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
From the home page, click on the EXECUTIONS tab (located above Incoming), then locate and open your execution.
Navigate to the results by clicking on the RESULTS tab and click ANALYSE CHROMATOGRAPHY RESULT.
Select a different dataset
By default, the visualization displays the results of the execution of the simulation that you selected from the EXECUTIONS 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.
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 will either display the loaded volume (CV) or fractions that you collected during each chromatography stage. Use the Chromatogram X-axis drop-down to select between the options.
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.
Visualization Controls
To adjust the visualization of the plots, 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.
Select Wavelengths for Analysis
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. Select wavelengths for pathlength correction, background subtraction and calculating protein concentration.
Upper and Lower Pathlength Wavelength: 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.
Protein Absorbance: Select the wavelength that you used to measure the absorbance of the protein of interest.
Select Dilutions for Average Dilution Adjustments: Choose which dilutions will be used for calculating average dilution adjustments.
Select Blank Correction: Check this box if you instructed the liquid handler 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.
Adjust Physical Properties for Analysis
K-Factor: A reference value obtained by measuring the absorbance of assay buffer at nm (or an alternative infrared wavelength with absorption maximum) and nm (a reference wavelength which eliminates the background of plastic, etc.) with a cm cuvette, then performing a subtraction. The K-Factor of pure water (0.173) is used by default, unless explicitly specified by the user in the Analysis Setup.
Adjust Sample Analysis Parameters: If you haven’t added the molecular weight and/or molar extinction coefficients when defining your sample or wish to modify them, you may do so here. To edit, click on the table cells and enter your desired values. Results will automatically recalculate based on your values. To learn more about adding the parameters in the workflow builder, click here.
Response and Filter Selection
Select Response: Use the drop-down buttons beneath the Plate View header to select the data that you want the pseudo-chromatogram and collection plates to display.
Optional: Use the slider to set the upper and low range of the response. This also filters the collection plates so that they only display the wells with a value within the range that you select.
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.
Filter Responses: By default, the pseudo-chromatograms and collection plates display the fractions/loaded volumes (CV) that you collected during every chromatography stage.
Filter using the check boxes so that they only display those that you collected during the chromatography stages that you select.
By default, the pseudo-chromatogram and collection plates display any fraction with a value for the data that you select.
Toggle between loaded volume (CV) and fraction to adjust the Chromatogram X-axis. 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. Additional adjustments may be made using the visualization controls. To learn more, click here.
In the collection plates, a darker colour indicates a higher value for the data 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 liquid handler to dilute the fractions.
Select the Dilute By SubComponent element.
In Target Subcomponent Concentrations, specify the concentration to which you want the liquid handler 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 Export.
Unmeasured Collection Plate Data: If you have collected fractions in deep well plates, aliquoted into shallow plates, and then measured the shallow plates, you will have unmeasured collection plate data. Synthace will use a combination of the planner data and the measured shallow well plate data, to understand the contents of the original collection plate. For example, estimates of the eluted volume in the collection plates enables you to cherry-pick from the deep well plates rather than the shallow ones.
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 2.70 nanomoles (nMol). The cumulative quantity of the fractions that you collected from RoboColumn 2 is 2.95 nMol. If you set the range to 2.75 to 3.10, the bar chart and data table will include RoboColumns 2-4, but not RoboColumn 1.
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 Stages 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.