liquid handling robot by Hamilton Company in action

When looking at how to teach pipetting as part of our Automated Liquid Handling Guide, the same general process applies when developing liquid handling methods across all labs, industries, and applications. Even though the details change, the liquids fundamentally need to be defined and optimized for use on the automated liquid handler. The methodology explored below walks you through the high level step by step liquid handler setup process needed to develop optimized liquid handling performance, including best practices for common liquid handling activities.

automated pipetting machine filling samples of liqud

Before You Begin Liquid Handler Setup

Make sure that the following items are ready to go before getting started:

  • The automated liquid handler needs to be installed, set up, and ready for use.
  • The liquids for the application should be known, available, and brought to the desired temperature for the application.
  • The tips and labware for the application should be known and available.

Step 1: Understand Properties of Liquid

The first step in the methodology is a careful examination of the physical properties of the liquid to be transferred. The purpose of the examination is to determine how the liquid behaves so you can get a sense for how to teach the automated liquid handler to handle it. For example, you could use a hand pipettor to pipette the liquid and get a feel for how it transfers before testing it in the automated liquid handler.

You can also refer to related reference documentation to get the specifics. Common resources include the MSDS (Material Safety Data Sheets) and online resources like

For a detailed list of physical properties to analyze and their implications, view Liquid Properties.

Step 2: Select a Predefined Liquid Class

All Hamilton software platforms come with predefined liquid classes. These liquid classes can be implemented in methods or used as a starting point for developing a more precise liquid class for a specific application.

When selecting a liquid class for use in a method or developing a new liquid class, always choose the liquid class with the closest match to the liquid. A suitable liquid class allows the automated liquid handler to know how to interact with the liquid and prevent mishandling.

For example, if developing a new liquid class for Master Mix, use the existing water definition as a starting point since both liquids are part aqueous and will have similar properties.

Examples of Synonymous Liquid Classes

Aqueous SolutionVolatile Organic SolventsInvolatile Organic SolventsViscous LiquidBlood Products
Water Ethanol DMSO Glycerol Serum
PBS Isopropanol Alkane Beads Plasma
Master Mix Acetone Oil Whole Blood
TRIS Formaldehyde Red Blood Cell

Step 3: Run Test and Visually Inspect Pipetting

Once the predefined liquid class is selected, it’s time to test the liquid transfer on the robot. The predefined liquid class is used as a baseline to speed up the process of testing.

  1. Set up the automated liquid handler with the appropriate source and destination labware.
  2. Fill the labware with the liquid that you intend to transfer. If the liquid in question is costly or in short supply, use water for initial testing and optimization.
  3. In the automated liquid handler’s software, build a simple method to transfer the liquid that will mimic the step in the actual process. Focus on one transfer step in the process before testing the next. Make sure that the method settings are defined as you want them for the actual transfer.
  4. Run the method and observe the transfer. Look for the following:
    — Is the aspirate height or liquid level submerge depth too high/low?
    — Are there any droplets on the end of the tips after aspiration?
    — Is the dispense height or liquid level submerge depth too high/low?
    — Are there any droplets on the end of the tips after dispense?
    — Are the channels properly following the liquid level during aspiration and dispense? Should following be turned off?

Step 4: Optimize Parameters Until Pipetting Appears Acceptable

Continue to run the simple method, observe the pipetting, and make adjustments based on what you see. The goal is to make sure that the pipetting looks correct. For example, there should be no dripping from the tip and no bubbles on dispense.

Start by making modifications to method settings such as enabling cLLD on the aspirate or adjusting the fixed height.

If the method settings are optimized, but the transfer still appears inconsistent, you can then focus on modifying the liquid class settings to improve performance. Follow these steps to adjust the liquid class:

  1. Save the liquid class under another name. Now it can be modified to work for the specific application.
  2. Change one liquid class parameter at a time to see its effect on the liquid transfer.
  3. Inspect transfers.
    — Visually inspect for consistent transfers.
    — Spot check with a handheld pipette to give an indication of consistency and if the transferred volume is short or in excess
  4. Once the transfers look consistent, move on to step 5.

Step 5: Verify Volumes and Adjust Correction Curve Accordingly

Once the pipetting appears to be acceptable, the transferred volumes can be quantified to determine the precision and the trueness of the liquid transfers.

Before measuring, it is important to know your application’s pipetting requirements to make sure that the final optimizations meet the need. If the requirements are unknown and you want to minimize the amount of variability that pipetting contributes to your application, you can strive to match the specifications set for the pipetting device you are using.

You might measure volumes gravimetrically to make sure the transfers are both accurate and precise. Then, you can continue to adjust liquid class settings until optimal precision is achieved.

When precision is achieved, the correction curve of the liquid class can be adjusted to ensure trueness of all volumes of interest for your application. For example, if you are transferring a volume of 300 μL, but are measuring a value of 295 μL, then you can increase the corrected value in the liquid class by an additional 5 μL. Continue to adjust until the proper target volume is achieved.

graph teaching automated pipetting techniques showing relationship between targeted volume and corrected volume

Once verification is complete, make sure the updated liquid class is implemented in your method. The liquid class can then be used by lab technicians running liquid transfers for experiments. Periodically check the performance of the liquid transfers to make sure that no changes are needed. Keep in mind that the correction curve for any default liquid classes cannot be modified.

Download this information and much more with the Liquid Handling Guide.

A 36-page PDF about everything you need to know about Liquid Handling.

Additional Resources for Automated Liquid Handling

Check out our Automated Liquid Handling Platforms
See Hamilton Automated Plate Sealers and Small Devices
See the line of Hamilton Carriers and Pedestals
Check out the homepage of our Automated Liquid Handling Guide
Get our input on the process used to Measure Transferred Liquid Volume
The team at Hamilton gives recommendations for Best Practices for Liquid Handling Activities
Want to "own" the guide? Click for a PDF Downloadable Liquid Handling Guide
Read our tips to accurately Pipette Volatile Liquids
Learn how Liquid Properties affect automated liquid handling
Read our comparison of Manual Pipetting vs Semi Automation vs Automation
Hamilton Robotics highlights 10 Important Considerations for Accurate Automated Pipetting