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.
2
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
3
The HPLC ChemStation Software
Add Instruments
Access Instrument
and Software
Schedule ChemStation Tasks
4
Method and Run Control View
ChemStation Explorer
Sampling
Diagram
System
Diagram
Online Plot
Navigation Pane
Navigation Buttons
5
ChemStation Explorer Views
6
ChemStation Views
Views
Method and Run Control
Data Analysis
Report Layout
Verification (OQ/PV)
Diagnosis
Change
Views
Full menu or Short menu
7
View Preferences
•Allows you to configure the contents of the
ChemStation Explorer
•Specifies naming convention for sequence
data containers
•Specifies how signals are loaded
8
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

9
Instrument Parameters and Control: System
Diagram or Menus
Click on GUI for parameters.
Click here for instrument control.
Instrument control via menus or GUI
10
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.
11
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.
12
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
13
Method Information
Fill in any information you want stored with the method.
14
Pump Parameters
Time programmable composition, flow, and pressure.
15
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.
16
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 µl-1.5 ml
17
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.
18
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
19
VWD Parameters
VWD – G1314C
Set peak width to
narrowest
chromatographic
Peak width.
Time Programmable
20
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)
21
Run Time Checklist
Select items to execute during
the method.
Send your report to Excel
using a custom macro.
22
Saving the Method
Save a method by selecting Save Method or Save Method As from the
Method menu, or select the Save Method Tool.
23
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
24
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.
25
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.
26
Prepare the Instrument –Instrument Actuals
Allows you to review
your instrument
parameters, check
pump pressure
and module status at
a glance.
27
Prepare the Instrument - Instrument Actuals
28
Edit Signal Plot
Click Change
29
Run One Single Injection
To inject an individual sample, select Sample Info..., then Run Method.
30
Start Method
31
Follow Acquisition
32
Logbook Entries
Check how the run proceeded in the Logbook.
33
Directory Structure for Data Files
Instrument #
C:
Chem32
DATA
DEFAULT.D
METHODS
1
BACKUP
Core
SEQUENCE
TEMP
VERIFY
REPSTYLE
SPECLIBS
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
34
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.
35