T E C H N I C A L

N O T E

# 0 0 2

Art & Science of

Liquid Handling

MINIPREP

Series

Robotic Sample Processors

Many factors come into play when aspirating and dispensing fluids. These
include pump mechanics, tubing, aspirate and dispense speeds, and the airgaps that separate the sample from the system fluid. In addition, characteristics of the fluid itself are important (i.e. viscosity, volume, conductivity).
Each of these factors affect the accuracy and precision of liquid handling.

Z-Values

Liquid Class Database

Tecans Gemini software for applications development incorporates a
unique new tool to help developers of robotic applications manage these
factors. The Liquid Class Database allows the management of all factors
that influence liquid handling in an organized manner. Pre-programmed
liquid classes are provided. The user can copy, rename, then edit the
default settings to create new custom liquid classes. The Liquid Class
Database will not only save you time when developing your first robot
application, but it will allow you to apply your experience quickly to subsequent methods that you may develop.
This note provides fundamental concepts that will help you optimize your
automated liquid handling applications and includes the following topics:

Pipetting Modes
Z-Values
Liquid Level Detection
Airgaps
Aspiration and Dispense Speeds

Multi-pipetting includes additional parameters than will be discussed here.

Other tips are available, including disposable tips (DiTi). Liquid classes
include water, serum, alcohol (e.g. ethanol), and DMSO. Refer to the
MiniPrep manual for additional information.

Wash Procedures
Additional Precision and
Accuracy Considerations
Default Settings and Procedures

Pipetting Modes
Table 1 defines three different modes. Single pipetting was used to obtain
data presented in this note.

Z-values represent the vertical heights set for the pipette tip as it moves,
aspirates, and dispenses. Table 2 describes each Z-value.

Liquid Level Detection

Liquid detection mode defines the manner in which the MiniPrep attempts
to detect liquid in the container. Liquid detection is crucial for the instrument to ensure aspiration of the liquid and minimal carry over of liquid on
the outer surfaces of the tip. Choices include single, double or no (off)
detection. Double detection was used for this technical note.

Airgaps
Airgaps separate different liquids in the pipetting tips (separation airgap)
and tubing and prevent droplets at the end of the tip while the arm moves
over the worktable (trailing airgap). Pipetted liquids can have both leading
and trailing airgaps. Airgaps are treated together with their associated liquids, and dispensed together at the same destination.
Table 3. Airgaps and recommended settings for water
Example

Airgap

Software Definition

Settings

System Trailing
Airgap (STAG)

Airgap after system fluid

Default range:
4-10 l

Leading Airgap
(LAG)

Airgaps before aspirated

liquid volume

Default range:
1-10 l

Trailing
Airgap (TAG)

Airgap after last aspirated Default range:

liquid volume, prevents
1-5 l
droplet formation at end
of the tip

Table 1. Pipetting modes

Single Pipetting

Mode

Definition

Single

A single probe aspirates the liquid volume (one aliquot) at one

source position and dispenses the entire volume at one
destination position

Dual

A dual probe aspirates and dispenses the liquid volume (one

aliquot) through each barrel

Multi

A single or dual probe aspirates and dispenses multiple liquids

(multiple aliquots) multiple times

System
Fluid
STAG
LAG

Vol.

Table 2. Z-values and recommended settings

Example

Z-Value

Definition

Setting

Z-Travel

Height at which the tip travels

between positions within a rack

Set higher than any possible obstruction

Z-Start

Height at which the MiniPrep

activates Liquid Detection
Operation

Set just inside the neck of the vessel and just

above the liquid

Z-Dispense

Height in the vessel at which

the MiniPrep dispenses liquid

Set based on your needs, must be above the

liquid level

Z-Max

Lowest height in the vessel in

the Z-axis

For routine operation, set Z-max to 5 steps above

the bottom of the vessel

steps

~1600

Aspiration and Dispense Speeds

Wash Procedures

Liquid handling techniques employ the general rule: aspirate slow,

dispense faster. Viscosity of the liquid, syringe, tubing size, and tip
configuration may effect speeds. The choice of aspiration and dispense
speeds affects both accuracy and precision. Rapid aspiration speeds can
cause the airgap to break up which results in a significant dilution effect,
or can introduce bubbles into the system. Faster dispense speeds generally result in better break off at the tip. The MiniPrep has speeds ranging
from 1.2 seconds per stroke to 20 minutes per stroke.

Routine pipetting with steel tips requires both wash and flush steps to
ensure proper cleaning of the tips between pipetted liquids. Use flush
before starting a procedure as a means of priming and cleaning the system.
During the operation, wash the tips between pipetting steps since this will
aspirate a new STAG. When using a DiTi, you dont need to wash, but
flush the system between use to ensure hydraulic operation. After about 12
aspirate/dispense movements with a DiTi, the STAG is gone.

Table 4. Aspiration settings for water

Aspirate

Dispense

Definition

Settings

Speed: the flow rate at

the tip when aspirating
the liquid volume
Delay: a pause in the
syringe movement after
aspirating the liquid
volume; gives the
system time to return to
equilibrium

typical range is 10150 l/s

Speed: the flow rate at

the tip when dispensing
the liquid volume

100 ms is sufficient for

most applications,
default = 0 ms

When optimizing liquid handling procedure:

wash volume must be sufficient to empty the clean station
wash speed must be set to create turbulence to clean the outside of the tip
In general use a larger wash volume whenever possible. For pipetted liquid volume <30 l/s, >1000 l is suggested; for pipetted liquid volume
>50 l use 2500 l. Several small washes are more efficient than a single
large one.
A Miniwash Pump (an option), a high velocity membrane pump, pushes
liquid through the tip at the flow rate of ~180 ml per minute, and provides
fast and effective washing to enable faster processing times. When using
the Miniwash, set the minimum wash volume to 5 ml. This activates the
pump for 1 to 2 seconds.

Additional Precision and Accuracy Considerations

typical range is 100600 l/s

In general, a decrease of aspiration speed will increase precision and

accuracy. Low speeds are needed when pipetting small volumes
(<25 l), liquids with high viscosity or low boiling points. Viscous
liquids require an appropriate aspiration delay.
CAUTION: Retracting the tip from the liquid too quickly can result in
aspiration of air instead of liquid.
During liquid aspiration a vacuum builds inside the system tubing. The
faster the liquid is aspirated, the stronger this vacuum pulls. The
aspiration delay gives the system time to return to equilibrium.
When optimizing the dispense speed, consider the following when
dispensing from air:
Calculation of the total volume to be dispensed
This total includes all volumes in a pipetting sequence including the
airgaps (LAG, TAG).
The type of liquid
Different viscosities and compositions require different liquid handling
parameters.
Foaming and droplet formation
Liquids such as detergents must be dispensed slowly to avoid foaming
and droplet formation on the outside of the tip.
Small volumes
Relatively higher dispense speeds are required for volumes < 20 l.
Large volumes
Larger volumes lose accuracy when dispensed with high speeds due to
dilution effects and breaking of airgaps. Larger volumes should be
dispensed at relatively slower speeds.

Below is a brief description of other parameters. Please refer to the

MiniPrep manual for more information.
System Liquid should always be deionized water
The MiniPreps digital pump uses a stepper motor to drive the plunger.
A mechanical tolerance, called Backlash, exists between the gears in the
drive train. Backlash compensation is built into the firmware, at fixed
settings. It ensures the plunger moves to the correct position.
After detecting liquid, the tip submerges a set number of steps in the
liquid. In normal liquid, set the Submerge Steps to 5. When foaming is
present, use double detection and increase the submerge steps between
10 and 25 steps.
Syringe Selection: generally small syringes maximize accuracy and
precision. Match syringe size with appropriate tubing (e.g. for syringe
< 1 ml use smaller inner diameter tubing, not standard 0.06 inch tubing).
Other factors to consider are the smallest and largest volumes
pipetted, accuracy requirement, speed, and breakoff. If the application
requires multiple aspirations or multiple dispenses, then a larger syringe
will allow more aliquots.
NOTE: For the best precision and accuracy, the syringe size should not
exceed 100 times the smallest sample volume. For example, a 10 l
sample requires a 1000 l syringe or smaller.
Use Tracking to avoid dispensing directly into the liquid or to avoid
foaming while dispensing. Using deionized water for diluting samples or
reagents may interfere with liquid detection, due to minimization of
active ions, which is critical to liquid detection.
NOTE: Dispensing with tracking overrules the Z-dispense setting.
The Breakoff indicates the number of steps not performed at the end of
the plunger strokes. You can set the breakoff from 0-24. Typically, set
the breakoff to 10 (the default).
Serial dilutions are prepared in destination racks. To ensure a constant
volume of liquid for all dilution steps, including the one at the end of the
sequence, insert a waste line at the end of the dilution series. The waste
line aspirates the requested volume from the last tube and dispenses it to
the waste position.
Pre-Dilutions are prepared in separate racks. Specifying a maximum
mix volume minimizes carry over. For mixing, a default 50% of the
volume dispensed to a serial or pre-dilution well is re-aspirated and
dispensed. One mixing cycle is best.

Figure 1. Water liquid class screen example from software.

Default Settings
Specifications on precision and accuracy for the MiniPrep are based on
gravimetric measurements of a single pipetting process using a standard
tip, 1 ml syringe on a standard XL 3000 diluter with 3 port valve, and
water pipetted. Description of gravimetric procedure is given below
(a photometric procedure is available too).
Table 5. Precision and accuracy for standard tip*
Volume dispensed
from air (l)

Precision1 (CV)

Accuracy2
+/-

10
100

< 3%
<1.0%

<5%
<1.0%

* Each volume was dispensed 540 times from air. A matrix composed of
three XL 3000 diluters, three syringes and three standard tips was used
(20 dispenses per combination of diluter-syringe-standard tip). Within each
volume range at least three volumes were measured, low, medium and high.
_

Standard deviation (Stdev): s =

(x x)

Figure 2. Calibration example.

n1

Water Liquid Class, single pipetting

Coefficient of variation: CV % = (stdev / average) *100

2 Accuracy

%:

standard tip, 1mL syringe, standard XL3000, 3 port valve

120

Set value Actual value

*100
Set value

Gravimetric Calibration Procedure:

1. In the instrument software define your balance (Mettler AT261 or
equivalent) as a destination rack.
2. The Worktable should also include source (tube racks) and wash station.
3. Use the Application Software to compose a model process (method) for
each liquid transfer using the same volumes and liquid handling
parameters. For statistical reasons, every single distribution step should
be repeated at least 15 times.
4. Run the model process in single step mode. Only 1 tip can be used at a
time. After each dispense read the actual mass from the balance display.
Values from your balance can be collected with a special data
acquisition software.
5. Calculate average, precision (CV) and accuracy of your data. Keep in
mind to correct the volume by the density of the liquid.
6. Determine a calibration curve. An example of a linear regression line is
seen in Figure 2.

Measured Volume (L)

100
80
60
40
20

0
0

20

40

60

80

100

120

Expected Volume (L)

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