A-
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.
- 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
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.
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
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
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]
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
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
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.
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].
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].
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
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].
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.)
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
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].
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.
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.)
Graph selection
list
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.
- 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.
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].
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].
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.
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].
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.
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
<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
Section Non
Conversion factor
Elution curve factor 1.00 Calculation of differential and integral TOSOH
Differential curve factor 1.00
Section Non
Conversion factor
Elution curve factor 1.00 Calculation of differential and integral TOSOH
Differential curve factor 1.00
Detection control No
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
[LogM]
2
6.00 8.00 10.00
[min]
[LogM]
2
6.00 8.00 10.00
[min]
<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].
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.
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.
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.