Adjusting Pipetting Variables to Optimize Methods

LCO Keith Albert


Keith Albert, Senior Applications Specialist, gives an overview on how you can adjust pipetting variables to help optimize automated liquid handler (ALH) methods.


Keith will be using the Artel MVS Multichannel Verification System as well as the Beckman Coulter Biomek automated liquid handler (ALH) during this video.

Liquid Class Settings Explained

Simple adjustments to some of the pipetting variables, sometimes referred to as liquid class settings, directly affects volume transfer accuracy.

Challenging liquid transfers, such as those involving different liquids with varying viscosity or low surface tension properties, are common in molecular biology assays, and experienced manual pipette operators often adjust their technique without thinking. They do things like slowing down the pipetting speed, pausing more (or less), or adding a blowout. But when an assay is transferred to an automated system, those adjustments need to be explicitly programmed into the liquid handler to ensure accurate and precise liquid handling.

Volume Transfer Accuracy

When volume transfer is inaccurate, the reagent concentration in the assay will also be inaccurate, resulting in experimental reagent concentrations that do not match the theoretical values, as demonstrated below.

Improving Liquid Class Settings

When pipetting by hand, nearly every action taken, such as how fast or slow the plunger is released when aspirating or if a blowout is used to get all the liquid out of the tip, are in the form of liquid class settings that can be adjusted on most ALHs.

Example of Liquid Class Settings

Adjustable settings may include the use of air gaps, offset volumes, aspirate and dispense rates, tip immersion depths and more.

Adjusting Liquid Class Settings on the ALH

Run the Method, and Observe

First, start by running the method and visually observe it. Observing the liquid transfers is a critical part of the optimization process.

If you notice that liquid is dripping or falling out of the tip, you might consider adding a trailing air gap after the sample aspiration.

Do you notice bubbles? This might indicate you need to change the pipetting technique.

Measure and Assess

Next, measure and assess the volume transfer(s). We use the MVS to measure the well by well and tip by tip volume values within minutes of a transfer.

There are other methods for measuring, but whatever method you employ to measure volumes, remember that it is important to know both accuracy and precision of the critical volume transfers. Pairing observations with data allows for a more comprehensive understanding of the liquid handler pipetting behavior, especially as adjustments are made to the pipetting variables.

Albert, K, Bradshaw, J. JALA Tutorial, Importance of Integrating a Volume Verification Method for Liquid Handlers: Applications in Learning Performance Behavior.

Below is a data set from the published JALA article which includes volume measurement data collected after liquid class settings were sequentially adjusted, all with the goal of optimizing the method on a 96-tip automated liquid handler for a 10-μL wet transfer into a 96-well plate.

Experiment 1 (Column D)

This experiment employed an arbitrary aspirate and dispense rates of 5-μL/s , and no air gaps. The result is a CV < 4%, but the accuracy is -30% with less then 7 μL transferred.

Experiment 2 (Column E)

In experiment 2, a 5-μL leading air gap and blow out volume was introduced. The results drastically improved to within 10% of the target volume (9.18 μL average volume measured).

Experiment 3 (Column F)

In experiment 3, a trailing air gap was introduced, but it did not have any real impact on the accuracy. However, it did improve the repeatability as noted by the slightly improved CV value.

Experiment 4 (Column G)

Finally, because the measured volume in experiment 3 was low by 0.88 μL, the last experiment added that additional volume as an offset volume. Ultimately, this resulted in an optimized volume transfer in just minutes.


The process of method optimization by adjusting liquid class settings relies heavily on a reliable volume measurement process.

As always, we recommend that the liquid handler manufacturer’s default settings are NOT altered – but instead, are saved as a new pipetting template that can be adjusted so that you can always go back to the original default settings, if needed.

In some cases, a method can be optimized over a narrow range by adjusting the calibration or correction curve values, as we demonstrated previously. By optimizing liquid class settings within a method, a liquid handler can be better prepared for assay work.

If there is anything we can do to help to support your work, we are here to help.