Agilent Technologies Optimizing 1100 Highest Performance Technical Note
Introduction
This document will help you to
efficiently prepare an Agilent
1100 Series HPLC system for best
performance. It describes how to
achieve
Optimizing the Agilent 1100 Series
System for Highest Performance
Technical Note
• highest sensitivity,
• highest precision,
• low flow rates,
• lowest delay volume,
• lowest carry-over, and
• best results in normal phase
chromatography.
We first describe a short standard
check-out procedure, which is recommended before starting to
optimize the instrument. This
ensures that the instrument has
no defects which may cause ghost
peaks, increased noise or unstable retention times.
In the second section, we then
explain how to optimize the performance of an Agilent 1100
Series HPLC system.
1
Agilent Technologies
Innovating the HP Way
Standard Check-out
Procedure
What is needed for the standard check-out procedure
• Column:
125 × 4 mm Hypersil ODS, 5 µm
(Agilent part number 7982618-
564) Use a new column or a column of known history.
• Solvents:
bi-distilled water, acetonitrile
• Isocratic standard sample
(Agilent part number
01080-68704)
Checking the equipment for
leaks and contamination
1. Fill one solvent bottle with bidistilled water (channel A) and
the other one with acetonitrile
(channel B).
2. Open the purge valve and purge
both channels with 5 ml/min for
at least 10 min. Check outlet
tubing of the purge valve. If no
air bubbles are observed purging can be stopped.
3. Adjust compressibility and
stroke under INSTRUMENT,
MORE PUMP. Use the Help key
to find appropriate compressibility values for the different
solvents. With the quaternary
pump you only need one compressibility value, which should
be set to 100. For the binary
pump set A to 46 and B to 115.
The stroke should be set to
auto for both A and B.
4. After the purge process, and
after installing the column, set
the flow rate to 1 ml/min and
close the purge valve.
5. Watch for leaks. Leave the thermostatted column compartment
open and check each connection for leaks.
6. Watch pressure in the ONLINE
SIGNAL screen. The ripple
should be very regular, no spiking or pressure drops should
occur. If they do occur and you
can detect no leaks, we recommended contacting the Agilent
service organization for possible repair.
7. To check for contamination in
your system, select the following settings on your instrument.
This is especially important if
sensitivity in gradient runs is an
issue.
No peaks should be observed if
these parameters are set. If, however, the first run does show
peaks, run the gradient a second
and a third time. Should peaks
continue to occur, clean the
instrument using different solvents or/and passivate it with 65 %
nitric acid.
1.
Parameter Setting
Flow rate 1 ml/min
Detection wavelength 210 nm
Stop time 30
Column compartment
temperature 36 ºC
Gradient start with 0 % B and
go to 100 % B in
25 min.
Blank run, no injection no vial number in
SAMPLE INFO
screen
Checking for baseline noise
Before running the system the
detector lamp should have been
switched on for at least half an
hour. Select the following settings
on your instrument:
Using the Agilent ChemStation
choose the PERFORMANCE AND
NOISE report and set six 1-minute
ranges in the SYSTEM
SUITABILITY screen which is
located under the REPORT menu.
The instrument then calculates the
noise automatically. The noise
measured over one minute at six
different parts in the chromatogram should be close to the
following specifications:
• variable wavelength detector
(VWD):
± 0.75 x 10
-5
AU at 254 nm
• diode array detector (DAD):
± 1 x 10
-5
AU at 254 and 750 nm
These specifications are based on
a cell with a pathlength of 10 mm,
a response time of 2 s, a flow of
1 ml/min methanol and a slit width
of 4 nm for the diode array
detector.
Figure 1 shows how to evalutate
the signal noise based on the
ASTM method. The Agilent
ChemStation software evaluates
ASTM noise automatically.
If the noise drastically differs from
the specifications, we recommend
contacting the Agilent service
organization for possible repair.
Figure 1
Signal noise based on the ASTM method
Parameter Setting
Flow rate 1 ml/min
Detection wavelength 254 nm
Stop time 30
Column compartment
temperature 36 ºC
Isocratic: 100 % water
Blank run, no injection no vial number in
SAMPLE INFO
screen
Interval
1 min
123456
Time [min]
Drift
Peak to peak
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