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)