Rev.

A-

High-performance GPC System HLC-8320GPC

EcoSEC-WorkStation
Quick Reference Manual

Tosoh Corporation
Bioscience Division

No part of this document may be reproduced in any form without prior permission.
1. Introduction
Thank you for purchasing the High Performance HPLC system HLC-8320GPC and
EcoSEC-WorkStation.
This is a quick reference manual covering the basic settings necessary for SEC analysis
as well as sample analysis methods, calibration curve creation, molecular weight analysis,
and report output.

For more detailed information about operation, refer to:

EcoSEC-WorkStation Operators Manual
This manual is also installed on the PC. Open the [Help] section of the
application to view it on the PC screen.
High-performance GPC System HLC-8320GPC Operators Manual

This quick reference manual is based on the following analytical conditions.

For a different column size or solvent, the settings must be changed.

- Analytical conditions -
Column TSKgel SuperMultipore HZ-M 2 (4.6 mm I.D. 15 cm 2)
Solvent THF
Flow rate 0.35 mL/min
Temperature 40C (pump oven, column oven)
Injection Volume 10 L
Sample Polystyrene SRM706 (NIST)
Concentration 0.2 wt%
Marker PStQuick MP-M (diluted to 1.0 mL)
Instrument HLC-8320GPC, UV-8320
Detection RI: Polarity +, Response 0.5 s
UV: Wavelength 254 nm, Polarity +, Response 0.5 s
1-1 Analysis flowchart

The system operates as shown below from power-on to power-off. This manual is based
on the order of the following steps.

- Analysis -
2-1 Logging on to the Acquisition Control Program

2-2 Setting Analytical Conditions

Set the flow rate, oven temperature, and detection conditions.

2-3 Setting Warmup Conditions

Set the flow rate and standby time for warmup.

2-4 Setting Shutdown Conditions

Set the flow rate at shutdown and the time until power-off.

2-5 Creating a Method

Set the data processing conditions.

2-6 Creating a Sample Queue

Set the run time, data acquisition time, and sample record.

2-7 Specifying a Saving Database

Specify the database in which analysis data is to be saved.

3-1 Warmup (Power-on)

Turn on the power to warm up the system.

3-2 Loading Samples

Load samples onto the sample table for analysis.

3-3 Executing Analysis

Analyze molecular weight markers and samples.

3-4 Executing Shutdown (Power-off)

Shut down the system.
- Analysis -
4-1 Logging on to the Analysis Program

4-2 Analyzing Data

Execute chromatogram peak edit and molecular weight calculation.

4-2 Selecting a Chromatogram Database

Select a database in which chromatogram data is to be saved.

4-3 Selecting Chromatogram Data

Select data from the chromatogram list for analysis.

4-4 Editing peak data

Execute peak editing(automatic peak detection or manual peak edit).

4-5 Executing Calculations

Execute calculations on edited peak data.

4-6 Creating a Calibration Curve

Create a calibration curve from the results of molecular weight marker analysis.

4-7 Executing Molecular Weight Calculations

Execute peak editing and molecular weight calculations on an unknown sample.

4-8 Outputting a Report

Check molecular weight calculation results on a preview screen and print them.
1-2 Logon dialog box
The EcoSEC-WorkStation comprises five application programs.
From the Start menu of Windows, select [Programs][EcoSEC][EcoSEC] to open the
EcoSEC Logon dialog box.
To logon to the application, the users and their authoritys must be registered in advance
by the system administrator. For details on registration and authority settings, refer to
Section 3, Basic Knowledge Before Use, in Chapter 1 of the EcoSEC-WorkStation
Operators Manual.

Note
Once activated, the Logon dialog box is shown as a key icon in the task bar at the bottom
right of the screen. For subsequent or later activation, click the key icon.

Analytical Method Validation

Report Layout

Data Management

Analysis

Acquisition Control

To logon to one of these applications, enter your user name and password, select the
appropriate application, and click [Log on].

EcoSEC-WS manages analysis files and data in directories created for individual logged-
in authorized users.
For example, if a user logs on to the project workspace called ProjectCSC with the
username Level1user, the following directory is automatically assigned:

C:\PolyScape\Data\ProjectCSC\Level1User

and analysis data is saved in a database created in this directory.

2. Pre-analysis Settings
2-1 Logging on to the Acquisition Control Program
From the EcoSEC Logon dialog box, log on to the Acquisition Control program. Enter the
user name and password, select the Acquisition Control program, and click [Log on].

When the user logs on to the Acquisition Control program, a monitor screen is displayed.
During analysis, this screen consists of a graph display field to display chromatograms
and other data in real time, icons for switching the screen display, and a toolbar for
instrument operation.

Status
indicators
Toolbar

Screen icons

Graph
display

Instrument Control buttons

2-2 Setting the Analytical Conditions
Operation of the instrument and setting of parameters are carried out in the Instrument
Control screen of the Acquisition Control programn.
To show this screen, click the [Instrument Control] screen icon:

At the bottom of the Instrument Control screen are tabs entitled [Flow Diagram],
[Instrument Parameter], [Warmup], and [Shutdown]. Click the tabs to switch between
these.

Clicking the [Flow Diagram] tab displays the operating status of each component unit and
the channel connections based on the instrument configuration diagram. When the
mouse pointer is positioned over one of the component units, the component is
surrounded by a blue square. Clicking the mouse will bring up the operating menu for
that unit.

To change items on the operating menu, see Section 6.2, Setting the User-Defined
Menu. It is recommended that frequently executed commands or changes in parameters
be added to the menu in advance.
The settings can be changed by selecting [Detail setting][Instrument Parameter] from
the operating menu. Alternatively, click on the [Instrument Parameter] tab.

For example, select [Instrument Parameter] and make the following changes:
Sample flow rate (mL/min) to [0.350]
Reference flow ratio to [Equal]
Column and pump oven temperature (C) to [40]
RI detector: Balance value (mV) to [30.000], Response (sec) to [0.5]
UV detector: Wavelength (nm) to [254], Balance value (mV) to [30.000], Response
(sec) to [0.5]

2-3 Setting the Warmup Conditions

Click the [Warmup] tab to display the Warmup screen.

For example, insert the following settings and click [Apply] to confirm.
Running time to complete warming up process - [15] min
Warmup Flow Rate - [50]%
Start Temperature Control - All checked
Execute purge - Checked, Purge volume - [30] mL
Lamp ON - Checked

2-4 Setting the Shutdown Conditions

Click the [Shutdown] tab to display the Shutdown screen.

For example, insert the following settings and click [Apply] to confirm.
Waiting time before shutting down process starts - [15] min
Running time to complete shutdown: [15] min
Shutdown Flow Rate - [50]%
Stop Temperature Control - All checked
Power off after shutdown completion: Checked

To return to the Monitor screen, click the [Monitor] icon.

2-5 Creating a Method
An analytical method consists of four items:
Analytical Conditions 1 - Set the analytical conditions and peak detection conditions,
enter comments.
Analytical Conditions 2 - Set the molecular weight fraction and distribution list range.
Peak Editing Conditions - Set the peak editing conditions by specifying commands.
Calibration Condition - Set the conditions and approximations for creation of a
calibration curve.

The parameters of the method do not need to be changed from their initial values
because they can be changed during analysis.
The method is set in the Method screen of the Acquisition Control program.

Click the [Method] icon.

Here, the only modification is of the comments in Analytical Condition 1. (Checked items
are included in the report).

After editing the method, select [Method][New] to open a name input window. Enter a
name (e.g., Standard Polystyrene) and click [OK].
To overwrite the existing data, select [Method][Save].

To return to the monitor screen, click the [Monitor] icon.

2-6 Creating a Sample Queue
Set Sample Queues in the Sample Queue screen of the Acquisition Control program. To
navigate to this screen, click the [Sample Queue] icon.

The Sample Queue screen is used to set times and sample attributes for analytical
procedures.
Click [New] at the upper right or select [Sample Queue][New Sample Queue] to display
the New Sample Queue window. Enter a name for the procedure, e.g., Polystyrene
analysis, and click [OK].

Enter the various settings sequentially.

Analysis times
Enter the items as shown in the examples.
Item Meaning Example
Run Time (min) Auto sampler injection interval 30.1 min
Start Time (min) Data acquisition start time 0.0 min
(time after sample injection)
End Time (min) Data acquisition end time 30.0 min

Sampling Interval Data acquisition interval 100 msec

(msec)
- Creating a sample record -
The following items may be entered into the sample queue worksheet.
Enter the items as shown in the examples.
Cup Sample Name Injection Repeat Type UV wavelength
Volume [nm]
[L]
1 Blank 10 1 Unknown 254
2 PStQuick MP-M 10 1 Unknown 254
3 SRM706 (NIST) 10 1 Unknown 254
4 Blank 10 1 Unknown 254
5 PStQuick MP-M 10 1 Unknown 254
6 SRM706 (NIST) 10 1 Unknown 254

When analyzing multiple samples with the same name, we recommend that you use the
Sample Queue Wizard on the Sample Queue screen for easy input into the worksheet.
Click the [Sample Queue Wizard] key to display the Sample Queue Wizard window. After
entering the items, click [Create and add record]. When the name input window appears,
enter a name (e.g., Molecular weight analysis of standard polymer). The settings are
automatically written into the worksheet.

- Specifying a method -
A method can be specified for samples of Standard or Unknown processing types.
Since every sample setting used here is Unknown, the Standard Polystyrene method
created in Section 2-5 is used as an Unknown method.
Item Meaning Example
Standard Method Method for processing Default Method
standard samples
Unknown Method Method for processing Standard Polystyrene
unknown samples

Note
The use of standard and unknown methods enables calibration curve creation by
standard polystyrene analysis (standard method) and molecular mass calculation by
unknown sample analysis (unknown method) in a series of worksheets.
After entering all of the items, click [Error Check].
If there is no conflict between the settings, the total analysis time is displayed with a
message There is no error. Click [OK].

If there is a conflict between settings, an error message is displayed.

The message below is displayed when the run time is specified as being shorter than the
data acquisition end time.

To return to the monitor screen, click the [Monitor] icon.

2-7 Specifying a Database
A name for the saved database may be set by setting [Auto Save] on the Other Settings
screen of the Acquisition Control program.
Click the [Other Settings] icon.

When [Auto naming] is selected, the time at which the analysis is conducted ([YEAR-
MON-DAY], [MON-DAY-HOUR], or [DAY-HOUR-MIN]) is designated as the database
name. When [Manual input at every analysis start] is selected, a database name input
window is displayed when the analysis is executed. (In this example, the database is
named SRM706.)

Note
A database is created at the time of analysis. If [YEAR-MON-DAY] is selected under
[Auto naming] and a database of the same name already exists, the user may click [Add
to existing DB] or [Store to new DB]. When [Manual input at every analysis] is selected,
the input window is displayed every time analysis is executed.
To return to the monitor screen, click the [Monitor] icon.
3 Executing Analysis
3-1 Warmup (Power-on)
Click the [Power] button at the top left of the Acquisition Control program screen to turn
on the power of the HLC-8320GPC. (The main power switch on the right side of the
instrument should be turned on in advance.)

Click the [Power] button

The upper status indicator changes from [Power Off] to [Ready].

To execute warmup, click the [Warmup] key on the toolbar of the monitor screen or select
[Analysis][Warmup] from the menu.

During warmup, the upper status indicator on the monitor screen changes to [Warmup].

The power of the instrument is turned on and the display backlight is illuminated.
The LEDs in the control section illuminate sequentially during warmup.
1) Warmup LED 2) Temperature control LEDs 3) Flow Key LEDs

2) Temperature control LEDs

3) Flow keys
1) Warmup key

3-2 Loading Samples onto the Auto Sampler

Press the [Rack Eject/Insert] key in the control section of the instrument to eject the
sample rack.

Rack Eject/Insert key

Place the sample cups at positions 1 to 6 on the sample rack, as in the following
example:
No. 1 - Blank
No. 2 - PStQuick MP-M
No. 3 - Polystyrene SRM706 (NIST)
No. 4 - Blank
No. 5 - PStQuick MP-M
No. 6 - Polystyrene SRM706 (NIST)
Remount the sample rack on the instrument and press the [Rack Eject/Insert] key.
When the rack is inserted, the LED lamp on the [Rack Eject/Insert] key is illuminated.

3-3 Executing Analysis

Click the [Analysis] key on the toolbar of the Acquisition Control program or select
[Analysis][Start Analysis] from the menu. When the message [Start analysis?] is
displayed, click [OK] to start analysis.

During analysis, the upper status indicator on the monitor screen changes to [Running].
The lower status indicator indicates the data acquisition status (during acquisition,
[Acquisition] is displayed). The individual settings of the data acquisition start and end
times may result in a period of no data acquisition. In this case, [Ready] is displayed on
the lower status indicator.

At the same time, the LED of the [ANALYZE] key in the control section of the instrument
is illuminated.

Analyze key
Note
While a sample queue is under analysis, editing is not allowed for the current or
subsequent record but only for the records following those. In this example, six records
have been set, so after [Start Analysis] has been selected, only the third and subsequent
records can be edited.

- Stopping analysis -
To stop the analysis process, click the [Analysis] key on the toolbar during analysis, or
select [Analysis][Stop Analysis] from the menu. The process will stop when analysis of
the current sample is completed. When analysis is stopped, the upper status indicator
changes to [Stop Analysis].
- Delaying execution -
To delay analysis, press and hold the Shift key and click the [Analysis] key on the toolbar.
The Delay Start window appears. Enter the time period by which the start of the analysis
is to be delayed and click [OK]. Analysis will begin after the specified time. During the
delay time, the upper status indicator changes to [Delay].

3-4 Executing Shutdown (Power-off)

To shut down the instrument, click [Shutdown] on the toolbar of the monitor screen, or
select [Analysis][Shutdown] from the menu.

When shutdown is executed, the upper status indicator on the monitor screen changes to
[Shutdown].

During shutdown, the LEDs in the control section of the instrument illuminate and switch
off sequentially.

1) Shutdown LED on 2) Temperature control LEDs off 3) Flow key LEDs off
after the set time, the instrument is powered off.

1) Temperature control LEDs

3) Flow key
2) Shutdown key

Note
When [Shutdown] is selected during analysis, shutdown is executed after analysis.
Data Analysis
4-1 Logon to the Analysis Program
Logon to the Analysis program using the EcoSEC Logon dialog box. Enter the user name
and password, select the Analysis program, and click the [Log on] key.

When the Analysis program is opened, the Data Analysis screen is displayed. The main
items in this screen are a method selection field, an analytical and chromatogram data
selection field, a field showing calculation results, and a graph display field. (A graph can
be selected by checking the appropriate box at the graph selection list.)

Chromatogram data selection Calculation

and analytical data view Toolbar results

Graph selection
list

Method selection Graph

display
4-2 Selecting a Chromatogram Database
The analytical data field displays all analytical data from the database specified in the
chromatogram data selection box.

Note
The database can be changed by clicking [Browse] in the chromatogram data selection
box or selecting [Chromatogram][Browse Chromatogram Database] from the menu of
the Analysis program, which brings up the [Select Chromatogram Database] window.

Note
When [Chromatogram][Change Project] is selected from the menu of the Analysis
program, the list of projects saved in C:\PolyScape\Data is displayed and the appropriate
project may be selected. The project can also be changed in the chromatogram database
selection window.

4-3 Selecting Chromatogram Data

Analytical data is listed by database. When the user clicks on the required data, the
calculation results and the graphs are displayed. Here, PStQuick MP-M is selected to
create a calibration curve.
4-4 Peak Edit
Peaks are automatically detected in chromatogram data based on the peak detection
conditions specified in a method. If inappropriate peak detection occurs, two correction
techniques are available: 1) Change parameters for automatic peak detection or 2) edit
peaks manually.

1)automatic peak detection with different parameters

Peaks are detected automatically based on the peak detection conditions specified in a
method.
Display the Method screen by clicking [Method] in the Analysis program screen, and
change the peak detection conditions under Analytical Condition 1.

- Detection Sensitivity -
This parameter is used to set the level of change (inclination) in the baseline that is
recognized as a peak. For single peaks, optimum peak detection can be achieved by
varying the detection sensitivity. If the sensitivity is set low, peaks of various shapes can
be detected, but fine baseline changes or noises may also be detected as peaks. If the
detection sensitivity is set high, broad peaks become difficult to detect.
- Baseline Sensitivity -
This parameter is used to select vertical detection or skimming for the troughs of multiple
non-separable peaks. If the baseline sensitivity is set high, skimming is promoted
because peak start point and end points are assigned to each peak. If the baseline
sensitivity is set low, vertical editing is promoted because multiple peaks are handled as
a group. Since the purpose of SEC analysis is molecular weight calculation of the eluted
components, priority is given to vertical editing rather than skimming.
- Minimum Area, Minimum Height, Minimum Width -
These parameters exclude peaks which are smaller than the set values from calculation.
When unnecessary minute peaks (noise) are detected, they can be excluded from
calculation by setting appropriate values.
2) Peak Edit
In peak edit, peak start and end points may be set freely in the analyzed chromatogram
data.
Click [Peak Edit] in the Analysis program screen to display the Peak Editor screen.

The peak edit commands are displayed in conjunction with the chromatogram. Select any
command and edit the baseline by moving the mouse on the chromatogram.

For an example of peak edit, see Chapter 3, Section 6 of the EcoSEC-WorkStation

Operators Manual.
1) Select [Delete All] to delete the baseline generated by automatic peak detection.
2) Select [Draw Base], click the start position of the peak, and move the mouse to the
peak end position while pressing the left mouse button (dragging). If there is more
than one peak, repeat this operation for each peak.
3) For vertical editing of non-separable peaks, select [Draw Base] and left-click the
appropriate trough.

4-5 Executing Calculations

After automatic peak detection with parameter modification or manual peak edit in the RI
and UV chromatograms, click [Calculation]. In the Calculation screen, select [Auto Peak
Edit] for automatic peak detection with parameter modification or [Edited Peak] for
manual peak edit, and click OK. The results of the calculation are displayed in the results
field and the graph display field.

Note:
For recalculation without changing the peak information (e.g., when changing the
calibration curve), select [Skip Peak Edit].
If the calculation results are acceptable, click [Save Data]. Enter the necessary
information in the window and click [OK].

Example of PStQuick MP-M peak edit.

4-6 Creating a Calibration Curve
Create a calibration curve from a peak-edited PStQuick MP-M chromatogram.
Select [Method] in the Analysis program screen to display the Method screen, and click
[Calibration Condition].

Delete any numeric values in the calibration curve data.

Click [Set the Elution] to display the Setting Calibration Data dialog box. Click [Create
from Memory Data] to register the elution time in the calibration curve data.

Note
To create a calibration curve from several chromatograms, click [Registration] on the
Setting Calibration Data dialog box and specify the appropriate chromatograms. When
Select the Chromatogram dialog box is closed and [Create from Another data] is selected
in the Setting Calibration Data dialog box, all peak elution times of the registered
chromatograms are registered in the calibration curve data.

PStQuick MP-M includes four types of standard polystyrene as molecular weight markers.
Enter these values into the molecular weight field of the calibration curve data to display
the calibration curve in a graph on the right. (The red line indicates the percentage
difference from the approximate curve at each calibration point.)
F-80 Mw 706,000
F-10 Mw 96,400
A-5000 Mw 5,970
A-500 Mw 495

Note
If you are using a column of high separation efficiency in a low molecular weight area,
standard polystyrene A-500 and A-300 may elute as peaks at every degree of
polymerization. In this case, enter a molecular weight value suitable for the degree of
polymerization.
Dimer Mw 266, Trimer Mw 370, Tetramer Mw 474, (+104, subsequently)
Example of calibration curve creation

After creating calibration curves for the RI and UV detectors, click the [Analytical
Condition 1] tab and change [Analytical Condition][Calculation Type] to [Molecular
Weight].

Since a new method for molecular weight calculation has been created, select
[Method][Save As] from the menu of the Analysis program. In the method name input
window, enter a folder name (e.g., STD PS) and reason for the change and click [OK].

4-7 Calculating Molecular Weight

Click on the analytical data to calculate its molecular Weight. Calculations are carried out
for the selected chromatogram and the results are displayed in the results field and the
graph display field.

Here, select the appropriate data (e.g., SRM706 (NIST)).

Next, select a pre-created method (e.g., STD PS) from the method selection field.

Either automatic peak detection with parameter modification or manual peak edit may be
selected. Here, manual peak edit is selected.

Click [Peak Edit] on the Analysis program screen to display the Peak Editor screen.

As the peak edit commands are displayed along with the chromatogram, select any
command and edit the baseline by moving the mouse on the chromatogram.

For an example of peak edit, see Chapter 3, Section 6 of the EcoSEC-WorkStation

Operators Manual.
1) Select [Delete All] to delete the baseline generated by automatic peak detection.
2) Select [Draw], click the start position of the peak, and move the mouse to the end
position while holding the mouse left button (dragging).

After automatic peak detection with parameter modification or manual peak edit for the RI
and UV detectors, click [Calculation]. In the Calculation screen, select [Auto Peak Edit]
for automatic peak detection with parameter modification or [Edited Peak] for manual
peak edit, and click [OK]. The results of the calculation are displayed in the results field
and the graph display field.
If the calculation results are acceptable, click [Save Data]. Enter the appropriate items in
the window and click [OK].

Example: SRM706 (NIST) peak edit results

Example: SRM706 (NIST) molecular weight calculations

If the calculation results are acceptable, click [Save Data]. Enter the appropriate items in
the window and click [OK].
4-8 Outputting a Report
Click [Report] in the toolbar of the Analysis program to display the Report Form screen.

In the Chromatogram Report box, click [] and select [(CH.1+CH.2)

Title+Chromatogram+Mol.Result+Comment] for the report form. To set this as the default
for printing, click [Set as a permanent format].
Click [Preview] to display the report contents in MS Word format. After checking, the
contents may be printed.
To change the fixed scale of chromatogram, check the box for the time or output axis,
enter a numeric value, and click [Preview] again. It is also possible to print the scale of
the graph screen in the Analysis program. Click [Fixed Scale of Chromatogram] to set
numeric information automatically.

By selecting a format in the Report Form screen, method and instrument reports for
specified analytical data can also be output.
Example of Chromatogram report
Chromatogram report

<Header>
Data acquisition
Title 2007/12/25 13:32:25
date time
Sample Calculation date
SRM706(NIST) 2007/12/25 15:25:50
name time
Database
SRM706.chd Sampling time [min] 0.000 - 15.000
name
Data name RSLT0000 Serial number 0
Method
STD PS Cup number 1
name
Molecular Weight
Channel RI UV Calcuration type
Molecular Weight

RI[mV]

6.145 / 312798
312666
-- RI --
-64
Peak No.
40.000
Elution time
Molecular weight of peak top
30.000 1 / 6.087 -- UV --
Peak No.
20.000 Elution time -6
Molecular weight of peak top

10.000

0.000 5.000 10.000 15.000

[min]

<Result of molecular weight calculation> (RI)

Peak 1 Base Peak
[min] [mV] [mol] Mn 49,895
Peak start 5.242 29.662 1,392,036 Mw 279,882
Peak top 6.145 46.260 312,666 Mz 437,407
Peak end 10.213 29.964 375 Mz+1 564,638
Mv 279,882
Height [mV] 16.543 Mp 312,667
Area [mV*sec] 1203.144 Mz/Mw 1.563
Area% [%] 100.000 Mw/Mn 5.609
[eta] 279882.14851 Mz+1/Mw 2.017

<Result of molecular weight calculation> (UV)

Peak 1 Base Peak
[min] [mV] [mol] Mn 3,348
Peak start 5.140 -657.267 1,497,865 Mw 253,928
Peak top 6.087 -643.008 312,798 Mz 439,013
Peak end 11.030 -657.209 87 Mz+1 567,125
Mv 253,928
Height [mV] 14.250 Mp 312,799
Area [mV*sec] 1148.317 Mz/Mw 1.729
Area% [%] 100.000 Mw/Mn 75.850
[eta] 253928.19455 Mz+1/Mw 2.233

<Analytical Comment>
Column TSKgel SuperMultiporeHZ-M X2
Column no. M0006,M0007
Flow rate 0.35mL/min
Example of Method report
Method report

<Header>
Database name SRM706.chd
Method name RSLT0000

<Acquisition and control condition>

Start time [min] 0.000 End time [min] 15.000 Sampling interval [msec] 100

<Analysis condition> (RI)

Calculation type Molecular Weight
Output type Area Alpha B 1.0000 Kappa B 1.0000

Correction of internal standard peak No

Section Non

Molecular mass fraction No

Conversion factor
Elution curve factor 1.00 Calculation of differential and integral TOSOH
Differential curve factor 1.00

<Analysis condition> (UV)

Calculation type Molecular Weight
Output type Area Alpha B 1.0000 Kappa B 1.0000

Correction of internal standard peak No

Lag time correction No

Section Non

Molecular mass fraction No

Conversion factor
Elution curve factor 1.00 Calculation of differential and integral TOSOH
Differential curve factor 1.00

<Smoothing condition> (RI)

Smoothing condition No

<Smoothing condition> (UV)

Smoothing condition No

<Peak detection condition> (RI)

Minimum area [mV*sec] 10.000 Sensitivity [mV/min] 3.000
Minimum height [mV] 0.000 slope [mV/min] 1.000
Minimum width [sec] 0.000

Detection control No

<Peak detection condition> (UV)

Minimum area [mV*sec] 10.000 Sensitivity [mV/min] 3.000
Minimum height [mV] 0.000 slope [mV/min] 1.000
Minimum width [sec] 0.000

Detection control No
 Detector condition RI : Pol. (+) , Res. (0.5s) / UV : 254nm , Pol. (+) , Res. (0.5s)
Concentration 0.2 wt.%
Injection volume 10uL
Pressure 3.5MPa
Column temperature 40 C
System temperature 40 C
Buffer THF

<Calibration condition> (RI)

Formula of approximation Linear: At+B
Correction Non

[LogM]

2
6.00 8.00 10.00
[min]

Calibration data (RI)

Time Molecular Error [%] Weight Mark Data Coefficient
[min] weight name
5.615 706,000 -6.36598 1 STD RSLT0001 A= -7.179730e-001
6.875 96,400 2.97484 1 STD RSLT0001 B= 9.907026e+000
8.613 5,970 11.45788 1 STD RSLT0001
10.038 474 -5.73878 1 STD RSLT0001 Correlation -1.000
10.197 370 -4.14855 1 STD RSLT0001
10.425 266 0.62539 1 STD RSLT0001

<Calibration condition> (UV)

Formula of approximation Linear: At+B
Correction Non

[LogM]

2
6.00 8.00 10.00
[min]

Calibration data (UV)

Time Molecular Error [%] Weight Mark Data Coefficient
[min] weight name
5.557 706,000 -6.42444 1 STD RSLT0001 A= -7.185293e-001
6.817 96,400 3.07807 1 STD RSLT0001 B= 9.868714e+000
8.552 5,970 11.30966 1 STD RSLT0001
9.980 474 -5.19919 1 STD RSLT0001 Correlation -1.000
10.135 370 -4.28383 1 STD RSLT0001
10.362 266 0.36065 1 STD RSLT0001
Example of Instrument report
Instrument report

<Header>
Database name SRM706.chd
Method name STD PS
Acquisition date time 2007/12/25 13:32:25
Operator User Level 1
Software version Software version for report 1.00 Data acquisition version CD-ROM 1.01

<Pump>
Column 1
Sample
Pressure upper limit Pressure lower limit flow rate
25.0 0.0 0.350
[MPa] [MPa] [mL/min]

Reference
Pressure upper limit Pressure lower limit flow
25.0 0.0 Equal
[MPa] [MPa] ratio

Column 2
Sample
Pressure upper limit Pressure lower limit flow rate
25.0 0.0 1.000
[MPa] [MPa] [mL/min]

Reference
Pressure upper limit [MPa] 25.0 Pressure lower limit [MPa] 0.0 flow ratio 1/4

<Sampler>
Manual wash volume [mL] 20.0 Air detection level 691 Tube volume [uL] 88
Auto wash volume [mL] 1.0 Injection volume [uL] 10 Presuction volume [uL] 150
Sampling speed [uL/sec] 5 Number of Repeat Air volume [uL] 1
Wash speed [uL/sec] 40 Cup number 1 Loop volume [uL] 100

<Column oven>
Column
Preset temperature [C.] 40 Gas sensor [mV] 300

Pump
Preset temperature [C.] 40 Gas sensor [mV] 300

<RI Detector>
Balance [mV] 0.030 Response [sec] 0.50 Temperature [C.] 40
Polarity + Range [uRIU/FS] 256

<UV detector>
Balance [mV] 0.030 Response [sec] 0.50 Wavelength [nm] 254
Polarity + Range [ABU/FS] 1
[C.] [MPa]
45.000

3.000

40.000

2.500

35.000
0.000 5.000 10.000 15.000
[min]
5. List of Frequent Operations
5-1 Changing the Acquired Signal
In the Acquisition program, select [Other Settings][Auto Save][Acquired Signal], and
choose either [RI+UV] or [RI].

5-2 Setting Auto Balance

In the Acquisition program, select [Other Settings][Auto Balance], and choose [No
Action], [Auto balance just before data acquisition], or [Auto balance just before injection].

5-3 Editing Peak Information in Results Area of Analysis Application

Right-click on the chromatogram. From the popup menu, select [Graph Settings]. You
can select the peak information to display and the number of digits displayed.

5-4 Editing a Report

By using the Report Layout program of EcoSEC-WorkStation, an original report format
may be created. (Refer to Chapter 5 of the Operators Manual for details on layout
editing.) This section explains how to modify a preset format.
Log on to the Report Layout program from the EcoSEC Logon dialog box.
Click [Open] to display the Open Report screen.
When a report type is selected, the stored report formats are listed.
For example, select [Chromatogram Report] and [(CH1+CH2)
Title+Chromatogram+Mol.Result+Comment] and click [OK] to display the stored report
layouts by print items.

To modify peak addition information, select [Chromatogram]. The current settings will be
displayed in the Item field. Edit the check-box settings for peak information. (In this
example, [Peak No.], [Elution time], and [Baseline] are set as peak information for
output.)
In addition to peak information, the number of digits displayed in the calculation results
field and the font can also be changed.

By right-clicking any record, a print item can be cut, copied, or pasted, or lines can be
deleted or inserted to create your own layout.
After editing, save the layout and terminate the Report Layout program.

5-5 Creating a Project

A project may be created using the [Project] command at the upper left of the monitor
screen in the Acquisition Control program.
When [Project][New project] is selected, a project name input window opens. Enter a
new project name and click [OK].
Although it is convenient to create projects by analytical sample themes, the settings
must be redefined when [New project] is selected since the various settings for analysis
are managed by projects.
6. Useful Functions
6-1 Registration of Molecular Weight Marker
Log on to the Analysis application and select [Options][Mw. Registration] to display the
Molecular Weight Definition dialog box. Check one of the boxes under [List] to add the
registered molecular weight values of Tosohs Standard Polystyrene to [Detail]. To add
registered molecular weight values to calibration curve data, double-click the molecular
weight field during calibration curve creation and select [Setting Values] from the popup
menu.

6-2 Setting the Menu Editor

Frequently used commands (to control oven temperature, pump, flow rate, etc.) can be
added to a popup menu and executed on the flow diagram and monitor screens.
To add a command to a popup menu, select [Options][Menu Editor] to display the
Menu Edit screen in the Acquisition control program. Select the appropriate object and
add the command. (When the tab of the object is clicked, the command is displayed.)

6-3 Automatic Column Check Function

This function checks column deterioration by analyzing a standard sample. The
theoretical plate number is set as a criterion for a specific peak, and a warning is issued if
the result is lower than the threshold (%).
Select [Options][Column Check] in the Acquisition Control program to display the
Column Check screen. Enter the appropriate values and check the [Column check] box
for check.

6-4 Result Exporter

The Analysis program lists analytical data saved in a specified database. By selecting
multiple analytical data, the peak information of individual analytical data points can be
statistically calculated.
To select a continuous set of analytical data in a database, hold down the Shift key and
click the first data point followed by the last data point. To select non-continuous
analytical data, hold down the Ctrl key and click on the individual data points. (In this
example, the data points RSLT0001 to RSLT0004 are selected.)

Select [Lump Work][Result Exporter] to list the peak information for the specified
analytical data.

In the Result Exporter screen, click [Calculate] to obtain and display the average,
variance, standard deviation, and coefficient of variation for the individual peak data
points.

The results can be saved as text by clicking [Save As] or transferred to MS Excel for
display as a spreadsheet.

6-5 Automatic Launch Using the Weekly Timer

The EcoSEC-WorkStation can be set to launch on a specific day of the week. Log on to
the Acquisition Control program and click [Timer] in the Other Settings screen. Check the
box for the appropriate day of the week and enter a time for automatic launch. (In this
example, the instrument is set to turn on and execute warmup at 08:00 on Monday.) By
setting the weekly timer for automatic launch, the time required for instrument and
column stabilization time can be allocated automatically.

Note
The weekly timer automatically triggers execution of the set commands only when the
instrument is in a standby or power-off state. If at the set time the instrument is warming
up, undergoing operations (including manual acquisition), or shutting down, an error
notice is issued and the current operation continues.

6-6 Auto scaling without Negative Peaks in the Graph Display

When the RI detector is used, negative peaks are often observed due to elution of gas
dissolved in the sample solution. The ordinary auto scaling graph display supports all
chromatograms regardless of scale setting. However, the settings can be changed to
compensate for negative peaks, thus zooming positive peaks.
To achieve this, select [Graph][Avoiding Negative Peak] in the Analysis program.