After the plate reader has measured the absorbance of each fraction, SynthaceHub sends the raw data from the plate reader to Synthace. Synthace combines the raw data with the chromatography stages and metadata that you specified in your workflow to produce a set of corrected, structured data.
Example: In your workflow, you specified the concentration, molecular weight and molar extinction coefficient of the sample. Once Synthace knows the absorbance of the fractions that contain the sample, it uses this information to calculate the mass, mass concentration and yield of the sample.
Synthace uses the set of normalized, structured data to generate a visualization of the results.
Open the visualization
On the WORKFLOWS tab, locate and open the simulation.
Beneath the Data visualizations heading, click Chromatography results.
Tip: Synthace also displays this link on the Results tab.
Select a different dataset
By default, the visualization displays the results of the execution of the simulation that you selected on the WORKFLOWS tab.
If you want to display the results from a different execution, complete the following steps.
Click Select Purification Data.
Locate and select the execution that you want.
Click Load.
Raw Chromatograms
On this tab, Synthace displays a series of pseudo-chromatograms.
Each one matches the absorbance measurements that the plate reader took at a specific wavelength to the chromatography stage during which you collected each fraction.
Wavelength
In your workflow, you selected the absorbance protocol that you wanted your plate reader to use.
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The absorbance protocol that you selected determined the wavelengths at which your plate reader measured the absorbance of each fraction.
By default, the visualization displays a pseudo-chromatogram for each wavelength.
Use the check boxes beneath the Wavelength heading to select the wavelengths for which you want the visualization to display a pseudo-chromatogram.
Axes
Each pseudo-chromatogram is divided into sections.
There is one section for each chromatography stage in your workflow.
On the x-axis, the pseudo-chromatogram displays the fractions that you collected during each chromatography stage.
On the y-axis, the pseudo-chromatogram displays the absorbance of each fraction at the stated wavelength.
Column Name
By default, the pseudo-chromatograms display the absorbance of the fractions that you collected from every robocolumn.
Use the check boxes beneath the Column Name heading to filter the pseudo-chromatograms so that they only display the absorbance of the fractions that you collected from the robocolumns that you select.
Controls
Click a control to highlight it. If a control is highlighted, you can perform its action. To learn more, click here.
Raw Purification Data
On this tab, the visualization displays the raw data from the execution and the plate reader.
Use this tab to review the raw data in more detail.
If you want to download the raw data, use the check boxes on the left to select the columns that you want to download, then click Download.
Downstream Analytics
On this tab, if you have already used a LabChip® to analyse some fractions, Synthace adds the concentration, purity and size measurements from the LabChip® to the absorbance measurements that the plate reader took at 280nm.
Analyse
On this tab, the visualization displays a pseudo-chromatogram and a series of collection plates.
Analysis Setup
Use the Analysis Setup to prepare the analysis.
Pathlength correction: The shape of the wells that contain the fractions, the volume of the fractions in the wells, and the shape of the menisci of the fractions all affect the absorbance measurements. To adjust the analysis to account for these effects, select two near-infrared wavelengths that are appropriate for the liquid that contains the fractions. Synthace uses the wavelengths that you select to perform a pathlength correction.
Tip: For water, 900nm and 975nm are appropriate.
Background subtraction: Noise from the plate reader and light scattering also affect the absorbance measurements. To adjust the analysis to account for these effects, select a wavelength that is considerably different to the one that measured the absorbance of the protein of interest. Synthace subtracts the absorbance measurements that the plate reader took at that wavelength from the analysis.
Example: If the wavelength that measured the absorbance of the protein of interest is 280nm, you might want to select 340nm.
Analysis: Select the wavelength that measured the absorbance of the protein of interest.
Blank Correction: Check this box if you instructed the Tecan Freedom EVO® to collect a volume of one or more liquids to use as a blank. Synthace subtracts the absorbance measurements that the plate reader took of each blank from the analysis.
Data
Use the drop-down buttons beneath the Analysis Setup button to select the data that you want the pseudo-chromatogram and collection plates to display.
The pseudo-chromatogram displays the data that you select on the y-axis: a higher peak indicates a higher value for the data that you select.
In the collection plates, a darker colour indicates a higher value for the data that you select.
Controls
Click a control to highlight it. If a control is highlighted, you can perform its action. To learn more, click here.
Filters
By default, the collection plates display the fractions that you collected during every chromatography stage.
Use the check boxes to filter the collection plates so that they only display the fractions that you collected during the chromatography stages that you select.
By default, the collection plates display any fraction with a value for the data that you select.
Use the slider to filter the collection plates so that they only display the factions with a value within the range that you select.
Cherry pick, pool and normalize
If you want to cherry pick the fractions that the collection plates display onto a new plate, complete the steps that follow to create a new workflow.
Select the type of workflow that you want Synthace to create and the number of wells on the destination plate, then click Render Workflows.
Cherry-pick: Select this workflow if you only want to cherry pick the fractions.
Cherry-pick and pool: Select this workflow if you want to cherry pick all of the fractions that you collected from the same robocolumn during the same stage into the same well.
Cherry-pick and normalize: Select this workflow if you want to cherry pick the fractions, then dilute them to the same concentration.
Cherry-pick, pool and normalize: Select this workflow if you want to cherry pick all of the fractions that you collected from the same robocolumn during the same stage into the same well, then dilute them to the same concentration.
Click Cherry-pick selected fractions in Synthace to open the builder.
Note: The text of this link will be different if you selected a different type of workflow.
If you selected a type of workflow that includes normalization, tell Synthace the concentration to which you want the Tecan Freedom EVO® to dilute the fractions.
Select the Dilute By SubComponent element.
In Target Subcomponent Concentrations, specify the concentration to which you want the Tecan Freedom EVO® to dilute the fractions.
Select the Cherry Pick From Well To Well element.
Open the cherry picker.
Use the cherry picker to select the plate type of the destination plate, then simulate the workflow.
Tip: To learn more about the cherry picker, click here.
Sample Data
If you click Sample Data, Synthace displays the set of corrected, structured data that it used to create the pseudo-chromatogram and the collection plates.
If you want to download the set of corrected, structured data, use the check boxes on the left to select the columns that you want to download, then click Download.
Cumulative Data
If you click Cumulative Data, the visualization displays a bar chart and a data table.
In the bar chart and data table, the visualization cumulates the data for every fraction that you collected from each robocolumn.
Axes
On the x-axis, the bar chart displays the robocolumns that you used. On the y-axis, the bar chart displays the data that you select from the drop-down button.
Filters
By default, the bar chart and data table include every robocolumn that you used.
Use the upper slider to filter the bar chart and data table so that they only display the robocolumns from which you collected some fractions that have a cumulative value within the range that you select.
Example: The cumulative quantity of the fractions that you collected from RoboColumn 1 is 3.8 nanomoles (nMol). The cumulative quantity of the fractions that you collected from RoboColumn 2 is 2.7 nMol. If you set the range to 2.9 to 4.6, the bar chart and data table will include RoboColumn 1, but not RoboColumn 2.
By default, the values in the bar chart and data table include every fraction.
Use the lower slider to filter the bar chart and data table so that they only include the fractions that have a number within a specific range.
By default, the values in the bar chart and data table include the fractions that you collected during every chromatography stage.
Use the check boxes to filter the bar chart and data table so that they only include the fractions that you collected during the chromatography stages that you select.
Controls
Click a control to highlight it. If a control is highlighted, you can perform its action. To learn more, click here.