User Guide 20: Impact of Pipetting Techniques on

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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 requirements in terms of precision and accuracy the application
primarily calls for a precise device with high-quality tips
and also, sufficient practical experience, correct handling
and cleanliness of the dispensing systems.
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 dispensing 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 blowout 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).
During the process of dispensing the aspirated liquid is dispensed 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
Liquid uptake
1. Press operating button down to 1st stop
2. Let operating button move up completely
1. Press operating button dow
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.
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.
REST POSITION
1st STOP
2nd STOP
2.
UserguideNo
No020
020| page
| page33
Userguide
Optimumhandling
handlingofofmanual
manualpipettes
pipettes
Optimum
Table
Table
Table
1 11
Volume
(ml)
Volume
(ml)
Volume
(ml)
Optimum
immersion
depth
(mm)
Optimum
immersion
depth
(mm)
Optimum
immersion
depth
(mm)
0,1
0,1
0,1
- 1-- 11
1 11
Table 1
1100
100
1-1100
22
-23-- 33
Volume (μl)
101
1000
101
-- 1000
101
-0,1
1000
-1
Optimum immersion depth (mm)
-24-- 44
12 2
1-- 10000
1001
- 10000
10000
1001
-100
1001
23- 3
-3 6-- 66
The
pipette
should
be
held
vertically
during
aspiration.
On
The
pipette
should
held
vertically
during
aspiration.
On
e pipette
should
bebe
held
vertically
during
aspiration.
On
The pipette should be held vertically during aspiration.
aspiration
of
the
liquid
the
hydrostatic
pressure
of
the
liquid
spiration
of
the
liquid
the
hydrostatic
pressure
of
the
liquid
piration of the liquid the hydrostatic pressure of the liquid
On aspiration of the liquid the hydrostatic pressure of the
column
in
the
tip
falls
as
the
angle
of
inclination
of
the
olumninliquid
in
the
tip
falls
the
angle
of
inclination
of
the
lumn
the
tip
falls
as
the
angle
inclination
the
column
inas
the
tip
fallsof
as
the
angle ofofinclination
of
pipette
increases.
This
will
result
in
an
increased
aspiration
pipette
increases.
This
will
result
in
increased
aspiration
pette increases.
will
result
anan
increased
aspiration
the pipetteThis
increases.
Thisinwill
result
in an increased
aspiration
volume
[1]. The
deviation
in
volume
can be estimated
volume
[1].
The
deviation
in
volume
can
be
estimated
for
olume[1].
[1].
The
deviation
volume
can
estimated
for
lume
The
deviation
inin
volume
can
bebe
estimated
for
for
any
pipette
volume
and
angles
of
inclination.
With
an anany
pipette
volume
and
angles
of
inclination.
With
an
angle
pipette
volume
and
angles
inclination.
With
angle
ynypipette
volume
and
angles
ofof
inclination.
With
anan
angle
gle of 45° diverging from the vertical the extra volume with
of
45°
diverging
from
the
vertical
the
extra
volume
with
of
45°
diverging
from
the
vertical
the
extra
volume
with
45°
diverging
from
the
vertical
the
extra
volume
with
a aa
a 1000 μl pipette is 2.9 μl or 0.29 %. For a 100 μl pipette at
1000
µl
pipette
is
2.9
µl
or
0.29
%.
For
100
µl
pipette
atwill
000µlµl
pipette
or
0.29
%.
For
100
µl
pipette
at
00
pipette
isis
2.9
µlµl
or
0.29
%.
For
aofaa
100
µlμl
pipette
an
angle
of2.9
60°,
an
extra
volume
0.53
or
0.53at
%
an
angle
of
60°,
an
extra
volume
of
0.53
µl
or
0.53
%
will
nangle
angle
of
60°,
an
extra
volume
of
0.53
µl
or
0.53
%
will
of
60°,
an
extra
volume
of
0.53
µl
or
0.53
%
will
result (Fig. 3).
The
fact that different people do not pipette exactly the
esult
(Fig.
3).
esult
(Fig.
sult
(Fig.
3).3).
same
volume
with
exactly
the
same
pipette
canthe
also
The
fact
that
different
people
do
not
pipette
exactly
the be
The
fact
that
different
people
not
pipette
exactly
the
e fact
that
different
people
dodo
not
pipette
exactly
explained by factors such as different angles of inclination
same
volume
with
exactly
the
same
pipette
can
also
be
amevolume
volume
with
exactly
the
same
pipette
can
also
me
with
exactly
the
same
pipette
can
also
bebe
when aspirating liquid. Although the effect is small in each
explained
by
factors
such
as
different
angles
of
inclination
xplained
factors
such
different
angles
inclination
plained
byby
factors
such
asas
different
angles
ofof
inclination
instance, the error may be much greater when all of the
when
aspirating
liquid.
Although
the
effect
is
small
in
each
when
aspirating
liquid.
Although
the
effect
small
each
hen
aspirating
Although
effect
isis
small
inin
each
differingliquid.
factors
are
addedthe
together.
nstance,
the
error
may
be
much
greater
when
all
of
the
nstance,the
theerror
errormay
maybebemuch
muchgreater
greaterwhen
whenallallofofthe
the
stance,
differing
factors
are
added
together.
differing
factors
are
added
together.
fering factors
are
added
together.
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for the liquid to rise in the tip.
Research
Research
Research
Research
2-4
3-6
Research
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101 - 1000
1001 - 10000
Research
Research
Research
Research
rrespective
of
the
dispensing
technique
used,
the
following
rrespective
the
dispensing
technique
used,
the
following
espective
ofof
the
dispensing
technique
used,
the
following
Optimum
handling
of
manual
pipettes
tems
should
be
taken
into
consideration
during
pipetting:
emsshould
should
taken
into
consideration
during
pipetting:
ms
bebe
taken
into
consideration
during
pipetting:
l
In
the
case
of
air-cushion
pipettes,
the
pipette
tip
should
l
the
case
of
air-cushion
pipettes,
the
pipette
tip
should
InIn
the
case
of
air-cushion
pipettes,
the
pipette
tip
should
Irrespective of the dispensing technique used, the following
be
selected
so
that
the
air
cushion
between
the
pipette
selected
so
that
the
cushion
between
the
pipette
bebe
selected
that
the
airair
cushion
between
the
pipette
items so
should
be
taken
into
consideration
during
pipetting:
piston
and
the
surface
of
the
liquid
is
as
small
as
possipiston
and
the
surface
the
liquid
small
possipiston
and
the
surface
ofof
the
liquid
isis
asas
small
asas
possil In
the
case
of air-cushion
pipettes,
the
pipette
tip
should
besmaller
selected
so
that
the
air
cushion
between
theand
pipette
ble.
The
smaller
the
tip,
the
lower
the
air
volume,
and
the
ble.
The
smaller
the
tip,
the
lower
the
air
volume,
and
the
ble.
The
the
tip,
the
lower
the
air
volume,
the
piston
and
the
surface
of
the
liquid
is
as
small
as
possible.
greater
the
accuracy
of
the
results
will
be.
greater
the
accuracy
the
results
will
be.
greater
the
accuracy
ofof
the
results
will
be.
The smaller the tip, the lower the air volume, and the
l
When
aspirating
the
liquid,
the
tip
should
only
be
iml
When
aspirating
the
liquid,
the
should
only
imWhen
aspirating
the
liquid,
the
tiptip
should
only
bebe
imgreater the accuracy of the results will be.
mersed
a
few
millimeters
into
the
medium
(Tab.1,
Fig.
3).
mersed
a
few
millimeters
into
the
medium
(Tab.1,
Fig.
mersed>aWhen
few millimeters
into
the medium
(Tab.1,
Fig.
aspirating the
liquid,
the tip should
only
be 3).3).
l
The
filled
tip
should
be
moved
up
against
the
wall
of
the
l
The
filled
should
moved
against
the
wall
the
The
filled
tiptip
should
bebe
moved
upup
against
wall
ofof
the
immersed
a few
millimeters
into
the the
medium
(Tab.1,
Fig.
3). ofof
vessel
to
avoid
residues
of
liquid
on
the
outside
of
the
tip.
vessel
avoid
residues
liquid
the
outside
the
tip.
vessel
toto
avoid
residues
liquid
onon
the
outside
ofof
the
tip.
>
The
filled
tip
should
be
moved
up
against
the
wall
of
l
Pre-wetting
the
tip
two
or
three
times
will
improve
the
lPre-wetting
Pre-wettingthe
thetiptiptwo
twoororthree
threetimes
timeswill
willimprove
improvethe
the the
vessel to avoid residues of liquid on the outside of the tip.
accuracy
and
precision
of
the
results.
accuracy
and
precision
the
results.
accuracy
and
precision
ofof
the
results.
> Pre-wetting the tip two or three times will improve the
l
Liquid
should
be
aspirated
slowly
and
evenly.
l
Liquid
should
aspirated
slowly
and
evenly.
Liquid
should
bebe
aspirated
slowly
and
evenly.
accuracy
and
precision
of the
results.
l
A
waiting
period
of
to
seconds
should
be
allowed
l
waiting
period
to
33
seconds
should
allowed
AA
waiting
period
ofof
1 11
to
3aspirated
seconds
should
bebe
allowed
> Liquid
should
be
slowly
and
evenly.
for
the
liquid
to
rise
in
the
tip.
for
the
liquid
rise
the
>
A
waiting
period
oftip.
1tip.
to 3 seconds should be allowed
for
the
liquid
toto
rise
inin
the
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measurement
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JB>PROBJBKQABSF>QFLK
deviation
deviation
deviation
¨Ë
¨Ë
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¨Ë
¨Ë
¨Ë
¨Ë
¨Ë
¨Ë
¨Ë
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¨Ë
¨Ë
¨Ë
¨Ë
¨Ë
¨Ë
0.2–0.4 %
0.2–0.4 %
0.2–0.4 %
Figure 3:
Influence of immersion depth and holding angle of pipette during
aspiration
Figure
3: of liquid:
Figure
Figure
3:3:
1. Pipette vertical, immersion depth of tip in liquid approx. 1 cm
Influence
of
immersion
depth
and
holding
angle
of
pipette
during
Influence
immersion
depth
and
holding
angle
pipette
during
Influence
ofof
immersion
depth
and
holding
angle
ofof
pipette
during
2. Pipette vertical, immersion depth of tip in liquid approx. 3 cm
aspiration
of
liquid:
aspiration
of
liquid:
aspiration
of
liquid:
3. Pipette at a holding angle of 30° to 40°, immersion depth of tip
1.
Pipette
vertical,
immersion
depth
of
tip
in
liquid
approx.
1 cm
cm
Pipette
vertical,
immersion
depth
liquid
approx.
1.1.
Pipette
ofof
tiptip
in in
liquid
approx.
11
cm
in
liquidvertical,
approx.
3immersion
cm
to 4 cm depth
2.
Pipette
vertical,
immersion
depth
of
tip
in
liquid
approx.
3 cm
cm
Pipette
vertical,
immersion
depth
liquid
approx.
2.2.
Pipette
vertical,
immersion
depth
ofof
tiptip
in in
liquid
approx.
33
cm
3.
Pipette
at
holding
angle
of
30°
to
40°,
immersion
depth
of
tip
Pipette
aa holding
angle
30°
40°,
immersion
depth
3.3.
Pipette
atat
a holding
angle
ofof
30°
toto
40°,
immersion
depth
ofof
tiptip
in
liquid
approx.
3 cm
cm
to
4 cm
cm
liquid
approx.
in in
liquid
approx.
33
cm
toto
44
cm
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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User Guide 20: Impact of Pipetting Techniques on

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