6 ml vials in 1/2 tray

G Microvials with sleeves

Injection volume
G 0.1 - 100 µl standard

G Up to 1500 µl with multi-draw

kit.
GUp to 900 µl in a single draw

using expanded injection

upgrade kit.

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Agilent 1100/1200 Injector Special Functions

G NEEDLE WASH
to reduce carry-over to the
absolute minimum Metering
device
G MULTI DRAW MODE
for injection volumes
greater than 100 ul
From pump
G SWITCH VALVE TO BYPASS To column
to decrease standard loop
delay volume (300ul) to a
minimum (bypass) delay To waste 4-port rotor
volume of 6.2 ul seal
G INJECTOR PROGRAM
for programming custom
Widest dynamic
injection steps injection range:
0.1 µl-1.5 ml

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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
VWD – G1314C

Set peak width to

narrowest
chromatographic
Peak width.

Time Programmable

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Column Thermostat Parameters

G 10 degrees below ambient to

80 degrees C (Standard).

G10 degrees below ambient to

100 degrees C (SL).

G Two separate heated zones for

two columns.

G Optional valve for column

switching applications.

G Compartment holds up to 30 cm
column.

G 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

1. Make certain the vacuum degasser is on

(if applicable).

2. Open the purge valve.

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

Instrument #
C:

Chem32

1 BACKUP Core REPSTYLE SPECLIBS

DATA METHODS SEQUENCE TEMP VERIFY

DEFAULT.D DEMO

005-0101.D

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

Logbook for Run

UV Spectra UV Signal
Chromatograms

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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.

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