1. Environment and materials

Environment

Draft free, constant temperature between 15 °C and 30 °C, max. ±0.5 °C deviation during the measurements.

Materials

Balance

Equipped with draft protection and evaporation trap.
Alternative trap: 4 containers filled with water in each corner of the windshield.

Number of required digits is determined by the nominal volume of your pipette:

Nominal volume	Digits (Readability)
0.5 µl ≤ V < 20 µl	Single channel balance: 6 (0.001 mg) Multichannel balance: 5 (0.01 mg)
20 µl ≤ V < 200 µl	5 (0.01 mg)
200 µl ≤ V ≤ 10 ml	4 (0.1 mg)

Weighing container

Preferably use a metal container to minimize build-up of static charges.  
Alternative: 1.5 ml microcentrifuge tube.

Test liquid

Distilled water

Pipette tips

Use pipette manufacturer’s recommended tips for best results.

Note: INTEGRA GRIPTIPS^® pipette tips snap on and never loosen, leak or fall off.

2. Before you start

Place pipette, tips and test liquid in the test room 2 hours before starting measurements to reach equilibrium with room conditions. 

Note date, ambient temperature and air pressure. If no barometer is available, search the internet for atmospheric pressure at a local weather station.

The use of correct pipetting techniques is crucial.

3. Leak test

Before performing any gravimetric measurements, test if the pipette is leaking:

1. Pre-wet the tip(s) by aspirating and dispensing the nominal volume 3 times. 
2. Using the same tip(s), aspirate the nominal volume.
3. With the tip(s) immersed 2 mm in liquid, hold the pipette vertically for 30 seconds then, with tip(s) immersed in liquid, dispense all remaining liquid from the tip(s).
4. If liquid level does not drop and no air bubbles are present, continue with validation. A decreasing liquid level indicates a leak. Contact the manufacturer to discuss further steps.

4. Gravimetric measurement

The weighing container should not be dry. Add some distilled water.

Number of measurements

Perform at least 4 measurements each at 100 % and at 10 % of the nominal volume. Start at 100 %.

Validate the first and a middle channel on multichannel pipettes.

Procedure of the gravimetric measurement

1. Tare the balance with the weighing container.
2. Load a new pipette tip.
3. Perform a pre-wet (see section 3).
4. Dispense liquid into the weighing container. Ensure to dispense along the inner container wall and finish by drawing the tip end along wall to remove residual liquid. 
5. Record the weight in a table.
6. Using the same tip, repeat steps 4 to 5 at least 4 times. Tare the balance after each reading.
7. Eject the tip and load a new tip.
8. Repeat steps 1 to 7 for the second test volume.
    

5. Data analysis

Step 1: Convert balance readings for each measurement (mg) to volume (µl) using the Z factor (table to determine the correct Z factor):

V_i = Single volume in µl
m_i = Single weighing in mg
Z = Z factor

Here is an example of how to convert balance readings for a 100 µl single channel pipette: Test volume: 100 µl Temperature: 21.5 °C Air pressure: 101.3 kPa  Measurement # Weight (mg) 1 100.08 2 100.10 3 100.11 4 100.22 5 100.15 Vi = 100.08 mg x 1.0032 → Vi = 100.40 µl Vi = 100.10 mg x 1.0032 → Vi = 100.42 µl Vi = 100.11 mg x 1.0032 → Vi = 100.43 µl Vi = 100.22 mg x 1.0032 → Vi = 100.54 µl Vi = 100.15 mg x 1.0032 → Vi = 100.47 µl

Step 2: Calculate the mean volume per test volume and per channel using the calculated V_i measurements:

V = Mean volume
n = Number of weighings

Using the same example as above, this results in a mean volume of 100.45 µl: V = (100.40 µl + 100.42 µl + 100.43 µl + 100.54 µl + 100.47 µl) / 5 → V = 100.45 µl

Step 3: Calculate the accuracy (systematic error) in % per test volume and per channel:

e_s = Systematic error in %
V_s = Selected test volume

Using the mean volume calculated above, this results in an accuracy of 0.45 %: es = (100 (100.45 – 100)) / 100 → es = 0.45 %

Step 4: Calculate the precision (random error) in % per test volume and per channel:

s_r = Repeatability standard deviation

CV = Coefficient of variation in %

Using the values from the example calculations above, this results in a precision of 0.05 %: sr = √(((100.40 – 100.45)2 + (100.42 – 100.45)2 + (100.43 – 100.45)2 + (100.54 – 100.45)2 + (100.47 – 100.45)2) / (5 – 1)) → sr = 0.05 CV = 100 (0.05 / 100.45) → CV = 0.05 %

Compare the calculated accuracy and precision with the manufacturer's specifications. If the calculated values are not within specifications, the pipette needs to be calibrated. If the pipette has passed the routine check, it is working as intended.

Note: INTEGRA electronic pipettes can be easily calibrated. Simply enter target volume and actual volume you have measured and then click calibrate.
 

Download those guidelines as a poster