Pipettes are indispensable tools in laboratory operations. When pipetting data issues arise, we often question the performance of the pipette, but pipetting technique also plays a critical role in the success or failure of an experiment. Neglecting pipetting technical guidance may result in significant time and financial losses. The proper use of pipettes not only affects the accuracy of experimental results but also the safety of laboratory personnel. This article compiles tips for using pipettes. By following these guidelines, the accuracy and reproducibility of your pipetting may improve by up to 5%.
How pipettes work:
Set the displacement volume, and the piston moves to the specified position.
Press the button, and the piston descends, expelling a certain volume of air. The volume of air expelled is equal to the displacement volume.
Release the button, and a partial vacuum is created inside the suction head. Under atmospheric pressure, the liquid enters the suction head.
Press the button again, and the air pressure inside the piston sleeve and suction head increases. The compressed air pushes the liquid out of the suction head.
10 Tips to Improve Your Pipetting Technique
Attaching the tip
When attaching the tip, make sure that it is securely fastened and there are no leaks. Press down firmly on the pipette handle and rotate slightly if necessary. Using original tips manufactured by the pipette manufacturer will ensure a better seal.
Incorrect operation: Striking the tip forcefully. This method can damage the tip and even wear down the pipette handle, thereby affecting its seal.
Range Setting
To achieve higher precision, the pipetted volume should be within 35% to 100% of the pipette's nominal range. When the pipetted volume is less than 35% of the pipette's nominal range, it is recommended to switch to a pipette with a smaller range.
Range adjustment: When adjusting from a smaller range to a larger range, rotate continuously in the direction of the desired range. Once the rotation exceeds one-third of a turn beyond the desired range, adjust back to the desired range. When adjusting from a larger range to a smaller range, rotate continuously directly to the desired range.
Tip immersion angle
The angle of the handheld pipette also affects accuracy. When pipetting, the angle deviating from the vertical direction should not exceed 10 degrees.
When pipetting, the tip should be immersed as vertically as possible into the sample, and should not deviate from the vertical position by more than 20°. For pipettes with microvolumes, pipetting at a vertical angle can improve accuracy by 2.5%.
Tip immersion depth
Immersing the tip to the appropriate depth can improve accuracy by 5%. If the tip is immersed too deeply, the air inside the tip will be compressed, causing excessive liquid aspiration; conversely, if the tip is immersed too shallowly, air will be drawn in, leading to bubble formation and inaccurate volume transfer.
Recommended immersion depths:
2–10 μL: 1–2 mm
20–200 μL: 2–3 mm
200–2,000 μL: 3–6 mm
Above 2,000 μL: 6–10 mm
Aspiration
When transferring different samples, maintain a consistent aspiration rhythm and press or release the button at a steady and consistent speed. Control the aspiration speed; if it is too fast, it may cause liquid or aerosol to enter the pipette, contaminating components such as the piston. After aspiration, hold the tip in the liquid for one second before slowly removing it. This is particularly important for high-volume pipettes or when aspirating viscous samples.
Dispensing and blowing liquid
First, press the piston to the first position to dispense liquid, pause briefly, then press to the second position to blow liquid.
Maintaining a consistent pipetting rhythm and speed will help you achieve ideal, more reproducible results and improve accuracy by 5%.
Select forward or reverse pipetting based on the type of liquid
Press the plunger to the first position, immerse the tip in the liquid, then release the plunger to aspirate the liquid. Remove the pipette from the liquid, press the plunger to the second position to dispense all of the liquid. For all solutions other than viscous or volatile liquids, standard (or forward) mode pipetting is more accurate and precise than reverse mode. Reverse mode typically results in over-dispensing. Therefore, it is recommended to assess the impact of over-dispensing in the experiment and adjust as needed.
Temperature changes
The ideal temperature for pipetting and calibration is 21.5°C ± 1°C. Ensure the liquid and equipment are equilibrated to room temperature before pipetting. Avoid operating in areas with drafts, direct sunlight, or significant temperature fluctuations, as these conditions may reduce pipetting precision. Prolonged hand-held use of the pipette may cause heat from the hand to warm the pipette, leading to thermal expansion of the internal air and resulting in inaccurate results. Therefore, the pipette should not be held in the hand for extended periods. When not in use, it is best to hang it on a stand or let it cool by holding it in the hand.
Rinsing
Be sure to distinguish between rinsing tips and situations where rinsing is not appropriate:
Before aspirating liquid, repeat aspiration and completely expel a certain amount of liquid at least 2-3 times. Rinsing the tip 2-3 times can form a homogeneous film inside the tip, which can improve accuracy by 0.2%.
Rinsing can adversely affect results when transferring hot or cold solutions, such as pipetting from an ice bath or when the solution temperature exceeds 37°C. Rinsing may result in an error rate of up to 5%. Therefore, do not rinse with high-temperature or low-temperature liquids!
Pipetting
Good pipetting technique can also improve accuracy by 1%. Before pipetting, lightly touch the side of the storage bottle with the tip to carefully remove any droplets from the outside of the tip, ensuring that they are far from the tip opening to prevent liquid from flowing out of the tip. After pipetting and before releasing the plunger, lightly touch the side of the container with the tip to remove any residual liquid from the tip. Surface tension will help expel residual liquid from the tip.
Pipetting Techniques for Special Samples
In most cases, pipettes are used to transfer aqueous solutions. However, you may occasionally encounter special samples such as highly volatile, high-viscosity, or high-density samples. If you frequently need to transfer such special samples, it is recommended to purchase a pipette with an external piston mechanism. Although the consumable costs are slightly higher, this ensures pipetting accuracy. If you only occasionally transfer special samples and have limited financial resources, please consider the following pipetting techniques, which may help improve pipetting accuracy.
Pipetting Volatile Samples
Before pipetting, be sure to rinse the pipette twice; after aspirating the sample, dispense it as quickly as possible.
Pipetting High-Viscosity Samples
Use reverse pipetting mode: when aspirating, press the aspirate/dispense button to the second position (second stop point), and when dispensing, press the button to the first position (first stop point). Additionally, a dwell time of 3–5 seconds is required during both aspiration and dispensing.
Transferring high-density/low-density samples
The accuracy values of pipettes are based on transferring pure water. If the density of the sample differs significantly from that of water, the accuracy will also be significantly reduced. Therefore, it is necessary to determine the density of the sample before pipetting and adjust the volume setting to the product of the sample volume and density. For example, if a sample has a density of 1.2 g/cm³ and 300 μL needs to be transferred, the volume should be set to 360 μL. This is only a rough adjustment method; for precise adjustments, auxiliary tools such as measuring devices or balances should be used for accurate calculations.
Transferring high-temperature/low-temperature samples
Do not rinse the tip before pipetting; use a new tip for each pipetting operation; complete both aspiration and dispensing as quickly as possible.
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