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How to apply DOE to Robocolumns experiments: Optimizing the purification process

DOE is a powerful tool for optimization. Learn how you can apply it to your miniturized purification processes

Updated over 6 months ago

In RoboColumn workflows, process optimization is important. For example, you may have a single sample to process, but want to explore the effect of:

  • Resin types to use

  • Optimal flow rates for the fastest execution

  • Loading volumes to fine-tune elution

Using Design of Experiments (DOE) is a very efficient way of approaching this type of experiment. To learn more about DOE, click here.
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In this tutorial, you will learn:

  • How to build a RoboColumn DOE workflow to investigate process development factors

  • How to describe the factors for a DOE design to be applied to the workflow

Get started

  1. Create a RoboColumn workflow in the builder. To learn how, click here.

  2. Rename the workflow. To learn how, click here.

  3. Select the Tecan liquid handler you want to use. To learn how, click here.

  4. Select a default plate type for input liquids. To learn how, click here.

How to build RoboColumn DOE workflow

A RoboColumn DOE workflow is very similar to a normal RoboColumn workflow; the same elements are used. The key difference is that you only want to describe the default conditions to use for a single RoboColumn.

  1. Configure the RoboColumn layout in the Define RoboColumn Plate element

    Only a single RoboColumn needs to be described. The number of robocolumns will automatically be scaled up depending on the number of runs in your final DOE design.
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  2. Configure the Chromatography actions in Run Chromatography Stage or Run Gradient Chromatography Stage elements.
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    Only a single set of actions needs to be defined. The entries for the number of fractions, load volumes, and flow rates/residence times will be used as a default set of conditions for the element and will be applied to all runs in your robocolumns DOE, unless this parameter is specifically overridden by a DOE factor later in the process.
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How to create a RoboColumn DOE design

RoboColumns DOE process parameters

Currently, the following elements and parameters can be treated as factors for RoboColumns DOE:
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Define RoboColumn Plate

Resin Type

Categorical: a set of possible resin types defined in the workflow.
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Resin Volume

Numeric discrete: a set of possible resin volumes. Note that this is limited by the type of RoboColumns you have.
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Run Chromatography Stage

Buffers/Samples to Load

Categorical: a set of possible liquids defined in the workflow. To see how this factor could be used, click here.
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Total Loading

Number of fractions into which liquid loading into RoboColumns (and any subsequent outflow collection) is split. This is constant for all Robocolumns and can either be described as a volumetric unit or a mass amount.

Each fraction corresponds to a load volume, determined by the Fractional Load parameter. The total volume of liquid loaded into any one RoboColumn by this element will be equal to Total Loading * Fractional Load, for the RoboColumn of interest.
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Fractional Load

Applies the apply the same load volume to each fraction loaded into any one RoboColumn, can be described as either a volumetric amount or loading time.
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Flow Rates/Residence Times

Numeric continuous: any flow rate or residence time. Practically this is restricted by the liquid handler limits (e.g. availability of a liquid class to enable the requested flow rate).
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Run Gradient Chromatography Stage


​Buffer A

Categorical: a set of possible liquids defined in the workflow. To see how this factor could be used, click here.
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Buffer B

Categorical: a set of possible liquids defined in the workflow. To see how this factor could be used, click here.
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Buffer B Start Percentage

Numeric continuous: between 0 to 100.
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Buffer B End Percentages

Numeric continuous: between 0 to 100.
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Total Load Volume

Total load volume to load into RoboColumns. Each fraction corresponds to a load volume, determined by the Fractional Load parameter.
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Fractional Load

This parameter will apply the fractional load to each fraction loaded into any one RoboColumn. Therefore, the total volume of liquid loaded into any one RoboColumn by this element will be equal to Total Load Volume * Fractional Load, for the RoboColumn of interest.
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Flow Rates / Residence Times

Numeric continuous: any flow rate or residence time. Practically this is restricted by the device limits (e.g. availability of a liquid class to enable the requested flow rate).
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Selecting parameters to use as factors in a DOE and setting their levels is almost identical to simple DOE workflows that create mixtures.

Defining DOE factors

  1. Simply switch the builder into DOE mode
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2. Toggle on the parameters you wish to investigate in your DOE
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3. Launch the design interface and define the factor setpoints, or levels, that you wish to test.

To learn how to define parameters as DOE factors and how to define their levels in more detail, click here.
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How to identify the RoboColumns workflow should be driven by DOE

When applying DOE to robocolumns workflows it is essential that at least one parameter in the Define RoboColumn Plate element must be set as a DOE factor - even if it is a constant.

If you do not wish to screen the Resin Type or Resin Volume in Each Robocolumn as a factor in your DOE, simply set Resin Type as a constant. Setting this parameter enables Synthace to recognize that the RoboColumns will be used in DOE.


To learn how to define constants for a DOE, click here.

Defining factors and levels to be shared across multiple chromatography stages

It is common in purification processes to reuse the same process factor across multiple stages. For example, you may want to vary flow rate but keep it constant between Load and Elution Run Chromatography Stage elements. To do this:

  1. Add a custom numerical factor with the flow rate. To learn how to add custom numerical factors, click here.

  2. In this example, enable Flow Rates / Residence Times as DOE factors in Load and Elution Run Chromatography Stage elements by switching your workflow into DOE mode and toggling the parameter on in the instance panel.

  3. Add a derived factor for each element’s Flow Rates / Residence Times and set it as being equal to the custom flow rate factor. To learn more about defining derived numerical factors, click here.

Calculating a DOE design

Once you have selected your factors and levels continue with these steps:

  1. Click on the Calculate Design tab
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  2. Select your design of choice. To learn more about DOE designs, click here.
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  3. Assess its quality. To learn more about DOE design quality, click here.

4. Once you are happy, verify the runs in your design match your expectations.

5. Click Simulate With Design.

6. Navigate to the simulation preview

Note: In the simulation preview, each RoboColumn in your RoboColumn plate will correspond to a run in your DOE design. Use the preview to help validate that your RoboColumns DOE is calculated as you would expect.


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Well done on making it to the end of this tutorial.


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To learn how to build more complex workflows where factors define the compositions of the liquids being loaded into the RoboColumns, i.e. your buffers and samples click here.


To learn how to structure your DOE data for analysis in external stats tools or how to analyze your Robocolumns with the chromatography analysis functionality in Synthace, click here.
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Check out the 🎓 Get Started: Introduction to Robocolumns DOE tutorial in the Synthace platform to see worked example workflows!
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Related Articles
Introducing the Robocolumns® elements
How to build miniaturized purification workflows in Synthace
How to apply DOE to RoboColumns experiments: Optimizing purification buffer compositions
How to access the data from your DOE miniaturized purification experiment
The DOE Elements
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