Manual for data analysis of SEC-MALS/RI（Wyatt DAWN HELEOS II (2023.01.27)）
0Saving data file (In case Astra 8.1.2 is already running and user want to continue theanalysis after the SEC-MALS measurement.)

0-1 Saving data fileIn the workspace area, right-click [Experiments] → [Sample name] and select [Save As].
After entering the save folder and file name, click [OK] to save the file (file type: ASTRA(*.afe8)).
Before save the data, create a folder under "C:¥ASTRA DATA¥", [e.g. "C:¥ASTRADATA¥20230220".][Then skip step 1 and proceed to step 2.]

1

Starting Astra 8.1.2, Opening the data file

1-1 Starting Astra 8.1.2
Click the Astra 8.1.2 shortcut icon on the desktop.

Menu barWorkspace

1-2 Opening the experiment file.
Select [File] → [Open] → [Experiment] from the menu bar to open the "Open"window, select an Experiment file (.afe6 or afe8) and click [開く(O) Open]2

Setting baselines

2-1 Baselines tab
In the workspace, double-click [Procedures] → [Baselines] to display the Baselines tab.

2-2 Specifying the Baseline range
Check the LS11 box to display the data from the 90° LS detector (on the right side ofthe graph area.) Click [Autofind Baselines] to automatically set the baseline range. Thendrag the left and right edges of the baseline (light blue) to set the start and end of thebaseline respectively.

Baseline Start

Baseline

Baseline End2-3 Confirmation of Baseline range
Click [Set All] to apply the baseline range of LS11 set in step 2-2 to all LS data. Thencheck whether the baseline range is appropriate for each of the LS1~18, UV and RI
data, and make adjustments to each data.
The displayed data could be changed by selecting the check boxes on the right side,
After checking and adjusting all data, click [OK].

*Common operations on the graph screen[Ctrl]+left drag : select a rectangle area and enlarge it.
[Ctrl]+right click : go back one step.
3Specifying the peak range.

3-1 Peaks tab.
In the workspace, double-click [Procedures] → [Peaks] to display the Peaks tab.

3-2 Specifying the peak range.
Click on the displayed peak range to select it. And the specified peak range can be
adjusted by dragging the left and right edge of the range respectively. Either delete thepeak range with the [Delete] key and specify the peak range by dragging. Afteradjusting the peak range, click [OK].

Peak range

4Alignment (Correction for misalignment of peak positions of LS, UV and dRI detectors.)

4-1 Alignment tab
Select [Experiments] → [Configuration] → [Alignment] in the menu bar to display theAlignment tab.

4-2 Execution of Alignment
Adjust the Alignment range by Dragging between the start of the peak and the end ofthe peak. [If necessary zoom-up the display range]. Click [Align Signals] and thenconfirm that the positions of each peak (LS, UV, dRI) are aligned. After confirmation,click [OK].

Alignment range

Before Alignment

5After Alignment

Band Broadening (Corrections for peak broadening in LS, UV and dRI detectors.)

5-1 Band Broadening tab
In the menu bar, select [Experiments] → [Configuration] → [Band Broadening] todisplay the Band Broadening tab.

5-2 Execution of Band Broadening
Ensure that dRI data (Optilab) is displayed. Specify the correction range by dragging arange that includes entire peak. [If necessary zoom-up the display range]. Click [PerformFit] and confirm that the width and offset of each peak (LS, UV, dRI) have improved.
After confirmation, click [OK].
OptilabBroadening range

Before Broadening

6

After Broadening

Normalize (Correction of intensity of multi angle LS detectors.)
Perform with standard sample before your own samples. Requires re-calibration forevery solvent because the correction factors depend on the refractive index of thesolvent.

6-1 Re-specifying the peak range
In the workspace, double-click [Procedures] → [Peaks] to display the Peaks tab. Narrow
the peak range to 1/2~1/3 of their peak height, and click [Apply].

Peak range (only 1/2~1/3 height)

6-2 Normalize tab
In the menu bar, select [Experiments] → [Configuration] → [Normalize] to display
the Normalize tab.

6-3 Normalize
Identify the peak to be calibrated in Peak Name and enter the approximate radius of
the sample in Radius. [BSA: 3.5 nm, OA: 3.0 nm?]Click [Normalize] to calculate new coefficients, then click [OK].
Open the Peak tab, restore the modified peak range and click [OK].

7

Selection of LS detectors to be used

7-1 Molar Mass & Radius from LS tab
In the workspace, double-click [Procedures] → [Molar Mass & Radius from LS] todisplay the Molar Mass & Radius from LS tab.

7-2 Selection of peak apexes in the control graphIn the control graph, move the blue bar symbol to the apex of the peak by left-clicking andusing the cursor keys. (If necessary zoom-up the display range).

Peak

Zoom up of Peak

7-3 Selection of LS detectors to be used
Click [+] in [Enabled Detectors] in the lower column of the screen to open the LS detector
selection field.
Each point in the results graph corresponds to the result of each LS detector,
To improve Molar Mass and the correlation coefficient at the bottom of the results graph, it
is necessary to add or exclude LS points that deviate from the approximate straight line bytrial and error.
After getting the reasonable results, click [OK].

All LS used

8

After selection

Report (output results with csv and PDF)

8-1 Displaying Report files
In the workspace, double-click [Results] → [Report (summary)] to display the Report(summary) tab.

8-2 Saving Report fileIn the menu bar, select [File] → [Print] to display the Print window. Select MicrosoftPrint to PDF in the printer selection and click Print to output the Report file as PDF.
8-3 Saving csv text fileIn the workspace, select [EASI Graph] to display the EASI Graph tab.

8-4 Checking the settings.
In the upper part of the EASI Graph tab, set that Display: "molar mass", Distribution: "vs.
time or volume" and Abscissa: "mL".

8-5 “EASI Graph options” window
Right-click on the EASI Graph tab and select [Edit] to open “EASI Graph Options”
window.
In "Chromatograms" area,
Check 1st box and select "Rayleigh ratio"
Check 2nd box and select "Differential refractive index"
Check 3rd box and select "UV absorbance".
And then click [Export].

8-6 Saving results with csv format
Select csv format as file type (even if Excel format is selected, the output file could not
be opened with excel and other editor software), enter folder and file name and click"[保存(S)]:Save" to save results.
Users can copy data to their own USB memory from the MALS PC.

Csv file after revising column name.

In csv file.
Column A:

volume (ml) [volume value of LS]

Column B:

xxx (sample name) [signal of LS]

Column C:

volume (ml) [volume value of dRI]

Column D:

xxx (sample name) [signal of dRI]

Column E:

volume (ml) [volume value of UV]Column F:

xxx (sample name) [signal of UV]Column G:

volume (ml) [volume value of Mw]

Column H:

xxx (sample name) [Mw]

Data are arranged every two columns. (Signals of LS, dRI and UV are scaled.)[It is better to rename each data name of the 1st row to make it easier tounderstand later].