Agilent Technologies Optimizing Technical Note
Optimizing the Agilent high-throughput
analysis system for high performance
and precision
Abstract
The Agilent 220 micro plate sampler—integrated into the Agilent 1100
Series LC system—is the ideal tool to analyze large numbers of struc-
turally distinct compounds. It offers the full potential of well plate
technology:
• High sample capacity to increase sample throughput and shorten
response times. This is especially useful for drug discovery, combi-
natorial chemistry, medicinal chemistry and natural product analysis.
• The flexibility of having a sampler mode for high-speed sample
analysis and a fraction collector mode for sample isolation and
purification.
• An automated injector program for sample preparation such as
derivatization, dilution and mixing.
• The ability to store and inject over 4,600 samples (using 384-well
plates) for unattended high-throughput.
Technical Note
Equipment
All experiments were carried out
on the Agilent 1100 Series HPLC
system consisting of
• Agilent 1100 Series vacuum
degasser,
• Agilent 1100 Series binary
pump,
• Agilent 1100 Series thermostatted column compart
ment,
• Agilent 1100 Series diode
array detector, and
• Agilent 220 micro plate
sampler.
The system was controlled using
the Agilent ChemStation (version
A.07.01) and the micro plate sampling software (version A.03.01).
Injection principle
In contrast to the Agilent 1100
Series autosamplers the Agilent
220 MPS works with a fixed-size
sample loop injection (figure 1).
While the valve is in loading position the amount of drawn sample
is injected into the fixed size sample loop capillary and the surplus
of sample is flushed through the
loop into waste. To achieve
optimal performance overfilling of
the sample loop is recommended.
The overfill volume depending on
loop size is shown in table 1.
The reason why overfilling is necessary is the hydrodynamic behavior of fluids as they pass through
tubing. A process called laminar
flow takes place under conditions
in which molecules close to the
tubing walls are slowed by frictonal forces. The result is a bulletshaped profile in which the molecules in the center of the stream
travel roughly twice the velocity
of those at the tubing wall3.
The surplus of sample volume also
ensures that the dead volume of
the injector port capillary of
approximately 4.2 µl is completely
flushed with sample. After switching the valve into the injection and
run position the amount of sample
in the loop is applied to the
system.
To apply sample amounts smaller
than the loop volume the Centered
Loop Fill or the Partial Loop Fill
& Inject commands can be used.
For the Centered Loop Fill technique a sandwich of 1-µl air gaps
and sample is injected into the
sample loop. The volume drawn
before the air gaps and the sample
volume is calculated by the software to position the sample volume in the middle of the sample
loop. The precision for this technique is lower than for complete
loop fill because only the
Introduction
The Agilent 220 micro plate sampler (MPS)1is an essential part of
Agilent’s system for combinatorial
chemistry and high throughput
HPLC analysis. It combines high
sample capacity, high speed and
sampling/fractionation capabilities in one system. In combination
with the Agilent 1100 Series HPLC
system using mass selective
detection it is a complete system
that fulfills the requirements of
combinatorial chemistry and highthroughput analysis, offering
robustness, ruggedness, sensitivity, selectivity and speed. The integrated system plus the single software platform simplifies system
setup, operation and management
of large amounts of data.
Details of the Agilent 220 MPS
and how it works in an Agilent
system for combinatorial chemistry or high throughput screening
is described in another Agilent
technical note2. In this note we
explain how to further optimize
the hardware and software setup
of the Agilent 220 MPS to achieve
higher performance and precision.
Loop Volume [µl] Sample Volume [µl]
535
10 40
20 55
50 95
Figure 1
Injection principle of the Agilent 220 micro plate sampler
6
5
4
3
2
1
6
5
4
3
2
1
Loading position Injection and run position
to column
to column
from pump
from pump
waste
waste
injection port
4.2 µl dead volume!
sample loop
(fixed size)
sample loop
(fixed size)
Table 1
Sample loop sizes and recommended sample
volumes
0
1
2
3
4
5
0 20406080100
Sample Volu me [µl]
RSD Area [%]
5 µl Sample Loop
20 µl Sample Loop
mechanical precision of the dilutor syringe determines the injected sample volume. For Partial
Loop Fill & Inject the sample volume plus a relatively large rinse
volume is drawn from the sample.
The rinse volume is used to rinse
the injection port and the sample
loop before the actual sample volume is pushed into the sample
loop. The precision is higher than
for Centered Loop Fill but also
more sample volume is required.
Parameters to optimize
Parameters influencing the performance of the sampler in sampling
mode are:
1. Drawn sample volume
(complete loop fill)
2. Draw speed
(complete loop fill)
3. Size of air gap
(complete loop fill)
4. Dilutor syringe size and sample
loop size (centered loop fill)
1. Drawn sample volume
(complete loop fill)
Although the size of the sample
loop determines the injected sample volume the amount of drawn
sample volume also influences the
precision of the measurement3.
Figure 2 shows the precision of
peak areas for ten measurements
for different drawn sample volumes
For the drawn sample amount of
5 µl the precision of peak area is
very high because 5 µl is just
enough sample to fill the injection
port capillary (dead volume
4.2 µl). Increasing the sample vol-
ume also increases the precision
of peak area. When the recommended sample volume is used
the precision is usually optimized.
More sample volume does not
increase the precision any further.
If not enough sample volume for
sufficient overfill of the sample
loop is available, a lower precision
of peak area results.
2. Draw speed
(complete loop fill)
The draw speed for the sample as
well as the inject speed influences
the precision of the measurement
as shown in figure 3.
Figure 3
Precision of peak area for different draw and inject speeds (5-µl sample loop, complete loop fill)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0.5 ml/min 1 ml/min 2 ml/min 3 ml/min
Draw Speed
RSD Area [%]
Figure 2
Precision of peak area for different sample volumes (5 and 20-µl sample loop, complete loop fill)