Adjustment - altering the pipette so that the dispensed volume is within the specifications.
Air Displacement Pipettes - are meant for general use with aqueous solutions. In air displacement pipettes, a certain volume of air remains between the piston and the liquid.
Aspirate - to draw up the sample.
Blow-out - to empty the tip completely.
Calibration check - checking the difference between the dispensed volume and the selected volume.
Dispense - to deliver the sample.
Positive Displacement Pipettes - are used for high viscosity and volatile liquids. In positive displacement pipettes, the piston is in direct contact with the liquid.
Air Displacement Pipette, used for standard pipetting applications, is highly accurate.
However, conditions such as temperature, atmospheric pressure as well as the specific gravity and viscosity of the solution may have an effect on the performance of air displacement pipettes.
The most important factor in pipetting accuracy is the liquid temperature. The figure below shows the change in volume when the liquid has a different temperature than the pipette and air. If the temperature of the liquid, pipette and air is the same, the accuracy is not significantly affected.
Density is the mass/volume ratio of the liquid. The density varies according to the
temperature and air pressure. Typically, the density of water is 0.996 kg/dm3, for ethanol 0.79 kg/dm3 and for sulfuric acid (H2SO4) 1.85 kg/dm3.
The geographic altitude affects the accuracy through the air pressure. The air pressure
decreases at higher altitudes and the conversion factor Z decreases as well. The boiling point of some liquids can also change to quite close to room temperature, increasing the evaporation loss dramatically.
Positive Displacement Pipette, is used for applications like PCR and other DNA amplification techniques. The micro-syringe tips used in positive displacement pipettes are disposable. This helps to avoid sample-to-sample cross-contamination (also known as sample carry-over), and contamination due to the aerosol effect.
Check your pipette at the beginning of your working day for dust and dirt on
the outside. If needed, wipe with 70% ethanol.
Set the volume within the range specified for the pipette.
Hold the pipette so the "grippy finger rest" rests on your index finger.
To maximize accuracy, the pipette, tip and liquid should be at the same
Check that you are using tips recommended for this pipette. To ensure
accuracy, use only high-quality tips made from contamination-free
Tips are designed for single use. They should not be cleaned for reuse as their
metrological characteristics will no longer be reliable.
*Pre-rinsing (1-3 times) the tip with the liquid to be pipetted may improves
accuracy, especially when using positive displacement tips.
Avoid turning the pipette on its side when there is liquid in the tip. Liquid might
go to the interior of the pipette and contaminate the pipette.
Avoid contamination to or from fingers by using the tip ejector and gloves.
Store pipettes in an upright position when not in use. Pipette stands are ideal for this purpose.
Check calibration regularly, depending on the frequency of use and on the
application, but at least once a year. If used daily, a three-month interval is
recommended. Follow the instructions for recalibration in the manufacturer�s instruction manual.
The reverse technique is used for pipetting solutions with a high viscosity or a tendency
to foam. Reverse pipetting is only possible with air displacement pipettes
This technique is intended for repeated pipetting of the same volume. Can only be used with electronic and repeator pipettes.
Use forward technique steps 1 and 2 to fill the tip with blood (do not pre-rinse the
tip). Wipe the tip carefully with a dry clean cloth.
Filter or wide orifice
Tween 20, 10% solution
Bronidox L, 10% (preservative)
10 x PBS, 0.1M NaCl, 3M
For genomic DNA wide orifice should be used to avoid mechanical shearing.
A contaminated pipette or contaminated tips can cause contamination of samples.
Samples or aerosols from samples can enter the cone of the pipette.
The remains of sample A can mix with next sample B inside the tip and may cause
a false test result.
One of the most important factors in accurate Pipette measurement is the Tip. The Pipette and the Tip form a System. The best Pipetting results are naturally achieved by those manufactured together to ensure complete compatibility between Pipettors and Pipettor Tip.
Many tips on the market look fine, but when studying them more carefully, it appears that the price really reflects quality. Many of the cheap tips may have flashes, protrusions, and scratches, air bubbles, be bent or contain impurities. All these influence Pipetting results. For example, if the tip is not straight this alone may result in a 10% error in Pipetting accuracy (ISO 8655-2). Moreover, if the tip orifice is not accurately centered, the dispensing of the liquid might be affected significantly. If premium grade pure polypropylene is not used or the mold and processing are not up to the highest standards, part of the liquid stays inside the tip as a droplet, which causes an error in the Pipetting result.
GUIDELINES FOR SELECTING TIPS
All tips should be:
Calibration of Pipettes
Calibration of Pipettes, officially means determining the difference between the dispensed volume and the selected volume. Adjustment means altering the pipette
so that the dispensed volume is within certain specifications. During factory calibration, performance is checked with different weighings at both the maximum volumes of the range and at the minimum or 10% of the maximum volume, whichever is higher. When you purchase a Pipette, you should choose one that is designed to permit recalibration and adjustment for different temperatures and various viscous liquids.
Calibration of Pipettes in a Quality System
The main objective of pipette calibration in a quality system is to ensure that measurements are made with the intended accuracy. Very often error limits are taken
from the manufacturer�s specifications, while far less accuracy is required to perform
the work. If these limits are not easily obtained, or vary, another option is to set the limits according to accepted standards (DIN 12650 or ISO 8655). However, if the laboratory work requires the highest accuracy, the manufacturer�s limits should be used. Basically every user should define their own limits, according to the application used and the ambient conditions.
The scale graduation value of the balance should be chosen according to the selected pipette volume.
Note: check the calibration of your balance regularly using known weights.
Water, distilled or de-ionized, "Grade 3", conforming to ISO 3696. The test water is
held in the calibration room for at least 2 hours before calibration to reach equilibrium
with the test room conditions.
Tests are performed in a draught-free room at a constant (+/- 0.5C) temperature of 20C to 25C. Relative humidity is recommended to be above 45%. Especially with volumes under 50 vl, the air humidity should be as high as possible to reduce the evaporation loss effect.
- The pipette, the water and the air in the test room should be at the same
- A new tip should be pre-wetted 1 to 3 times to improve the accuracy.
- Always pipette water from a reservoir, do not take it back from the balance.
- Check the calibration regularly, depending on the frequency of use and on the
application, but at least once a year. If used daily, a three-month interval is
Incoming Inspection procedure
Sources of pipette error:
Conversion of mass to volume
V = (w + e) x Z
V = volume (vl)
w = weight (mg)
e = evaporation loss (mg)
Z = conversion factor for mg/vl conversion
Note: evaporation loss can be significant with low volumes. To determine mass loss, dispense water into the weighing vessel, note the reading and begin timing with a
stopwatch. Check how much the reading decreases during the 5 seconds. Compare
this to the pipetting time. Typically, the pipetting time might be 5 seconds and the mass loss up to 2 mg. If an evaporation trap or lid on the vessel is used, an evaporation correction is unnecessary.
The conversion factor Z is for calculating the density of water suspended in air at
the test temperature and pressure. See the conversion Table 1 on page 15.
Accuracy (systematic error)
Accuracy is the difference between the dispensed volume and the selected volume of a pipette.
Accuracy is expressed on the calibration certificate as a relative value:
Precision (random error)
Precision refers to the repeatability of the pipettings. It is expressed as standard
deviation (s) or coefficient of variation (cv). In addition to the features of the pipette,
laboratory practice and user experience are the main factors affecting precision.
Error according to DIN 12650 (F-value)
The DIN standard does not give individual limits for accuracy and precision, but
uses a combined error limit: the F-value.
F = | A | + 2 x s
The relative F-value is calculated:
F% = | A% | + 2 x cv
Table 2 shows the error limit according to the DIN 12650 standard for single-channel
air displacement pipettes. For multichannel pipettes, these limits are doubled. With variable volume pipettes, the nominal volume is the maximum volume. The absolute vl limit of the nominal volume applies to every selected volume throughout the volume range.
For example, for a 20 - 200 vl pipette, the error limit is 2.0 vl for every selected volume. If the nominal volume of the pipette is between those in the table, the relative error limit F% of the nearest volume is used. If the nominal volume is exactly between the two volumes in Table 2, the relative error limit F% of the lower volume is used.
DIN 12650 error limits for single channel air displacement pipettes
Error-free pipetting requires both precision and accuracy. A number of factors can
effect these specifications, which are the main quantitative parameters for evaluating
What are accuracy and precision?
For example when the set volume is 20 vl:
Accurate, but not precise: The mean volume is the correct (set) volume, but
separate pipettings differ from the set volume.
Precise, but not accurate: There is no variation between the separate pipettings,
but the mean volume differs from the set volume.
Accurate and precise: The mean volume is the set volume and there is no variation between different pipettings.
1. Pipette vertical, tip immersed about 1 cm into the liquid.
2. Pipette vertical, tip immersed about 3 cm into the liquid.
3. Pipette at a 30 � 40 degree angle; tip immersed about 3-4 cm into the liquid.
Please, follow these instructions carefully in order to avoid damage to tips and pipettes.
Note: Autoclaving has a limited spectrum of action and will not destroy RNA for example. It is also important to note that certain parts of a pipette - for instance the piston and the handle cannot be autoclaved without altering accuracy and precision
What is the difference between air displacement and positive displacement pipettes?
Both types of pipettes have a piston that moves in a cylinder, or capillary. In air displacement pipettes, a specified volume of air remains between the piston and the
liquid. In positive displacement pipettes, the piston is in direct contact with the liquid.
This keeps air from entering the tip, preventing contamination effectively. Air
displacement pipettes are meant for general use with aqueous solutions. Positive
displacement pipettes are used for high viscosity and volatile liquids.
How can I pipette viscous liquids?
You can do so using an air displacement pipette with standard or wide orifice tip
(reverse pipetting, slowly). An alternative to this is to use a positive displacement
How can I prevent liquid dropping out of the tip when pipetting volatile compounds?
If you use air displacement pipettes, aspirate and dispense the liquid a few times
keeping the tip in the liquid. By doing so, the air inside the pipette will be saturated
with vapor of the volatile compound. It is recommend using positive displacement
pipettes for highly volatile compounds, since the built-in piston tip is in direct contact with the liquid.
How accurately can I pipette warm or cold liquids.
The pipettes are calibrated by weighing distilled or de-ionized water of 20 - 25C.
With warm liquids, you will get a smaller mass with a certain volume, with cold liquids,
you will get a higher mass.