How
To
Calibrate
Your
Micropipette
?
How To Calibrate Your Micropipette?
Dr. Tamer Soliman
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
Principle
1
•
Micropipettes
instruments.
are
precision
laboratory
•
They require routine maintenance and calibration
to ensure accuracy and precision.
•
Using gravimetric methodology this is determined
by using an analytical balance and weighing the
amount of water delivered by the pipette. For the
purpose of calibration, milligrams and micro liters
are considered to be equivalent.
•
Calculation of the standard deviation and the
coefficient of variation are used to determine the
accuracy and precision of each pipette.
How To Calibrate Your Micropipette?
Dr. Tamer Soliman
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
Frequency
• Before pipettes are newly placed into service
• Periodically; suggested interval is quarterly or
according to manufacturer guidelines
• After cleaning
• Anytime a pipette becomes dismantled and
repaired
2
• As a means of troubleshooting assay problems
How To Calibrate Your Micropipette?
Dr. Tamer Soliman
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
Requirements
• Micropipettes for calibration
• Analytical balance with maintenance completed
• Pipette tips for appropriate pipette volumes
• Weigh boats
• Worksheet for Micropipette Calibration
• Room temperature water in a clean glass beaker
(Some labs use Peanut Oil)
3
• Thermometer
• Calculator with statistical program, computer with
statistical software, or standard calculator.
How To Calibrate Your Micropipette?
Dr. Tamer Soliman
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
Calibration
The complete calibration consists of testing both
accuracy and precision of all micropipettes outlined in
this procedure.
Guidelines for Acceptable Calibration Results:
• Precision is based on the guidelines suggested by
the manufacturer in the pipette package insert.
• Acceptable accuracy ranges for pipettes should be
based on the manufacturer’s recommendations.
4
• Accuracy ranges of a good guide:
plus or minus 4% for pipettes 5 – 50 µl
plus or minus 2% for pipettes 51 – 1000 µl
How To Calibrate Your Micropipette?
Dr. Tamer Soliman
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
Calibration
For variable micropipettes,
calibration should be done at 3 different levels
Example
200 µl
= High-volume setting
5
Variable Micropipette
20 – 200 µl
100 µl
= Mid-volume setting
20 µl
= Low-volume setting
How To Calibrate Your Micropipette?
Dr. Tamer Soliman
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
Procedure Flowchart
Un-Calibrated
MicroPipette
Calibration
Calculate
both Precision
and Accuracy
Using Good Pipetting Technique
Re-measure
Measure
High-Volume Setting
Re-measure
Measure
Mid-Volume Setting
Re-measure
Measure
Low-Volume Setting
Re-calibrate
If Not OK
6
Perform
Routine Maintenance
Adjust
At low-Volume Setting
Using Pipette Calibration Tool
If Not OK
Re-calibrate
If Not OK
Remove the pipette from service
How To Calibrate Your Micropipette?
Dr. Tamer Soliman
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
Procedure
For Pipettes 5 – 100 µl
1. Record ambient room temperature to determine
water density to be used in calculations.
2. Place a plastic weigh boat and pipette tip on the
balance and tare the balance (adjust to Zero).
3. Place the tip onto the pipette requiring calibration.
Prewet then Pipette room temperature reagent grade
water into the tip using good pipetting technique.
4. Turn the pipette into a horizontal position and remove
the tip taking care not to lose any of the water.
7
5. Weigh the tip and water on the same plastic weigh
boat and record the result.
6. Repeat steps 10 – 20 times (depending on the criteria
for precision established by your laboratory).
How To Calibrate Your Micropipette?
Dr. Tamer Soliman
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
Procedure
For Pipettes 100 – 1000 µl
1. Record ambient room temperature to determine
water density to be used in calculations.
2. Place a plastic weigh boat on to an analytical balance
and tare the balance.
3. Prewet then Pipette room temperature reagent grade
water into the tip using good pipetting technique.
4. Dispense the grade water onto the weigh boat.
8
5. Allow the balance to equilibrate and record the
reading.
6. Repeat steps 10 – 20 times (depending on the criteria
for precision established by your laboratory).
How To Calibrate Your Micropipette?
Dr. Tamer Soliman
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
Procedure
For a multi-channel Pipette
• It is important to check each channel, since they
can develop problems independently.
• A practical procedure would be to verify one
channel, using 10 data points, at each of 3 different
volume settings. Then verify that the other channels
are performing properly, by using fewer data points
at one or two volume settings.
9
How To Calibrate Your Micropipette?
Dr. Tamer Soliman
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
Calculation
Formula
measured water Weight
(pipetted) water Volume =
(using Analytic balance)
water Density (sp. gravity)
(at the measured room temperature
and atmospheric pr.)
Density of Water
at 20ºC = 0.998
at 25ºC = 0.997
at 30ºC = 0.996
10
How To Calibrate Your Micropipette?
Dr. Tamer Soliman
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
Calculation
Precision
• Precision is a measure of how close together a
number of values are. The closer together they are,
the better the precision.
• Imprecision is the opposite meaning of precision.
• Imprecision is either:
o Absolute Imprecision
expressed as SD (standard deviation) of 10 samples.
11
SD =
Σ ( xi – x̄ )2
(n–1)
o Relative Imprecision
expressed as CV (coefficient of variation) of 10
samples. SD
CV =
x̄ (mean of measured pipetted volumes)
How To Calibrate Your Micropipette?
Dr. Tamer Soliman
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
Calculation
Accuracy
• Accuracy is how close a result comes to the "true"
value
• Inaccuracy is the opposite meaning of accuracy.
• Inaccuracy is expressed as the deviation of the mean
of 10 samples from the set point volume.
• Inaccuracy can be expressed in either:
• Absolute units such as microliters, or
• Relative units such as percent.
12
Inaccuracy =
‫نسبة الخطأ‬
Accuracy =
Set Volume – Mean of pipetted volumes
Set Volume
Mean of pipetted volumes
Set Volume
x 100
How To Calibrate Your Micropipette?
Dr. Tamer Soliman
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
Micropipett Calibration Worksheet
Ambient Temperature:
Date:
H2O Density:
µl
Volume:
Micropipette ID:
Actual Precision:
Allowable Precision:
Accuracy Range: +
%
Calculated Accuracy:
Acceptable?
%
Acceptable?
Yes or No
13
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Sum (Σ)
Mean (x̄)
Pipette readings
Difference from the mean
(Difference)2
(xi)
(xi – x̄)
(xi – x̄)2
How To Calibrate Your Micropipette?
Dr. Tamer Soliman
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
Micropipett Calibration Worksheet
Worksheet for Variable Micropipette (20 – 200 µl) Calibration
[ At Water Density (specific Gravity) = 1.000 ]
14
At 20 µl setting
At 100 µl setting
At 200 µl setting
(Low Volume Setting)
(Mid Volume Setting)
(High Volume Setting)
Weight
Volume
Weight
Volume
1
0.0198 g
19.8 µl
1
0.0989 g
98.9 µl
2
0.0197 g
19.8 µl
2
0.0988 g
3
0.0199 g
19.8 µl
3
4
0.0199 g
19.8 µl
5
0.0197 g
6
Weight
Volume
1
0.1995 g
199.5 µl
98.8 µl
2
0.1992 g
199.2 µl
0.0991 g
99.1 µl
3
0.1989 g
198.9 µl
4
0.0987 g
98.7 µl
4
0.1995 g
199.5 µl
19.8 µl
5
0.0990 g
99.0 µl
5
0.1993 g
199.3 µl
0.0198 g
19.8 µl
6
0.0989 g
98.9 µl
6
0.1995 g
199.5 µl
7
0.0197 g
19.8 µl
7
0.0988 g
98.8 µl
7
0.1994 g
199.4 µl
8
0.0199 g
19.8 µl
8
0.0991 g
99.1 µl
8
0.1995 g
199.5 µl
9
0.0199 g
19.8 µl
9
0.0987 g
98.7 µl
9
0.1990 g
199.0 µl
10
0.0197 g
19.8 µl
10
0.0990 g
99.0 µl
10
0.1991 g
199.1 µl
Sum (Σ)
0.1980 g
198.0 µl
Sum (Σ)
0.9890 g
98.9 µl
Sum (Σ)
1.9920 g
1992.0 µl
Mean
0.0198 g
19.8 µl
Mean
0.9890 g
98.9 µl
Mean
0.1992 g
199.2 µl
How To Calibrate Your Micropipette?
Dr. Tamer Soliman
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
Procedural Notes
• Avoid handling pipette tips with your fingers. Use a
lint free wipe or forceps whenever possible.
• Two readings are required. Two fresh pipette tips
must be used to confirm accuracy.
• Peanut Oil can be used to prevent evaporation of
water. Simply place a small amount of the oil in the
weigh boat in procedural step 2.
15
• One weigh boat may be used for multiple readings
with a tare performed between each weight
measurement. Multiple pipette calibrations will
require the boat to be emptied and re-zeroed.
Reminder: The balance will not allow the tare of a
negative reading. The balance must be re-zeroed.
How To Calibrate Your Micropipette?
Dr. Tamer Soliman
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
Interpretation of Results
1. Pipettes not meeting the established acceptance
criteria for accuracy or precision should receive
routine maintenance before being calibrated
again.
2. If a pipette cannot meet the acceptance criteria it
should be removed from service until it can be
serviced by the manufacturer or an approved
vendor.
16
3. Pipettes that do meet the established acceptance
ranges should be labeled with a sticker that
records the actual accuracy of the pipette, the
date of calibration and the initials.