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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.

Maintaining the Computer System

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.

Accessories

Right-click drive letter

The HPLC ChemStation Software

Add Instruments

Access Instrument
and Software

Schedule ChemStation Tasks

Method and Run Control View


ChemStation Explorer

Sampling
Diagram

System
Diagram

Online Plot

Navigation Pane

Navigation Buttons

ChemStation Explorer Views

ChemStation Views

Views
Method and Run Control
Data Analysis
Report Layout
Verification (OQ/PV)
Diagnosis

Change
Views

Full menu or Short menu

View Preferences

Allows you to configure the contents of the


ChemStation Explorer
Specifies naming convention for sequence
data containers
Specifies how signals are loaded

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
Method information

Instrument control parameters

Data analysis parameters

Run Time Checklist

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

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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Agilent 1100/1200 Injector Special Functions

NEEDLE WASH
to reduce carry-over to the
absolute minimum

Metering
device

MULTI DRAW MODE


for injection volumes
greater 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 PROGRAM
for programming custom
injection steps

From pump
To column
To waste

4-port rotor
seal
Widest dynamic
injection range:
0.1 l-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

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


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:
C h em 32

DATA
D E F A U L T .D

M ETHO DS

BACKUP

C o re

SEQ UENC E

TEM P

V E R IF Y

REPSTYLE

S P E C L IB S

DEM O
0 0 5 - 0 1 0 1 .D

A C Q R E S .R E G

D A D 1 .U V

UV Spectra

D A D 1 A .C H

G L P S A V E .R E G

UV Signal
Chromatograms

L C D IA G .R E G

R U N .L O G

S A M P L E .M A C

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.

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