In pipetting, users often focus on volume settings and tip selection. However, a small preparation step performed before the actual transfer can also affect dispensing stability.
One example is pre-wetting the pipette tip.
Pre-wetting means aspirating the sample into the tip and dispensing it before beginning the actual transfer. Depending on the application, this aspiration and dispensing cycle may be repeated a few times before aspirating the intended volume.
At first, this may seem like an unnecessary extra step. After all, the liquid is simply aspirated and returned. However, under certain conditions, such as low-volume pipetting or handling volatile liquids, this short preparation step can help make dispensing more consistent.
The important point is not to perform pre-wetting automatically in every situation. It is to understand what the step is intended to do and use it when it fits the sample and the procedure.
What Does Pre-Wetting Actually Do?
Immediately after a new pipette tip is attached, the inside of the tip has not yet been exposed to the sample.
During the first aspiration, the air inside the tip and the inner surface are still in their initial condition. As a result, the behavior of the first transfer may not be exactly the same as the transfers that follow.
Pre-wetting exposes the inner surface of the tip to the sample before the actual transfer begins.
By aspirating and dispensing the same liquid in advance, the inside of the tip becomes conditioned with the sample and is brought closer to the state it will be in during routine dispensing.
Pre-wetting is not a cleaning procedure.
It is also not intended to fill the entire tip with liquid.
Its purpose is to prepare the internal condition of the tip and help reduce differences between the first transfer and the transfers that follow.
Why Conditioning the Inner Surface Matters
When liquid is aspirated and dispensed, not all of it necessarily leaves the tip in exactly the same way. Depending on the properties of the sample, a very thin liquid film or a small amount of residual liquid may remain on the inner surface.
During the first transfer with a new tip, the liquid contacts a dry inner surface. During the next transfer, however, the inside of the tip has already been exposed to the same liquid.
This means that the first transfer and the transfers that follow may behave slightly differently.
Pre-wetting helps reduce that difference by exposing the inner surface to the sample before the actual measurement begins.
This becomes especially important at low volumes. A small absolute difference that may be difficult to notice at a larger volume can become much more significant when only a few microliters are being handled.
Why Pre-Wetting Can Help in Low-Volume Pipetting
In low-volume pipetting, the total amount of liquid is small, so minor changes can have a larger relative effect.
Residual liquid on the tip surface, small air bubbles, differences in aspiration speed, and the timing of tip removal from the liquid can all affect the result.
By pre-wetting the tip, the actual transfer begins with the inner surface already conditioned by the sample. This can help create more consistent starting conditions.
However, pre-wetting does not replace good pipetting technique.
Aspiration should still be performed at a consistent speed.
The tip should be immersed to an appropriate depth.
The user should pause when necessary after aspiration.
The pipette should be held as vertically as practical.
Pre-wetting works best when it is combined with consistent basic technique.
Volatile Liquids Can Be Affected by the Air Inside the Tip
Pre-wetting is also an important consideration when working with volatile liquids.
In a standard air-displacement pipette, there is a column of air between the piston and the liquid. When a volatile liquid is aspirated, vapor from the liquid can affect the air inside the tip and alter pressure and volume conditions.
The internal condition of a new tip during the first aspiration may therefore be different from the condition after the same liquid has already been aspirated and dispensed once.
Pre-wetting can help condition the air space inside the tip with vapor from the sample before the actual transfer.
However, volatile liquids require care beyond pre-wetting. Waiting too long after aspiration or warming the sample with the user’s hand can also affect performance.
For that reason, pre-wetting should be considered together with aspiration speed, temperature, pipette type, and the procedure recommended for the sample.
How Many Times Should a Tip Be Pre-Wetted?
Pre-wetting is commonly performed by aspirating and dispensing the same sample a small number of times before the actual transfer.
There is no single number that is ideal for every application. The appropriate number of cycles depends on the liquid, the volume, the pipette, and the laboratory procedure.
The goal is not to repeat the action as many times as possible. The purpose is simply to bring the inside of the tip closer to the condition it will have during the actual transfer.
Repeating the cycle unnecessarily can increase working time and may also cause foaming or temperature changes. This may be especially undesirable for delicate or easily foamed samples.
For this reason, it is best to review the procedure or the pipette manufacturer’s recommendations and determine a suitable number of cycles based on actual performance.
Once the number of cycles is established, it should be kept consistent. If different users pre-wet the tip a different number of times, that difference itself can become another source of variation.
Pre-Wetting Should Also Be Performed Consistently
Pre-wetting is a preparation step, but that does not mean it should be performed carelessly.
Aspiration speed should not vary from cycle to cycle.
The liquid should not be dispensed so aggressively that it foams.
The tip should not be immersed too deeply.
The plunger should not be released suddenly.
The same controlled movement used during the actual transfer should also be used during pre-wetting.
It is useful to think of pre-wetting as creating the same internal conditions that will be used during the real dispensing operation.
The destination of the pre-wetting liquid should also be decided according to the procedure.
Can the liquid be returned to the sample vessel?
Should it be discarded?
Would returning it create a contamination risk?
The correct choice depends on the sample and the experiment. The procedure should therefore define not only whether to pre-wet, but also where the pre-wetting liquid should be dispensed.
Continued Use of the Same Tip Still Requires Attention
When repeatedly dispensing the same sample, pre-wetting once at the beginning does not guarantee that the tip condition will remain unchanged for the entire workflow.
Over time, the temperature of the liquid or tip may change.
Liquid may collect on the outside of the tip.
The condition of the liquid film and residual sample inside the tip may also change.
In addition, continuing to use the same tip for too long may not be appropriate from the standpoint of sample quality or contamination control.
Pre-wetting is not a substitute for proper tip replacement.
The timing of tip changes should still follow the sample type, the workflow, and the laboratory procedure.
When a new tip is attached, that tip begins again in an unconditioned state. If pre-wetting is appropriate for the application, the new tip should also be pre-wetted before use.
Is Pre-Wetting Necessary for Every Pipetting Task?
Pre-wetting can be useful, but it is not required for every pipetting task.
When handling ordinary aqueous liquids at relatively large volumes, the difference created by pre-wetting may be small. In some workflows, maintaining a simple and consistent standard procedure may be more important than adding another step.
Pre-wetting is especially worth considering in situations such as:
- low-volume pipetting,
- applications requiring high reproducibility,
- volatile liquids,
- noticeable differences between the first transfer and later transfers,
- repeated dispensing with the same tip,
- or procedures in which pre-wetting is recommended.
The important point is not to add pre-wetting automatically as a habit. The expected benefit should be clear.
When introducing pre-wetting into a procedure, it can also be useful to compare results with and without the step and confirm whether it actually improves dispensing stability.
Pre-Wetting Cannot Solve Every Pipetting Problem
If results remain inconsistent even after pre-wetting, the cause may be somewhere else.
Aspiration may be too fast.
Tip immersion depth may be inappropriate.
The waiting time may be too short for a viscous liquid.
The selected pipette range may not suit the target volume.
The tip may not be attached correctly.
The pipette may require inspection or calibration.
In these situations, increasing the number of pre-wetting cycles will not solve the underlying problem.
Pre-wetting is specifically intended to condition the inside of the tip. It is not a universal solution for every source of pipetting variation.
That is why inconsistent results should be evaluated by reviewing the entire pipetting process rather than focusing only on pre-wetting.
How WATSON Thinks About the Role of Pre-Wetting
At WATSON, pre-wetting is not viewed as a fixed ritual that must be added to every procedure. It is a practical technique that can help prepare the pipette tip under conditions where dispensing stability is especially important.
Its main roles are to:
- condition the inside of a new tip with the sample,
- reduce differences between the first transfer and later transfers,
- and create more consistent conditions in low-volume or volatile-liquid applications.
The important thing is not simply to memorize a number of cycles. It is to understand why pre-wetting is being performed, which samples may benefit from it, and whether it is necessary for the specific task.
Pipetting stability is never determined by a single action. The pipette, the tip, the sample, temperature, technique, and working environment all interact to affect the result.
Pre-wetting is one practical option for improving that overall combination and supporting more consistent routine liquid handling.