Do you have problems with the operation of air-displacement pipettes in your Quality Control Unit? Read on and stay tuned, we cover in this blog article a number of ways to mitigate this phenomenon.
Air-displacement pipettes are commonly used in industry quality control laboratories during for instance colorimetric assays, like ELISA, molecular biology. These pipettes require a different handling than normal glassware (e.g. volumetric flasks, graduated glass pipettes, etc.). As they are mostly used to aspirate and dispense relatively small volumes of a given liquid, the systematic and random error is usually rather large.
The United States Pharmacopeia (USP) – National Formulary (NF) general chapter <31> refers to plastic aspiration and dispensing devices as type B glassware; the general chapter will direct you to the International Organization for Standardization (ISO) Standard number 384. The limits that are listed in ISO 384 are much broader than required for type A glassware as stated in the USP compendial.
This means that the pipettes cannot be expected to outperform the specifications listed in the standards mentioned earlier. Nonetheless, one should aim to keep the systematic and random error as low as possible.
When pipetting, random and systematic error is important when a high level of reliability must be achieved.
Given the fact that a volume of air is used to aspirate a given liquid, the aspirated and dispensed volume is highly Subjected to physical parameters of the environment and that of the dispensed liquid.
This article may help you to mitigate the effect following factors, that are listed below.
Density and Viscosity of the liquid
Density and viscosity of a liquid
Air-displacement pipettes are usually used for aqueous solutions and are calibrated as such for solutions with a density close to 1.0 mg/µL. Pipetting solutions that have a lower or higher density than 1.0 mg/µL (at 20°C) will thus have an impact on the dispensed (delivered) volume.
Pipetting solutions that have lower density than water (<1.0 mg/µL) will increase the volume dispensed. Pipetting a solution that has a higher density will decrease the volume that is dispensed considerably. This as a heavier liquid is aspirated using the same volume of air, meaning that the air is, stretched out causing a decreased volume. Therefore, one must prevent pipetting solutions that have densities outside 1.0±0.1 mg/µL. The expected deviation within this range is likely below 0,0±0.2% (Eppendorf user guide No. 21 - June 2015). Instead, the use of a positive displacement pipette should be considered.
Another physical property that adversely affects the random and systematic error of an air displacement pipette is the viscosity or surface activity of a solution. This can be visually detected by droplets at the tip’s exit. These effects can be prevented using reverse pipetting, low retention pipette tips (in case of surface-active substances) or positive displacement pipettes.
Note: Positive displacement pipettes have a piston that is in contact with the pipetted solution. Therefore it is less affected by a liquid’s properties.
Variations induced by temperature
The dispensed volume is largely independent from pipette temperature when there is no difference in temperature between the solution, the internal pipette and the environment (see Lochner K H, Ballweg, T, Fahrenkrog, H-H: Untersuchungen zur Messgenauigkeit von Kolbenhubpipetten mit Luftpolster. Lab Med. 1996; 20, No. 7/8: 430–440). There is however a relatively large error for small temperature variations of the air-cushion while the pipette tip is immersed in the solution. This is caused by temperature differences between the air in the environment, the solution and the air-cushion. Inevitably causing differences in the aspirated or dispensed volume.
The longer an operator holds an air-displacement pipette, the warmer the pipette, and thus the air inside the pipette, becomes. When the operator then pipettes a liquid that is at room temperature, the dispensed volume is bound to be lower (Eppendorf user guide No. 21 - June 2015).
Necessary precautions should be taken to make sure that solutions that are to be pipetted with air-displacement pipettes are as close to the environment temperature as possible. Depending on the volume of the solution this might take between 15 to 60 minutes. If this is not possible, e.g. when pipetting culture media (e.g. for cell growth), one should try to calculate or determine the differences that are induced with the use of the solution at a given temperature to correct for the difference.
Barometric pressure of a given location is determined by its altitude. A pipette calibrated at about 0 to 100 meters above sea level would not yield the same results at an altitude of e.g. 1000 or 2000 meters above sea level. This effect however remains limited to systematic deviations below -3% (depending on the nominal volume and the volume pipetted). It is thus recommended to calibrate a pipette at an altitude close to the pipette’s location of use. (Eppendorf user guide No. 21 - June 2015)
Even when a pipette tip is pre-wetted (moistened before activities), differences in the environments’ relative humidity (%RH) will cause differences in the dispensed volume. Given the fact that a dry environment makes a solution more likely to evaporate in the pipette, which would cause the pipette to dispense lower volumes. Ideally, laboratory temperatures would be around 20°C with %RH above 50%. (see Extreme Pipetting IV: It’s Not the Heat, It’s the…; By Rumery, D.; Carle, B.). If you would like the best results that can be reasonably expected from air-displacement pipettes, make sure to have an appropriately controlled %RH and temperature in the laboratory.
Pre-wetting a tip would, at least partly, counteract the %RH problems that are described in the previous paragraph. Allow me to elaborate. When no pre-wetting is applied with aqueous solutions in a relatively dry environment (<50%), one would see that consecutive pipetting steps would become more accurate and precise, as it draws closer to an equilibrium of the air inside the pipette and the evaporation potential of the solution.
Pre-wetting allows the air in the pipette, even when in a low %RH environment, to become saturated with the liquid that is aspirated and dispensed repeatedly. When the air in the pipette is saturated, and you would start the pipetting step intended, almost no evaporation would occur. Which allows the pipette to be more accurate and precise (Eppendorf user guide No. 21 - June 2015). It would however save time when the %RH and temperature is at an appropriate level.
Air-displacement pipetting is affected by many different environmental factors and physical properties of the pipetted liquid, as described above. Surprisingly, the %RH has, together with the density of the liquid dispensed, the strongest adverse effect on the random and systematic error of a pipetted solution.
To limit the error on this type of pipettes most effectively:
One must control their laboratory environment, %RH and temperature, as much as possible. Laboratory workspace should have a relative humidity above 50% at 20°C for the best pipetting results. This may however be more difficult to implement as laboratories’ temperature is usually higher than 20°C and drier than %RH of 50%.
If you are using an external calibration lab, you should make sure that the location of the laboratories is at a comparable altitude to your own laboratory.
Make sure that the operators are appropriately trained to work with air-displacement pipettes, so that they know not to hold the pipette in between pipetting steps and to pause frequently to avoid fatigue.
Apply all these recommendations in your Quality Control unit’s laboratory, and you can mitigate the error of pipetting on the Quality tests and increase the validity in the test results.
QC project engineer @ pi
Eppendorf user guide No. 21 - June 2015
Extreme Pipetting IV: It’s Not the Heat, It’s the…; By Rumery, D.; Carle, B., Artel
United States Pharmacopeia (USP) – National Formulary (NF) general chapter <31>
International Organization for Standardization (ISO) Standard number 384
Lochner K H, Ballweg, T, Fahrenkrog, H-H: Untersuchungen zur Messgenauigkeit von Kolbenhubpipetten mit Luftpolster. Lab Med. 1996; 20, No. 7/8: 430–440