USERGUIDE No.
20 I May 2015
Impact of pipetting techniques on
precision and accuracy
Kornelia Ewald, Eppendorf AG, Hamburg, Germany
Introduction
The fields of application for dispensing systems depend
on the design of the system as well as the physical limits
inherent to the application. To fulfill the high require-
ments in terms of precision and accuracy the application
Dispensing techniques
The dispensing techniques used most frequently are
forward pipetting, reverse pipetting, dispensing, sequential
dispensing and diluting. While mechanical pipettes are only
suitable for forward and reverse pipetting, electronic dis-
pensing systems frequently cover all of the above functions.
Selection of the dispensing technique suitable in each case
may have a significant impact on the analysis results.
The technique of forward pipetting (Fig. 1) is suitable for
aqueous solutions which may contain low concentrations
of protein or detergents. Pre-wetting of the tip improves the
analysis result.
With viscous or foaming liquids or when dispensing very
small sample volumes the results may be greatly improved
by reverse pipetting. Here the liquid is aspirated with blow-
out and dispensed without blow-out. A residue of liquid
remains in the plastic tip, and it is subsequently discarded
or returned to the aspirating vessel (Fig. 2).
primarily calls for a precise device with high-quality tips
and also, sufficient practical experience, correct handling
and cleanliness of the dispensing systems.
During the process of dispensing the aspirated liquid is dis-
pensed in defined steps. This technique is often used when
processing long test series or filling microtest plates.
With sequential dispensing, different volumes of a solution
are dispensed one after the other in a specified sequence.
This technique is often used with serologic work processes
and similar applications.
When diluting liquids an initial volume is first aspirated,
followed by an air bubble and the second volume. Both
volumes are then dispensed in one step. This dispensing
technique increases throughput and reduces fatigue
compared with performing aspiration and dispensing twice
with manual pipettes – particularly in the case of sizeable
volumes and multichannel pipettes.
USERGUIDE I No. 20 I Page 2

Forward and reverse pipetting

Transfer Forward pipetting Reverse pipetting

1. Press operating button down to 1st stop 1. Press operating button dow
Liquid uptake
2. Let operating button move up completely 2. Let operating button move

Liquid discharge 3. Press operating button via 1st stop down to 2nd stop 3. Press operating button dow

Observation 4. No liquid is left in tip after action 4. Liquid is left in tip after ac

1. 2. 3. 4.
REST POSITION

1st STOP

2nd STOP

Reverse pipetting

1. Press operating button down to 2nd stop

2. Let operating button move up completely

3. Press operating button down to 1st stop

4. Liquid is left in tip after action (volume of blow-out)

1. 2. 3. 4.
REST POSITION

1st STOP

2nd STOP
 UserguideNo
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USERGUIDE I No. 20 I Page 3

Optimumhandling
Optimum handlingofofmanual
manualpipettes
pipettes

Table
1 11
Table
Table
rrespective
rrespective
espective of
ofofthe the
the dispensing
dispensing
dispensing technique
technique
technique used,
used,
used, the
the
the following
following
following
Optimum handling of manual pipettes Volume
Volume
Volume (ml)
(ml)
(ml) Optimum
Optimum
Optimum immersion
immersion
immersion depth
depth
depth (mm)
(mm)
(mm)
tems
ms should
emsshould
should be
bebe taken
taken
taken into into
into consideration
consideration
consideration during
during
during pipetting:
pipetting:
pipetting:
l
l In
InIn the
the
the casecase
case of of
of air-cushion
air-cushion
air-cushion pipettes,
pipettes,
pipettes, thethe
the pipette
pipette
pipette tip
tip
tip should
should
should 0,1
- 1-- 11
0,1
0,1 1 11
Irrespective of the dispensing technique used, the following Table 1
be
bebe selected
selected
selected
items so so
so
shouldthatthat
that the
be the
the
taken air
airair cushion
cushion
cushion
into between
between
between
consideration the
the
the
during pipette
pipette
pipette
pipetting: 1-
1-1- 100
100
100 -23-- 33
22
Volume (μl) Optimum immersion depth (mm)
piston
piston
piston andand
and
l In the
the the
the surface
surface
surface
case of
ofof
of air-cushion the the
the liquid
liquid
liquidpipettes, is
isis as
asas small
small
small
the pipette as
asas possi-
possi-
possi-
tip should 101
101 -- 1000
1000 -24-- 44
101 -0,1
1000-1 12 2
ble.
ble.
ble. The besmaller
The
The selected
smaller
smaller the so
the
the that
tip, tip,
tip, the
the the
the air cushion
lower
lower
lower the the
the between
air
air
air volume,
volume,
volume, theandpipette
and
and the
the
the 1-- 10000
1001
1001
1001 -100
- 10000
10000 23- 3
-3 6-- 66
greater
greater
greater piston
thethe
the and
accuracy
accuracy
accuracy the surface
ofof of
thethe
the of the
results
results
results liquid
willwill
willis
be. as
be.
be. small as possible.
101 - 1000 2-4
The smaller the tip, the lower the air volume, and the
l
l When
When
When aspirating
aspirating
aspirating the the
the liquid,
liquid,
liquid, the the
the tip
tiptip should
should
should only
only
only be
bebe im-im-
im- 1001 - 10000 3-6
greater the accuracy of the results will be.
mersed
mersed
mersed>aWhen a
a few
few millimeters
millimeters
few millimeters
aspirating the intointo
into the
the
the medium
liquid, medium
medium
the tip should (Tab.1,
(Tab.1,
(Tab.1, only Fig.
Fig. 3).
be 3).3).
Fig.
l
lTheThe
The filled
filled
filled tip
tiptip should
should
should
immersed bebe
a few be moved
moved
moved
millimeters upupup against
against
against
into the the the
the wall
wall
wall
medium of
ofof the
the
the
vessel
vessel
vessel to
toto avoid
avoid
avoid
(Tab.1, residues
residues
residues
Fig. 3). ofof of liquid
liquid
liquid on
onon thethe
the outside
outside
outside of
ofofthe the
the tip.
tip.
tip.
>
Pre-wetting The
Pre-wettingthe
lPre-wetting
l filled
the tip
tip
thetiptiptwo should
two or
twoororthree be
three moved
threetimes times up
timeswill against
will improve
willimprove the
improvethe wall
the the
the of
vessel to avoid residues of liquid on the outside of the tip.
accuracy
accuracy
accuracy andand
and precision
precision
precision ofofof
the the
the results.
results.
results.
> Pre-wetting the tip two or three times will improve the
l
l Liquid
Liquid
Liquid should
should
should
accuracy
be
bebe aspirated
aspirated
aspirated
and precision
slowly
slowly
slowly of theand and
and evenly.
evenly.
evenly.
results.
l
lAA A waiting
waiting
waiting period
period
period
> Liquid ofof
should of
1 11tobe to
to 33 seconds
seconds
3aspirated
seconds should
should
should
slowly and be
bebe allowed
allowed
allowed
evenly.

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for
for
for the the
the liquid
liquid
liquid
> A waiting to
toto riserise
rise inin
period inthethe
the tip.
1tip.
oftip. to 3 seconds should be allowed

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for the liquid to rise in the tip.

Resea

Resea
Resea

Resea
sea
Re

Re
Re
Re
The
The pipette
pipette
e pipette should
should
should be
bebeheldheld
held vertically
vertically
vertically during
during
during aspiration.
aspiration.
aspiration. On On
On
The pipette should be held vertically during aspiration.
aspiration
spiration of
of the
the liquid
liquid the
the hydrostatic
hydrostatic
piration of the liquid the hydrostatic pressure of the liquid pressure
pressure of
of the
the liquid
liquid
On aspiration of the liquid the hydrostatic pressure of the
column
lumn in
olumninliquid
in
thethe
thetip tip
tip falls
falls
falls
column as as
inasthe
thethe
the angle
angle
angle
tip fallsof of
of
as inclination
inclination
inclination
the of
of the
the
angle ofofinclination
the of
pipette
pipette increases.
increases.
pette increases.
the pipetteThis This
This will
will
will
increases. result
result
result in
in
Thisinwill an
anan increased
increased
increased
result aspiration
aspiration
aspiration
in an increased aspira-
volume
olume[1].
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tion
[1]. The
volume
The
The deviation
[1]. The
deviation
deviation ininin volume
deviation
volume
volume can
in
can
can be
volume
bebe estimated
estimated
estimated forfor
can be estimated
for
any
ynypipettefor
pipette
pipette any
volume
volume
volume pipette and
and
and volume
angles
angles
angles and of
ofof angles of
inclination.
inclination.
inclination. inclination.
With
With
With ananan With
angle
angle
angle an an-
gle of 45° diverging from the vertical the extra volume with
of
of 45°
45°
45° diverging
diverging
diverging from
from
from the the
the vertical
vertical
vertical the the
the extra
extra
extra volume
volume
volume with
with
with a aa
a 1000 μl pipette is 2.9 μl or 0.29 %. For a 100 μl pipette at
1000
00 µl
000µlµl pipette
pipette
pipette
an angle is
isis2.9 2.9
of2.960°, µl
µlµl or
an or
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volume For
For aofaa 100
100
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0.53 µl
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pipette
or 0.53at % atwill
at
an angle
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of 60°,
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result (Fig. 3).an
an extra
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extra volume
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volume of of
of 0.53
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0.53 µl µl
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or 0.53
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will
will
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esult
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Influence of immersion depth and holding angle of pipette during
aspiration
Figure
Figure
Figure 3: of liquid:
3:3:
1. Pipette vertical, immersion depth of tip in liquid approx. 1 cm
Influence
Influence
Influence of
ofof immersion
immersion
immersion depth
depth
depth and
and
and holding
holding
holding angle
angle
angle of
ofof pipette
pipette
pipette during
during
during
2. Pipette vertical, immersion depth of tip in liquid approx. 3 cm
aspiration
aspiration
aspiration of of
of liquid:
liquid:
liquid:
3. Pipette at a holding angle of 30° to 40°, immersion depth of tip
1.
1.1. Pipette
Pipette
Pipette
in vertical,
vertical,
liquidvertical,
approx. immersion
immersion
3immersion
cm depth
depth
to 4 cm depth of
ofof tip
tiptip in
in in liquid
liquid
liquid approx.
approx.
approx. 111 cm
cmcm
2.
2.2. Pipette
Pipette
Pipette vertical,
vertical,
vertical, immersion
immersion
immersion depth
depth
depth of
ofof tip
tiptip in
in in liquid
liquid
liquid approx.
approx.
approx. 33 3 cm
cm cm
3.
3.3. Pipette
Pipette
Pipette at
atat aa holding
holding
a holding angle
angle
angle of
ofof 30°
30°
30° to
toto 40°,
40°,
40°, immersion
immersion
immersion depth
depth
depth of
ofof tip
tiptip
in
in in liquid
liquid
liquid approx.
approx.
approx. 33 3 cm
cm cm to
toto
44 4 cm
cm cm
USERGUIDE I No. 10 I Page 4

References

[1] Lochner, K. H.; Ballweg, T.; Fahrenkrog, H.-H.: Untersuchungen zur Messgenauigkeit von Kolbenhubpipetten
mit Luftpolster. In: Lab Med 20 (1996), Nr. 7/8, S. 430–440.

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