Pipetting is one of the most common laboratory procedures. Inconsistent pipetting can lead to errors in sample preparation, affecting data quality and potentially jeopardizing the entire experiment. Improving pipetting techniques not only increases accuracy but also reduces the risk of contamination and ensures reproducibility. This article will outline key strategies for optimizing pipetting techniques, covering equipment calibration, handling procedures, and avoiding common pitfalls.

Pre-wetting pipette tips

Before performing pipetting operations, it is essential to pre-wet the tips. This is done by aspirating and completely dispensing liquid at least three times. If pre-wetting is not performed, the air space inside the tip can cause liquid evaporation, thereby affecting the accuracy of aspiration and dispensing and resulting in a significant reduction in delivery volume. This is because un-pre-wetted tips absorb moisture from the air, causing a decrease in liquid volume. By pre-wetting, a liquid film forms on the inner surface of the tip, reducing evaporation effects and improving the accuracy and consistency of liquid transfer. This step is particularly critical in pipetting low-volume or highly volatile solutions, helping to enhance the reliability of pipetting results.

Ensure temperature balance

Before performing pipetting operations, ensure that the liquid and pipetting equipment are at the same temperature as the environment. The pipetting volume of air displacement pipettes is affected by the relative humidity and vapor pressure of the liquid, both of which are closely related to temperature changes.

When the ambient temperature changes, the vapor pressure and humidity of the liquid also fluctuate, leading to instability in the pipetting volume.

By operating in a temperature-controlled environment, you can minimize errors caused by temperature changes, ensuring the accuracy and consistency of the pipetting process.

Check the cleanliness of the pipette tip and any residual liquid.

Before sampling, be sure to carefully wipe off any droplets on the outside of the pipette tip with a lint-free cloth, ensuring that the wiped liquid is kept away from the opening of the pipette tip to prevent liquid from leaking out. After sampling, before releasing the plunger, gently touch the side of the container with the pipette tip to help expel any residual liquid inside the tip. By utilizing the effect of surface tension, residual liquid can be more effectively expelled from the pipette tip, thereby reducing volume errors caused by residual liquid.

Use the standard pipetting mode

Correct pipetting should follow the standard pipetting mode: First, press the plunger to the first stop at the bottom, then dip the tip into the liquid and release the plunger to aspirate the liquid. After removing the pipette, press the plunger to the second stop to expel all liquid from the tip. For most liquids, the standard (or forward) pipetting mode provides higher accuracy and precision, especially when handling routine liquids. Compared to the reverse pipetting mode, the standard mode effectively prevents excessive liquid expulsion.

Vertical withdrawal of the pipette

When aspirating liquid, the pipette should be held vertically and withdrawn vertically from the center of the container. This technique is particularly important for small volumes of liquid (e.g., less than 50 µL), as tilting the pipette while withdrawing it from the liquid may cause variations in the volume aspirated. If the pipette comes into contact with the container wall, it may also trigger capillary action, leading to volume loss. Maintaining a vertical operation effectively prevents these issues, ensuring the accuracy and consistency of the pipetted volume.

Minimize contact with pipettes and tips

When operating, hold the pipette lightly and place it carefully back on the pipette rack or set it down between pipetting operations. Avoid touching the pipette tip or the container holding the liquid to be pipetted, as the transfer of heat from your hands may interfere with the temperature balance of the liquid, thereby affecting the accuracy of the pipetted volume.

Immerse the tip to the appropriate depth

Before aspirating liquid, ensure that the tip is immersed to the appropriate depth below the liquid surface. For large-volume pipettes (1–5 mL), the tip should be immersed approximately 5–6 mm into the liquid; for small-volume pipettes, it should be immersed 2–3 mm. If the immersion depth is too shallow, especially when using large-volume pipettes, air may be aspirated, thereby affecting the pipetting volume. If the tip is immersed too deeply, liquid may adhere to the exterior of the tip or cause the tip to contact the bottom of the container, further impairing the pipetting performance.

Use the correct pipette tips

Always use high-quality tips that are compatible with your pipette. In most cases, original manufacturer tips provide the best performance. If you choose tips from other brands, make sure they have been tested on the corresponding pipette model and meet performance standards. Mismatched tips and pipettes can result in inaccurate or imprecise pipetting, or both. High-quality tips feature excellent sealing properties, are made from high-quality materials, and have no manufacturing defects, ensuring reliable and precise liquid transfer.

Maintain consistent pressure and speed when pressing and releasing the plunger

During operation, press the plunger smoothly and stop it at the first stop point with a gentle and consistent force. After immersing the tip into the liquid, release the plunger at a constant speed to complete the aspiration. The key is to maintain a consistent rhythm of action—only through repeated and standardized operations can the reproducibility and reliability of the pipetting results be ensured. Avoid uneven force or fluctuating speeds to minimize errors and improve pipetting accuracy.