You are on page 1of 78

Manual book

CHROMATOGRAPHY
DATA HANDLING SYSTEM
Content

1.Introduction……………………………………………………………………………………………1
1.1 Forward………………………………………………………………………………………………………1
1.2 Function………………………………………………………………………………2
1.3 SY-8100 HPLC system of full control…………………………………………………………………2
1.4 Main technical specifications……………………………………………………………………………3
1.5 Running environment of chromatographic work station……………………………………………3

2. Installation of chromatographic work station……………………………………3

3. Integrated environment of chromatographic work station………………3


3.1 Forward……………………………………………………………………………………3
3.2 Description of integrated environment of SY-8100 chromatographic work station……………3
3.3 Controlling Menu ……………………………………………………………………………………5
3.4 Main menu ……………………………………………………………………………………………7
3.4.1 File menu … ……… … …… ……… … …… ……… … …… ……… … …… ……… … …… … 7
3.4.2 Edit Method Menu……………………………………………………………………………………9
3.4.3 Edit Sample Menu……………………………………………………………………………………15
3.4.4 Analyze Report Menu………………………………………………………………………………17
3.4.5 Data Analyze Menu…………………………………………………………………………………18
3.4.6 Tool Menu ……………………………………………………………………………………23

3.5 Prompt Button……………………………………………………………………………………23

4. Custom Report……………………………………………………………………………………24
4.1 Report Template ……………………………………………………………………………………25
4.2 Create a new template (as example for external standardization of HPLC system) …………25
4.2.1 Call the Data File……………………………………………………………………………………25
4.2.2 Custom Report Template……………………………………………………………………………25
4.3 Assigned Print Template Report………………………………………………………………………30
4.3.1 Load data in the report editor and assigned report template to print the report……………30
4.3.2 Call the spectrogram in the work station and select the printing template to print………32
4.4 Printing Items in the Report……………………………………………………………………………33
4.4.1 Header and Footer……………………………………………………………………………………34
4.4.2 Report Header……………………………………………………………………………………34
4.4.3 Quantitative Parameter………………………………………………………………………………35
4.4.4 Integration Parameter…………………………………………………………………………………35
4.4.5 Time Producer……………………………………………………………………………………36
4.4.6 Component Table……………………………………………………………………………………36
4.4.7 Queue Table ……………………………………………………………………………………37

ii
4.4.8 Result Table ……………………………………………………………………………………37
4.4.9 Chromatogram Options………………………………………………………………………………37
4.4.10Calibration Curve……………………………………………………………………………………38
4.4.11 HPLC Base Information……………………………………………………………………………38
4.4.12 “Insert” Menu……………………………………………………………………………………38
4.5 Multiple Spectrogram Report…………………………………………………………………………39
4.5.1 Brief Descriptions……………………………………………………………………………………39
4.5.2 Function Characteristic……………………………………………………………………………40
4.5.3 Creating multiple spectrogram report for different sample……………………………………40
4.5.4 Creating multiple spectrogram report of parallel experiment data……………………………44

5. Operation Procedures of Work Station……………………………………………46


5.1 External Standard Method……………………………………………………………………………46
5.1.1 Create a Method File Parameter to Perform the Experiment…………………………………46
5.1.2 Call the Method File Parameter Original Saved to Make the Experiment…………………51
5.2 Internal Standardization of Multiple Points …………………………………………51
5.3 Single Point Internal Standardization…………………………………………53
5.4 Normalization Method………………………………………………………………………………57
5.5 Calibrating Normalization Method…………………………………………………………………58
AppendixⅠ Technical Terms………………………………………………………………………………59
AppendixⅡ Usage of Multiplier and Divisor……………………………………………………………63
AppendixⅢ Flow chart of Data Processing………………………………………………………………68
AppendixⅣ Each component in Paste Report to word/wps file ……………………………………70

iii
Chromatography Data Handling System

Chapter One Introduction


1.1 Forward
Chromatography Data Handling System is compatible with any model of
chromatographic analytical instrument available in the market. The computer is
connected with the chromatograph by a series interface. Under the software control of
the chromatographic work station, the analog signal of the chromatographic peak
output from HPLC’s detector is transferred, data acquisition save and treatment and o
process analytical correction and quantity for the acquired and saved chromatogram.
Print out the chromatogram and analytical report and calibration curve.
Simultaneously, the instrument is directly controlled by the computer
This software is run under Windows operation system, structured in such a way that
all the queuing related to an analysis (starting from acquisition of raw data signal, to
integration of chromatogram, to calculation of components quantities, through to
preparation of analysis report) are incorporated in one source document called
Chromatogram file.

1.2 Function Characteristics

1) High accuracy data collector with remote START/STOP integration.


2) Windows in English. Easy to operate.
3) Due to the technical invention of spectrogram intellectual identification, the system has
outstanding treatment capability of the spectral peak.
4) Possess multi-medium capability, with auto-voice hints during the operation.
5) Multi-tasks operation. During data acquisition and treatment, it enables to run the other
application program, such as: word processing and game.
6) During data acquisition and treatment, watching VCD, listening to CD and sending out or
receiving the faxes at the same time.
7) Display the spectrogram dynamic status, Sizes of the spectrogram window is available for
step less adjustment in order to secure the observation to the peak or baseline conditions at
any time.
8) Input level and running time is displayed on rear time.
9) The setting of the sample queue is convenient for analytical batch samples and using an
auto-sampler.
10) The spectrogram data can be automatically saved on Disc and data files are automatically,
continuously named.
11) Manual integration. It is very convenient for adjusting initial and landing points of the peak
integration .
12) Batch facility and re-analysis functions make the analytical sample and the integral
calculation separately so as to secure the success for sampling only once time.
13) Display the calibration curve on the screen, available for printing out.
14) Result report displays in English.
15) Automatically read in RT(min) during component table edition, read in the component name

1
Chromatography Data Handling System

from the compound laboratory. It is good for saving time and labor.
16) Multi-scale mode and mesh control during printing the spectrogram.
17) Plotting scale of the screen spectrogram is arbitral controlled, it is convenient for the
direct-view during inputting parameters.
1.3 SY-8100 HPLC system of full control
1.3.1 HPLC system of full control
HPLC system controlled by computer is linked with START key of the instrument, also available
to input controlling parameters in work station to control the instrument running without the
manual operation on the controlling panel of the instrument.
1.3.2 All in one Data file structure
There are following four files available for user in the chromatographic work station software of
Version V5.01:
√Method file: Including peak integral parameter, Quantitate parameter, component table,
calibration curve, report format, method information and instrument conditions, etc.
√Date file: Including chromatograph data, sample information, peak table, analytical result and
methods. File extended name •DAT, file path at will.
√Report template file: Save and print the parameters required by specific report. File extended
name •film, file path is content of work station\REP.
√Sample queuing file: Save sample name, multiplier and divisor, etc. File extended name •SDL,
file path is at will and available to continuously analyze the batch samples.
There are the other three files contained in the data file, These three files are independent file
separately. It is convenient for user to call the files required. Therefore, data file structure of all in
one can open the method from the method file, or from sample data file.
1.3.3 Flexible custom report tool
A custom report editor is used for the user self to design the report content and format. It is
flexible to make the report type what you want. It is similar with the character processing
program. Change style of calligraphy, color and page edge space, dragging components
(component can firstly move to ‘copy to cut and paste plate’, then paste it to word or wps file),
and insert spectrogram, icon, system information and format, etc. Let the user to flexibly edit
report, adjust appearance and content of the report.
A report template is used to control printing format in chromatographic work station. The report
template file saves various parameters required by printing report, those parameters will directly
influence on the outlook of the report. The report template enables the user to make own design
with the custom report editor, the designed report can be also saved on the new template. File
extended name of system auto-acquiescence template is •flm, saving path is: installing content
of work station\REP.
1.3.4 Multi-spectrogram result report
The custom report editor also provides the analytical function of “multi-overlapping
spectrogram”. The parallel test should be made by many times for checking the reproducibility
and calculating the average value, this report system can directly calculate out “result mean
value”.
1.3.5 Prompt operation page
The page is very clear, easy operation. Such as: the editing pages of every parameter on the
method file are stored together in form of card on the editing window of the method file.

2
Chromatography Data Handling System

1.4 Main technical specifications


Performance of complete instrument
√Inputting channel number: 1
√ Inputting voltage range: -5mV-1V
√ Inputting impedance: >10MΩ
√ Integral sensitivity: 0.1µ V.S
√ Minimum resolution: 1µV
√ Dynamic range: 106
√ Linearity: ±0.1%
1) Five kinds of quantitative calculation method are used in common.
2) Identify 256 pieces of constituents using RT(min) or component table.
3) The calibration curve can do up to three orders, eight points and repeating 10 times at per
point.
4) Treatable peak number: 1024
5) Auto-time program.
6) Auto-deification and segment for complicated peak shape.
7) Auto-tracing baseline and calibration.
8) Make manual integration by user itself.
1.5 Running environment of chromatographic work station
Hardware
Better 586, CPU
Better 64M
One free series interface on the system board (COM1 and COM2)
VGA Color display (better use an accelerated card of graphics)
CD ROM
Mouse
Printer
Software
Better WINDOWS 2000 operation system

2. Installation of chromatographic work station

The chromatographic work station consists of two parts: Hardware and software. Hardware
includes computer, mouse, voice card card, printer, chromatographic data acquisition card and
concerned cables. Software includes WINDOWS, OFFICE and software of chromatographic
work station. This section only describes for installing procedures of work station software of
chromatograph.
3. Integrated environment of chromatographic work station
3.1 Forward
This section mainly describes functions and applications of each window, menu, button,
parameters and format in integrated environment of chromatographic work station.
3.2 Descriptions of integrated environment of SY-8100 chromatographic work station
Double-click the icon of chromatographic work station on the desk to enter into the integrated
environmental page of work station.

3
Chromatography Data Handling System

Running indicating lamp


Main menu Prompt button

Spectrum display window

Controlling Menu Information line

The main page of the integrated environment consists of five parts: main menu, spectrogram
display window, prompt button, controlling menu, running indicating lamp and information line.
Main menu
As same as the other application program, according to the different requirement of processing
data, all of the functional commands are classified, and respectively put them into eight menus.
Every menu has own group commands.
Prompt button
Some common used functions in the main menu are marked on these buttons with icons.
Therefore, these functions in common use can be directly selected by pressing the concerned
button without selecting it from the menu in order to speed up the operation.
Controlling menu
Directly control SY-8100 HPLC system on the work station panel for convenience of user.
Spectrogram display window
Plotting area——Actively display the real time spectrogram area, the sizes can be automatically
adjusted with the size change of the spectrogram display window. When the user shortens the
spectrogram display window to a certain extent, the system automatically delete the plotting
coordinate scale and RT(min) displayed to secure observing the spectrogram clearly even in a
small size window.
Time coordinate scale——It is used as X axle coordinate scale, unit is minute. The initial value is
automatically adjusted with the change of the sampling time. Modify the full scale time difference
value on “screen plotting parameter edition”. The minimum time is 1min.
Level coordinate scale——It is used as Y axle coordinate scale of the spectrogram plotting, in
unit: mV The full scale maximum value and minimum value can be modified on “screen plotting
parameter”.
RT(min) of peak——During starting sampling, automatically display the RT(min) of the peak

4
Chromatography Data Handling System

after appearing the chromatographic peak top. Select “no display the RT(min)” to close this
function in the “screen plotting parameter edition”.
Running indicating lamp
The red lamp is flash when the data acquisition is running while the green lamp is lit under the
preparation status.
Information line
Display currently inputting level, sampling time, current sample name and current time.
The above describes the main page. When you select some command in the main menu or click
once some prompt button, the corresponding window or dialogue frame will be appeared.
3.3 Controlling menu

3.3.1 When the connection of the instrument with work station is successful, a icon of “

appears. If it is not so, the connection is failed.


3.3.2 Functions of each key on the controlling menu.

Adjust detector zeroing

P D

On this page, click once “P” or “D” button to appear the following page:

A. Click “pump” on this window, appears the following window:

5
Chromatography Data Handling System

Respectively set four modes of pump, maximum and minimum pressures in “control mode”.
Set gradient purge in time program table and different ratio flow in different time section of pump.
B. Click “detector”, appear the following window:

Set the wavelength, sensitivity and spectral scanning on this window; If the wavelength program
table is required, “√” is made at the front idle form, set time and wavelength, press ENTER key
to make the detector entering into the wavelength program.
3.3.3 Other functions for controlling button

Blue digits show current real parameters


A. Click “Prog Reset” to force the current running program retrieval.

B. Click “ ”, Start or stop the pump; “ ” is in the starting status.

6
Chromatography Data Handling System

C. Click “purge A” or “purge B”, appear the following page:

Click “ENTER” key to purge the pump.

D. “ ”,is column.

E. Click ,appear the following page:

Refer to the main menu for detailed operation.

F. Click directly to read the screen report.


3.4 Main menu
This section mainly describes how to operate the menu and the commands.
3.4.1File menu
Click once “file” menu to appear the following window:

7
Chromatography Data Handling System

Since some main parameters will be used during running chromatographic work station
(integration, quantitative, component table, calibration curve, report format, method information,
screen plotting parameter and data recording parameter, etc.), those parameters are saved as
method file. Extended name of the method file is: .mth.
1) Open Method
Click “Open Method” item, appear “Select Method File”,Here select the original method file,
load the working parameters in to the internal memory from the method file.

2) Save Method
When the current parameters used may be required for the future, you can record them into a
method file as follows:
Click “Load Method” to appear ‘Select Method File’, definition [file name] and fill in the
description characters into Describe line.

Note: Extended name of method file name is .mth. If selecting an existed file name, a new
parameter will cover the original one in file.
3) Load Method From Data File
Click‘Load Method From Data File’item (it is another mode for calling the method file,it only
calls the method file with the data together). Appear the window of “Select Data File”,here select
the method file as shown in following figure:

8
Chromatography Data Handling System

4) Option
It is option when controlling starting the work station. Select “Option” in “File” menu. The system
will appear the following window; it shows “Last Time Method” under acquisition condition.

5) Exit
When Exit the chromatography work station is required after completing the analysis, Select
“Exit” in “File” menu and system appears the following dialogue frame:

3.4.2 Edit Method Menu


1) Control——Control if the report is automatically printed out after completing the data
acquisition as show in following figure:

9
Chromatography Data Handling System

2) Record Parameter——Record (Save) the path and file name during the data acquisition.
By the function of “Record parameter”,definite the data acquiesced to record the path and
file name on the disc during running the chromatography work station. When the data is not
required to be saved on the disc, this function is able to use for the definition.
a In “Edit Method” menu, select “Record parameter”, the system appears the following
dialogue frame.

b Use the mouse to select the driver, file path of the data record, use the keyboard to input
file name (file name can not exceed over four English alphabet) If it is not necessary to
save the date of the acquisition, select ”Not Save Data File”.
c Click “ENTER” button.
Explanation:
a The file name can be input maximum five letters.
b Create a new file directory with “file manager” of windows.
c During saving the data, the chromatography work station is automatically add series
number behind the file definite and extended name .DAT.
3) Displaying(put it with Data files Properties in the same window, see above figure).
When running the work station, “ recorder ” window always actively display the
chromatography signal, modify plotting X and Y coordinate scale using “Displaying”. Input up
and down limits and x axle full scale value(the difference between up and down limits values of
Y axle coordinate can not be more than 1mV,full scale value of X axle coordinate can not be
more than one minute).
4) Quantitate parameter
Quantitate parameter — — Definite the method of the quantitative calculation , including
quantifying (Area/Height), Calculation (Normalization/Normalization by calibrator/External
standardization/Internal standardization/ Internal standardization(curve), Calibration level and
Order. Click “Quantitate parameter” icon, appear the Online Method window:

10
Chromatography Data Handling System

Set well “Quantifying”, “Calculation” (external standardization), “Calibration level (eight


concentration standard sample, select 8 Level), “power”, “standard concentration unit” and
“sample result unit”.
5) Integration parameter
Integration parameter is used to control the parameter for the chromatography peak making the
integral calculation, including Width, Slope, Split, Min. area, Min.height, Lock time, Acquire time.
Among them, the selections of Width, Slope, drift will directly influence on the integral result.
width——Set it on the basis of minimum effective half peak width(unit: s)..
This parameter will influence the time interval of calculating signal slope. For example: set 5,
the determination time of the peak will calculate the slope value of the signal change for once time
at the interval of 5/10 seconds.
Slope——Peak determines the sensitivity. Determine the start point and end point of the
peak.(unit:μV/min).
Split——Determine the baseline change degree to realize the auto-split of the peak area (unit:μ
V/min).
Min. area——When inputting the min. area, if the peak area is less than this peak area in the
chromatogram, the program does not identify it, so the RT(min) is not marked .
Min. height——As same as the minimum peak area (unit:μV).
Lock time──No integration is between the peak from starting the analysis to locking time,
mainly used for appearing air peak, solvent peak, solution peak and negative peak ,etc without any
marks from a certain time of starting analysis.
Acquire time──Auto-acquire time for the analysis.(unit:min)。
As shown in the following figure:

11
Chromatography Data Handling System

6) Time procedure
The width, slope, split, etc. parameters control the detection and integration of the peak. These
parameter values used are all decided according to the chromatography peak conditions. When
there are multi-chromatography peaks in a chromatogram, with increasing the RT(min), the
chromatography peak is getting more width. When fixing integral parameter is unable to secure all
of the peak to obtain the correcting detection, use the time program to definite the different a time
to have the different integral parameter value so as to secure the correct detection for all of the
peaks (it acts as a partial part of the integral parameter and put them on the same window as
shown in the above figure).
Time──To change a time of integral parameter (unit: min).
Width, Slope, Split──As the same with integral parameter.
Lock──When the value is one hour, no admittance for the integration. When the value is
zero, return to the integration.
Explanation Selection item of “Use time procedure”, available for opening or closing the
time procedure.
7) Component Table
In Component Table, input the component name required to be analyzed, Remain Time (RT) ,
Band (%) and Calibration factor in the internal standardization.
Component —Component name to be analyzed. When using the internal method, don’t input
the component name while inputting “the internal substance” for system to definite the internal
substance peak.
RT——Appears peak time of compound to be analyzed (unit: Min.).
Band——Allowable deviation of RT(min) (unit: %)
Calib.——Series concentration of sample, such as: mg/ml, %.
a) In “Edit method” menu, select “Component table”, appears the following dialogue
window.

12
Chromatography Data Handling System

b) Input the value required for each parameter. Among them, the component can be input
directly by the keyboard, also select it in bottom menu using the mouse; RT can be
directly input or also can click “Auto Load Peak Data” button to read the Data from
current inter memory.
c) Click “ENTER” button.
Explanation:
a The component name in “item table” can be increased or deleted using character edition
tool(such as: open C:\WMSP\Xm·ini notebook ).
b The spectrogram in current inter memory is a recent data acquisition.
8) Report Type
Output a printing template for the report in “print format” window (report printing system has
superiority, detailed description for it shows later).
9) Calibration Curve
Click “Calibration Curve” to appear “Online Method window.

Definite spectrogram peak file required at each point. Use the mouse to point at sample point
number, and then click left key, it is covered with blue color, again click the right key to appear
available menu:

13
Chromatography Data Handling System

Select “Add” item, (it shows that several parallel experimental data are added at the same
concentration point for the mean calculation), appear the window of “Select Data File”.

And then here select the corresponding spectrogram file, appear the symbol “+” before the file of
the series point. After completing the definition for every point, click “re-calibrate”, the moment
appears the calibration curve.
Operation of deleting file
Firstly click the symbol of “+” at the front of the peak number to change into “-“ symbol and list
the file pat, click the right key to select the file, appear the right key to select the menu, as shown
in following figure:

Click “delete” to cancel the file, the moment the symbol of “+” is not shown at the front of the file
point number, click “reread peak data” button, Number 2 on the calibration curve is deleted as
shown in the following figure:

14
Chromatography Data Handling System

Peak icon at the right down angle of the window is pre-observation button of the spectrogram file,
see the following figure.

3.4.3 Edit Sample Menu


1) Single sample information edits
Click “single sample information edits”, in “Online Sample Information” table, mainly given
“sample name” file definition of test data and normal information as follows:

2) Sample Sequence Edits


Click “ample Sequence Edits” to appear the following window:

15
Chromatography Data Handling System

Sample name──Sample name, letters or digits.


Multiplier and divisor──transfer the result unit.
Current sample number──What the location number of the sample queuing for current sample.
Use Sample Sequence──Control the queuing if it is running.
Explanation:
a “Use Sample Sequence” to open or close the sequence.
b Click sample series number, press “change” button to change the current sample
number.
c Use “Ins”, “Del” and “Cls” buttons to inserting line and deletion.
d “File name” bar, the work station is automatically filled with during running sequence.
3) Open Sequence File
When a sequence is already saved in the sequence file, the sequence data in this file can be
transferred to inter memory. Select “Open Sequence File” in “Edit sample” menu, sees the
following dialogue frame:
Select a sequence file required. Change the driver and directory if it is necessary.

4) Save Sequence File


When the current using sequence is used for the future, record them to a sequence file. Select
“Open Sequence File” in “Edit Sample” menu, appear the following edition window. Input the file
name in the file name edition frame, or using the mouse to select saved file name, change the
driver and directory if necessary.

16
Chromatography Data Handling System

Explanation:
Extended name of queuing is “file name.SDL”. If selecting a saved file name, a new
parameter will cover the original parameter in the file.
5) Use Sequence──Active current sample sequence.
6) Reset Sequence──Definite the first sample in the sequence as the current sample
3.4.4Analyze Report Menu
1) Screen Report
Click “Screen Report” directly to observe the current data result.

2) Print Report
Click “Print Report” directly to print out.
3) Preview Report
Preview the report before printing.
4) Custom Report
Click “ Custom Report” to enter into the custom report editor,here edit the report format.

17
Chromatography Data Handling System

3.4.5 Data Analyze Menu


Data re-analyze /Manual Integration Fixes treatment provides the user with fast repeatedly
analysis treatment function. For a saved data, it is available for promptly reanalysis calculation. If
modifying integral parameter or time program, click “Re-integration and calculate”;If modifying
method file, click “Re-calculate” (“Re-integration” has not been made yet), since the data may be
treated by “Manual Integration”. If again “re-integration” is made, the result will be changed.
Click “Data Analyze menu” button to enter into “Data Analyze menu” page, click “Open Data”
icon, appear “Data Analyze” window, here select the sample data file on this window, and see the
following:

Still click “Re-calculate” button after calling the data file. Under the acquiesce condition, the
data file opened is only brought into the analytical result, not into method file, so click
“Re-calculate” button, use current acquiesced method file( current method file of work station).
This work station software is very flexible for calling the data file and method file as shown in list
table in fig. But don’t forget single-click “Re-calculate” button, current data file and method
file are combined into a new data file.
Again click ‘Screen Report” icon to observe the screen report table.
Manual integration
The manual integration means to re-adjust starting point and end point during treating spectrogram.
The integration is automatically made by the computer during running work station. Other peaks
and tails will cause the deviation of the peak area, so the moment it is necessary to make the
integration manually for the accurate integration.
As shown in the following figure, proper integration without modification.:

18
Chromatography Data Handling System

If it is not properly, manual integration is required.

Procedures of manual integration:


a. Firstly click the peak required for manual re-integration.
If appearing peak time of the re-integration is the peak of 1.78 minutes, use the curcor to click
the peak area, select the peak of required manual re-integration. At the same time of selecting
peak, Peak information display area will display the appearing peak time of the peak, peak area
and peak height, see the figure below:

19
Chromatography Data Handling System

Peak area
Peak information
display area

b. After selecting the peak of manual integration, first click “adjust starting point”, as shown
in the figure below:

Adjust starting point

c. Use the left key of the mouse to click some of point on the peak selected, select the start
point required for manual integration, see the figure below:

The position of the starting point

After clicking with the left key of the mouse, the starting point is changed as shown in

20
Chromatography Data Handling System

figure below:

d. And then again click “adjust landing point” as shown in figure below:

Adjust landing point

e. Click some of point on the selected peak using the left key of the mouse, select the landing
point required for the manual integration, see the figure below:

The landing point

f. After clicking it with the left key of the mouse, the landing point is changed, the

21
Chromatography Data Handling System

spectrogram of the re-integration is again appeared on the page as shown in figure below:

Peak area display area

Peak area

After completing the adjustment of “ peak starting point” and “peak landing point”,
click the chromatography peak area finished adjustment, the moment “peak
information display area “ display the peak information after the adjustment.
g. Operation of deleting the peak. Single-click “deleting peak “ button, and then single-click
the chromatography peak required for deletion, see the figure below:

Deleting the peak

h. Click “re-calculation”, and then click “screen report” to observe the peak area after the
manual integration.

If returning to the integral result before the operation of “manual integration”, directly click
“re-integration + calculation” buttons on the page of sample treatment of work station.

22
Chromatography Data Handling System

3.4.6 Tool Menu


Concerning with slop test button
This button is used for determining baseline odd peaks. As far as we know, the small odd peaks
caused by the noise of the chromatograph will be also detected by the program. Therefore, we
have to let the computer know the slope values of these odd peaks in order to the computer to
identify the odd peaks with the slope value below, while it is not peak appeared during
determining sample. It is determined if we don’t know this value. Normally it is 10~12,unit:μv/s.
But in rear chromatograph analysis, the value is normally 30(when the slope value starts to be
more than 30, the starting appearing peak is a starting point; when the slope value starts to be less
than 30, finishing peak is a landing point), and set it in “integral parameter edition” to make better
determination for the chromatograph peak.

3.5 Prompt button


The buttons in tool bar described in this section are mainly used to execute some common used
functions in the main menu so as to reach propose of the prompt operation. They all have prompt
keys (such as: Ctrl + Q to quit current program running.)

HINT: When moving the cursor to the button, for a while, the button function is displayed.

Quit(Ctrl+Q)
Display to quit the dialogue of work station, the program is closed.

Open the test method(Ctrl+L)


It is equal to “open”. This commend is used to open a method file, and call the file parameter into
internal memory.

Quantitate parameter(Ctrl+D)
Display the dialogue frame of the Quantitate parameter.

Integral parameter(Ctrl+J)
Display the dialogue frame of integral parameter.

Screen plotting parameter Ctrl+G)


Display the dialogue frame of screen plotting parameter edition.

Constitute table(Ctrl+I)

23
Chromatography Data Handling System

Display the dialogue frame of constitute table edition. Edit compound name required to be
analyzed, etc.

Data record parameter


Display the dialogue frame of data record parameter. Definite recorded (save disc) path and file
name during the data acquisition.

Calibration curve
Appear plotting frame of calibration curve. Observe the calibration curve shape.

Report parameter(Ctrl+B)
Display the dialogue frame of report parameter, definite content of printing report, scale and
configuration.

Screen report
Display edition record frame of screen report, it is available for printing out sample name and
analytical result,etc.

Queuing edition(Ctrl+Z)
Display edition frame of sample queuing, it is available for edit a big batch of sample queuing.

Re-treatment
Click the button here or select “re-analysis/manual integration treatment” item in “data
re-analysis” menu.

Slope test
Slope test for baseline (noise) peak

Control instrument
Display every parameter during running chromatograph.

“Start”, “Stop”button
“Start”button——When pressing “Start” button, the system starts data acquisition, meanwhile
the plotting area on the spectrogram display window will be automatically disappeared. The time
scale starts from “0”, running indicating lamp in red is flash, sampling time in the information line
starts to time after returning to “0”, “Start” button transfers into “Stop” button.
“Stop” button ——When using the mouse to press “Stop” button, the system stops data
acquisition. “Stop” button transfers to “Start” button.
NOTE: At the same time, only one of “Start” button or “Stop” button can be seen.
4. Custom report
The user itself can design the content and format of the required report. It is familiar with
character treatment program, therefore, characters, color, margin space and drivable assembly
(copy to cut and paste plate, and then paste to word or wps file). It is available to insert
chromatograph, icon, system information and format, etc. Let the user flexibly edit report, adjust

24
Chromatography Data Handling System

the configuration. There is no limitation for the content and layout and print out an ideal report.
The system provides user with more selections.
4.1 Report template
Report template is used to control the report file of printing format in the chromatograph work
station. All of parameters required are saved in the report template. These parameters will be
directly influence on the report configuration. The report template can be own designed using
“Custom Report” (custom report editor), the designed report can be saved as a new template. The
file extended name of system auto-acquiesce template is·flm, saving path is: Installation
directory of work station\REP.
Standard template——The system provides a complete standard report template, the user
can be used directly.
User template——User can call the data file from custom report editor, and then self design
the report template and save it as a new one.
4.2 Create a new template (as example for external standardization of HPLC
system).
4.2.1 Call the data file
On the page of “Data analyze” of work station, single-click the icon of “Open Data File” to open
the window of “Select Data File”, call a data file as shown in the figure below.

4.2.2 Custom Report Template


1) Open the custom report editor
After finishing the operation of calling data on the page of “Data Analyze”, click “Custom
Report” in “Analyze Report” menu to open the custom report editor.

25
Chromatography Data Handling System

2) Insert page Header and Footer


Select “Header/Footer” in “View” menu, appear the edition window of ““Header/Footer”,
single-click “ENTER” after defining the contents of the Header and Footer.

3) Insert Textbox
4) Select ‘Insert Textbox’ in the Insert menu, and then edit and insert the characters on the
edition window. See the figure below:

5) Edit Report Header


Click once “Report Header” icon, it changes into ╋ when the mouse is moved to this page,Hold
on the left key to move the mouse to draft the edge frame of the report heading, the moment, the
mouse is located in this range (click once the left key to change it into , the four angles of
the edge frame and the middle at each edge all have a size controlling point, under this status,
enlarging and reducing frame and removing the edge frame). Click once the right key to appear
the selection table:

26
Chromatography Data Handling System

Select “Edit Report Header”, appear “Report Title” window:

After definition the contents of each item and attribution, click once “Enter” key and adjust the
size of the edge frame, select the position to be located.
6) Edit Chromatogram
Click once “Chromatogram” icon, open “Chromatogram Options” edit window following the
operation above:

After definition the contents of each item and attribution, click once “Enter” key and adjust the
size of the edge frame, select the position to be located.

27
Chromatography Data Handling System

7) Edit the basic condition of chromatograph.


Click once “HPLC” icon, open “HPLC’s information” edit window following the operation
above:

8) Edit calibration curve:


The operation as the same as the above, open “calibration curve” editing window.

Respectively selecting card to process the edition, and then click “ENTER”.
9) Edit Result Table
The operation as the same as the above, open “ result table” editing window:

28
Chromatography Data Handling System

Click “Data option “card to select the item, and adjust the table width on the top of the table
format bar, then click “Table Type”, “Font color” “Print”cards to process the edition in sequence.
Click “ENTER” to adjust the size of the edge frame as following figure:

10) Adjustment of report outer appearance


Open “Page Setup” editing window to make the edition on “File” menu, then adjust the report
configuration.
11) Printing out
Click “Print” on the “file” menu to process print (it is able to preview before printing).
12) Report Template File Save
Any of report can be saved as a new template for the future use directly. Click once “Save” icon,
appear the below window:

29
Chromatography Data Handling System

Input file name and template description characters, press “ENTER” button to save a new report
template, the acquiesce extended file is ·flm.
4.3 Assigned Print Template Report

4.3.1 Load data in the report editor and assigned report template to print the report.
1) Enter (Custom Report) into the page of the editor as the below::

2) Click once : “Data” icon, appear the window:

30
Chromatography Data Handling System

Select the data file in the path frame, Press “ENTER”, the S data is called.
3) Click once “TPL” icon, appear the window:

Select the template file, Press “ENTER”, the report format of assigned template output can be
observed on the editing window, the printing report is just the report you have been seen.

31
Chromatography Data Handling System

4.3.2 Call the spectrogram in the work station and select the printing template to print.
Open “Data Analyze Method” window, call the spectrogram.

1) Click “Report Parameter” icon on “Data Analyze Method” window, appear the method
information window, select “report format” selection card as the below figure:

Click once “selecting template” button, appear “report template selection” window:

Here select the template file, after pressing “ENTER” button, click “re-calculate” button on the
page of “again treatment”. This operation is to assign a new report template of the spectrogram

32
Chromatography Data Handling System

data, the original method file is changed, so the parameters of method file of new assigned report
template are used to calculate again. This way, the original parameters of the spectrogram can be
changed. After so, click “custom report” button on the “analysis report” menu to enter into the
report editor. A report format of assigned template output is displayed on the page of the editor.
The report printed out is the report observed.
4.4 Printing items in the Report
The following items can be available to print out in the report; the printing content can be
selected by the user, provided with the icons.
Printing item Contents
Header and footer User can edit headline and page footing
according the requirement.
Report header Some important information concerning
with this experiment.
Quantitate parameter The information is concerned with
quantitative calculation method.
Integration parameter Each integral parameter value
Time Producer Content of time program
Component table Concerned information of constituents
Queue table Queuing information
Result table Content of analytical result
Chromatogram Options Display spectrogram
Calibration curve Display calibration curve
HPLC Base Main information of chromatograph
Information system
“Insert” menu Available to insert straight line, icon and
character.
NOTE: Each item mentioned above in exception with headline and page footing, the other
items can be all moveable components. The open methods of the editing window are as follows:
(As example as editing report head):
Click “report head” icon, it is changed into ╋ when the mouse is moved to the page, hold
on the left key to move the mouse to draw the edge frame range of the report head, then the mouse
is located at the range. Click once the right key, appear selectable menu:

Select “Edit Report Header”, appear editing window of “Report Title”.


Select “Delete” not to print this item.
Select “Copy to Clipboard” function, the block firstly copy to clipboard, and then paste it to
WORD or WPS file. The operating procedures are as follows:
Click selecting item of “Copy to Clipboard”.
Enter WORD or WPS file, move the courser to the position of the component required and then
click once the right key to appear “right key menu”, click once “paste”, so the component is pasted
to WORD or WPS file.

33
Chromatography Data Handling System

4.4.1 Header and footer


Open the editing window of “header/footer” on “view” menu

It is fixed position component and multiple frames are selectable item, it can select if printing this
item. Input the character content below the blank bar of header and footer. For the characters,
word type, word size and color can be changed, For marking-off, line type, color and line size can
be changed.
4.4.2 Report Header
Report header is a moveable component of multiple line characters, available to select if printing
out some item, providing with prompt button. The editing window of “Report Title” is as follows:

1) The header of the report table is selectable, the content can be edited, and alignment
mode is selectable.
2) The contents of the report header include analyst, Analyst Unit, Print Time, Data File,
Method File, Analyst Note, Sample Name, Syringe Volume, Sample Source, Criterion of
analyze Method, Quality of Analyze Method, all of them are selectable.
3) Dividing into 1-3 rows, Users can adjust the position and sequence at will. Selecting
frame is available to make the selection and the content for the selecting frame can be
also selectable. Its method: click the right side of the black triangle symbol of required
selecting frame to appear a table (as shown in the following figure), and then select the
required item. If content of some item is longer, the space (position) at the selecting item
of the right side can be remained without putting the other item so as to remain enough
position to avoid overlapping characters. Auto-hints will be indicated if overlapping is
caused.

34
Chromatography Data Handling System

4) Line space is adjustable, Font, word number and color of character can be changed; Line
shape, color and size of marking-off can be changed according to the requirement.
4.4.3Quantitative parameter
The Quantitative Parameter is a moveable component of multiple line characters with a
prompt button as shown as following window:

The contents of the Quantitate parameter include Quantitative parameter, quantitative method,
curve type, Standard Sample Consistence Units, Sample Result Units. The editing method and
attribution are as same as “Report Title”.
4.4.4 Integration Parameter
The integral parameter is a moveable component of multiple line characters with a prompt button
as shown in the following:

35
Chromatography Data Handling System

The contents of the integral parameter include Stop Time, Width, Slope, Drift, Split, Minimum
Area, Minimum Height, Time of changing Parameter. The editing method and attribution are as
same as “report head”.
4.4.5Time Producer
The time producer is a moveable component of multiple line format with a prompt button. It is
available to enlarge and shorten the format and set the edge frame as shown as the following:

The contents of the time producer on the top of the window include: S/N, Time, Width, Slope,
Drift, Locked (time). The method for adjusting the row distance is to move the mouse to vertical
line of the first line format to adjust the row distance of the format. Click “Table Type” card to
select the format type required.
Click “Font_Color” card to select what you want. As shown in the following:

4.4.6 Component Table


The component table is a moveable component of multiple line format, include: S/N, Ret time,
peak name, relative, calibration factor and concentration of standard sample. The editing method
and attribution are as same as “report head”. See the following window:

36
Chromatography Data Handling System

4.4.7Queue table
The queuing table is a moveable component of multiple line format, including S/N, sample name,
multiplying factor, file name and dividing factor.. The editing method and attribution are as same
as “report head”. See the following window:

4.4.8 Result table


The result table is a moveable component of multiple line format, the contents in the format can be
selectable for printing, enlarging, shortening, and setting the edge frame. A prompt button is
provided. The “result table” is displayed below editing window:

Click “Data Option” card, select the required printing content, it displays on the top of the window,
and then adjust the row width. The result table includes S/N, Peak Name, Ret Time, Height, Area,
Result (see the above figure). The other editing method and attribution are as same as “Time
Table”.

4.4.9 Chromatogram Options


The Chromatogram Options is a moveable component, available for enlarging and shortening,

37
Chromatography Data Handling System

with a prompt button. See the below window:

4.4.10 Calibration Curve


The Calibration Curve is a moveable component, available for enlarging and shortening, with a
prompt button, see then below window:

1) Click “Print” selection item as shown in the above figure. It is available to select printing
“All the Peak Graphs” or “This Peak Graph” (assign the component ate the list table of the
right side).
2) Click “attribution” item as shown in the following figure. The definition of the point and
attribution of line can be made on this window.
4.4.11 HPLC Base Information
Input Instrument name, Det Name, Mobile Phase, Injector, Column, Column’s Flow, Wavelength
and Remark. See the below window:

4.4.12 “Insert” menu


It is available to insert the line, Text Box and Graph Box in the report. Click inserting content on

38
Chromatography Data Handling System

the “insert” menu as shown in the following operation (as example for inserting icon).
1) Click once “insert” menu, appear the menu:

2) Select “Graph Box”, mouse moves to the page to change into ╋, hold on the left key to
move the mouse to draw the edge frame range of the report head, appear the editing window:

It is OK to select the Graph file. The line, Text Box and Graph Box can be moved. The attribution
of line, Text Box and Graph Box can be definite by user self.
4.5 Multiple spectrogram report
4.5.1 Brief descriptions
The custom report editor described above is very similar with the character processing program. It
is available for editing icon, format and characters, each component as an independent body is
moveable, enlarging and selectable for printing. The user can tailor made the printing template that
means assigned printing template. This editor can provide the analytical function of
“multi-overlapping spectrogram”. Users can put several spectrograms and several result tables into
one report in order to save paper and compare data conveniently. Also the user can make several
parallel experiments for inspecting the reproducibility and mean value, our report system can
directly calculate “result mean value”, “variance coefficient” etc. This system provides more
choices to users to fill their requirements. The detailed operation procedures are described as
follows. Operation page is shown as follows:

39
Chromatography Data Handling System

4.5.2 Function characteristic


● “Channel”:10 parallel experiment data spectrogram are allowed loaded in the multiple
spectrogram report. Each position serial number of the spectrogram file saved is “Channel”
number.
● “Same” function: Single-click this button, the attributes of the multiple spectrogram group
according to the current channel number are the same, says, displaying effect is the same.
● “Volts Ruler”(Time Ruler):Rulers can select to be print or not. Displaying “Volts Ruler” or
“Time Ruler” can according to the collect data scales (“according to the screen drawing
parameters”), also can input custom “High” and “Low” values. Rulers display the value. The
type, size, and color of the ruler figure and the border of the rulers both can be changed at
will.(as shown above fig.)
● Attributes such as: Integration line, Chromatogram Line, Grid, size and type of the border are
all independently, but can be edit respectively.
● There are 15 options can be selected to be marked, such as “Name”, “RETE times”, “height”,
“Area” and “result” etc.
● “Marks Model”: “Named Peaks” and “All Peaks” can be select to be the Marks Model.
● “Time”, “Volt” both can as the X-coordinate. X or Y coordinate can be changed over in the
coordinate.
● “Type”: If “Deverlay data” is selected, program displays several spectrogram
4.5.3 Creating multiple Spectrogram report for different sample

1) The operation procedures is as same as the single spectrogram,on the page of “Data Analyze
Method”, call in the data file, so to take up the data file in the channel 1.
2) Single-click “Custom Report” on the “Analyze Report” to open the editor,and edit, adjust the
appearance of the report to be print out (except Chromatogram options and Result Table, the
operation procedures is as same as the single spectrogram).
3) Edit the Chromatogram Options
A. Click “CHRM” button, and drag mouse to report page to draw the frame range, loose

40
Chromatography Data Handling System

mouse then pop-up Chromatogram Options editor, click “Data Source” button, enter the
page of Chromatogram Options data source (shown as follows)(default state, current data
file in the editor take up the No. 1 channel)

B. Corresponding channel number: click “Open” icon (shown as above fig.) to pop-up a menu
(shown as follow fig.)

① Click “current Data”, to select current data file into the editor.
② Click “Open Data File”, to pop-up page of “Open Data File”,on this menu to select data
file(shown as follow fig.)

41
Chromatography Data Handling System

③ Select data file in turns into the report. Suggest put only one chromatogram in each
spectrogram frame because they are different each other, and tick on the left side frame to select
and display in the report. Also several chromatograms (says chromatogram group) can be put in
one spectrogram frame, and tick in the left side frame. Now take printing Channel 2
chromatogram for example, shown as follow fig. :

C. Edit Chromatogram Options respectively


Select the corresponding channel number in the Channel item on left top of the page of
Chromatogram Options, and then edit nine items such as: “Volts Ruler”, “Time Ruler”,
“Integration Line”, “Chromatogram”, “Grid”, “Border”, “Options”, “Marks Model” and
“Directions”.
D. About chromatogram group: also can according to the uniform attributes of data file of certain
channel number.
First select Channel N, and edit its attribute, then click “Same” button.
E. Complete editing attributes of every Chromatogram (about Chromatogram group: when
“Deverlay Data” is selected, display the chromatogram overlapped; when “Tile Data” is selected,
each chromatogram is sequential displayed.)

4) Edit Result Table


A. Click “Result” icon, and drag mouse to draw frame range on the report, to obtain the
Result Table of current data (channel 1) in the editor.

42
Chromatography Data Handling System

B. If Result Tables of any other data files is required, do the same procedures above, obtain
the Result Table of current data, and then modify on this table. Click right key of the
mouse in the Result Table range, pop-up optional menu, select “Edit” item (shown as
follow fig.)

C、Click “Edit” item to open the Result Table editor window. Select data file (corresponding to the
channel number) in the “Data Source” source column, then edit attributes, such as: “Data Option”,
“Justify Type”, “Table Type”, “Font-Color”, “Print”, “Width”. And adjust column width to obtain
the Result table. (shown as follows)

When complete selecting the Result Table and editing the components, adjust distribution of
the Table. As shown as follow fig. :

43
Chromatography Data Handling System

5) Print
6) Save the report Template
4.5.4 Creating multiple spectrogram report of parallel experiment data

The operation procedure is as same as the 4.5.3, except there is a little difference in selecting the
spectrogram file. Moreover, program can provide average replicates directly.
1) Select spectrogram file
A. Select spectrogram files needed in this report in turns. Put several chromatogram
(chromatogram group) in one spectrogram frame because they are parallel data. Work
station can calculate “Average Replicates” and “Variance coefficient” and so on according
to the data loaded into channels (no matter tick or not, so far as data have been loaded into
the channels).
B. The spectrogram files to be print should be tick in the multiple selecting frame to left side of
them. It is shown as follow fig.

C. Edit Chromatogram Options respectively


Select the corresponding channel number in the Channel item on left top of the page of
Chromatogram Options, and then edit nine items such as: “Volts Ruler”, “Time Ruler”,
“Integration Line”, “Chromatogram”, “Grid”, “Border”, “Options”, “Marks Model” and
“Directions”.

44
Chromatography Data Handling System

D. About chromatogram group: according to the data file same attributes of certain channel
number.
First select Channel N, and edit its attribute, then click “Same” button.
E. Complete editing attributes of every Chromatogram (about Chromatogram group: when
“Deverlay Data” is selected, display the chromatogram overlapped; when “Tile Data” is selected,
each chromatogram is sequential displayed.
2) Edit Result Table
The operation procedures is as same as 4.5.3,the program not only display Result Table of
each data file respectively, but also can provide the “Average Replicates”.
A. Click “Average Replicates” on the “View” menu (shown as below fig.)

B. Same as former operation, when mouse on the report change into “+”, and control the left key
to draw frame, then pop-up the edit window:

C. On “Average Replicates” menu, multiple selecting frame on front of every option for selecting
this item to be print or not. Workstation calculate the “RSD” of “RET time”, “Height”, “Area”,
“Result”, “CONV Result”, “50% WIDTH” and “Theoretical”, and provide select function for
users to select it whether is to be marked or not.
D. Edit attributes to complete the “Result Table”. It is shown as follow fig.

45
Chromatography Data Handling System

Concluding:
Different sample data and parallel experiment data are put simultaneously in one report is allowed
in the report system. There is no strict forbiddance. Users can select multiple spectrograms and
multiple Result Tables neatly. Users should pay attention to that: calculate average replicates of
the parallel experiment data is calculate the total quantity of the spectrograms file which had been
loaded into the channel. Make this point clearly, the operation is very easy.

5. Operation procedures of work station


5.1Exteral standard method

5.1.1Creat a method file parameter to perform the experiment.

The operation procedures are as follows:


1) Start the chromatograph and software of work station:

46
Chromatography Data Handling System

2) Edit data recording parameter


Click “Displaying” icon, appear the “Online Method” window, set the position saved the file.

3) Edit integration parameter


Click “integration” icon, appear the method edition window, here set the integral parameter value
and editing “time producer”. It is not necessary to set the acquiesce value.

4) Edit “Online Sample information” table


Click “Single Information” icon, edit “Online Sample Information” table, definite the file
“Sample” for the experimental data. See the below figure:

5) Standard sample analysis


After running the chromatograph table, input the standard sample. At the same time for the valve
sampling, the program automatically proceeds to the data acquisition on the spectrogram. And
mark the RT(min) of the peak. When all of the peaks are appeared, manually stop running the

47
Chromatography Data Handling System

program or set “stop time’ in the “integration parameter” table to auto-stop.


6) Sample analysis
Input the unknown sample. Its procedure is as same as the procedure 5 (this step can be made at
last). The data file (spectrogram) acquired by procedures 5 and 6 is automatically saved by the
system. The data file will be auto-continuously be named, such as: Test001.dat, Test002.dat,
Test003.dat…….
7) Draw Calibration Curve
a Edit Quantitate parameter
Click “Quantitate parameter” icon, appear the method edition window:

Set “Quantifying”, “Calibration (external standardization)”, “Calibration Level (8 pieces of


concentration standard sample, select ‘8 point’), “Order” , “ Criterion Units” and “Sample units”.
b Fill in component table
Click “component table” icon, appear the method edition window, fill with name of sample
Component, RT time and level, quantity series.

c Definite calibration curve


a). Click “calibration curve” icon, appear the method edition window:

48
Chromatography Data Handling System

b). Definite spectrogram peak file required by each point. Firstly move the mouse to the
number of standard sample point, then click the left key, this point is covered by the
blue frame, again click the right key, appear the right key to select the menu.

c). Select “Add” item (this operation shows that at the same concentration point,
several parallel experimental data can be added to proceed the mean calculation),
appear “Select Data File” window.

After that, here select corresponding spectrogram file, the file before the series point appears
“+”. After completing the definition for each point, click once “Re-calibrates”, the
calibration curve is displayed.
8) Look over the sample result
Enter into “Re-analyze” page, click “open data file” icon, appear “Select Data File” window to
select the sample spectrogram file as follows:

49
Chromatography Data Handling System

After that, click once “re-calculation” button, again click “screen report” icon, see the screen
report table:

9) Printing report
5.1.2Call the method file parameter original saved to make the experiment.

1) Using’ Load Method” mode


Select ‘Load Method’ “file” menu of work station, appear “ method file selection” window, here
select the method file as follows:

50
Chromatography Data Handling System

After calling the method file, directly input the sample analysis with passing through the
procedures of 1-7, as the same with the above operation of procedure 8 after completing the
experiment, directly look over the result concentration of sample.
1) Using ‘Load Method from the data’ mode
Select ‘Load Method from the data’ item in the “file” menu of work station (it is another mode for
calling the method file, it only calls the method file equipped with the data from the data), appear
“ Select Data File” window to select the method file here as follows:

As same as the method mentioned in section 5.1, directly input the sample for the analysis after
calling the method file without the operation from procedures 1-7. After completing the
experiment, the operation follows the procedures of 8 mentioned above, directly look over the
result content of the sample.
5.2Internal standardization of multiple points
The operation procedures are as same as the external standardization only except for filling the
component table during making the calibration curve.
The procedures are as follows:
1) Start the chromatograph and software of work station;
2) Edit the data recording parameter
Click “data record parameter” icon, appear the method edition window, set the position of file
storage.
3) Editing integral parameter

51
Chromatography Data Handling System

Click “integral parameter” icon, appear the method edition window, set the integral parameter
value and edit “time program table” here. If it is not set, the acquiescing value is available.
4) Edit “sample information” table
Click “sample information” icon, edit “sample information “ table, definite ‘sample name of the
experimental data file.
5) Standard sample analysis
After running the chromatograph table, input the standard sample. Click once “Start “ button, the
program automatically proceeds to the data acquisition on the spectrogram. And mark the RT(min)
of the peak. When all of the peaks are appeared, manually stop running the program or set “stop
time’ in the “integral parameter” table to auto-stop.
6) Sample analysis
Input the unknown sample. Its procedure is as same as the procedure 5 (this step can be made at
last). The data file (spectrogram) acquired by procedures 5 and 6 is automatically saved by the
system. The data file will be auto-continuously be named, such as: Test001.dat, Test002.dat,
Test003.dat…….
7) Draw calibration curve
a Edit Quantitate parameter
Click “Quantitate parameter” icon, appear the method edition window:

Set “quantitative mode”, “quantitative method (multiple point internal standardizationization)”,


“standard (5 pieces of concentration standard sample, select ‘5 point’), “power” , “ unit of
standard sample concentration” and “unit of sample result”.
b Fill in component table
Click “component table” icon, appear the method edition window, fill in name of sample
constituent analyzed, RT(min) and standard concentration series.

52
Chromatography Data Handling System

Respectively fill in the component and corresponding RT(min) and standard concentration at
each point, etc. During the analysis of the internal standardizationization, the internal
standardization can not input the compound name while input “internal standardization” so
that the system can identify the internal standardization. See the below figure when the cursor
moves to “internal standardization”:

NOTE: When using the internal method, the concentration of each internal
standardization are all the same. But this place,we have to input 1, 2, 3, 4, 5 in sequence.
This is only for avoiding the error during the system inspection, there is no any relation
with the concentration.
c Definite calibration curve
8) Look over the sample result
Enter into “Re-analyze” page, click “open data file” icon, appear “ Select Data File” window
to select the sample spectrogram file. After that, click once “re-calculation” button, again click
“screen report” icon and see the screen report table:
9) Printing report

5.3 Single point internal standardization


The operation procedures are as same as the external standardization only except for filling the
component table during making the calibration curve.
The procedures are as follows:
a) Start the chromatograph and software of work station;
b) Edit the data recording parameter
Click once “data record parameter” icon, appear the method edition window, set the position

53
Chromatography Data Handling System

of file storage.
c) Editing integral parameter
Click once “integral parameter” icon, appear the method edition window, set the integral
parameter value and edit “time program table” here. If it is not set, the acquiescing value is
available.
d) Edit “sample information” table
Click “sample information” icon, edit “sample information “ table, definite ‘sample name of the
experimental data file.
e) Standard sample analysis
After running the chromatograph table, input the standard sample. Click once “Start “ button, the
program automatically proceeds to the data acquisition on the spectrogram. And mark the RT(min)
of the peak. When all of the peaks are appeared, manually stop running the program or set “stop
time’ in the “integral parameter” table to auto-stop.
f) Sample analysis
Input the unknown sample. Its procedure is as same as the procedure 5 (this step can be made at
last). The data file (spectrogram) acquired by procedures 5 and 6 is automatically saved by the
system. The data file will be auto-continuously be named, such as: Test001.dat, Test002.dat,
Test003.dat…….
g) Draw calibration curve
a Edit Quantitate parameter
Click “Quantitate parameter” icon, appear the method edition window:

Set “quantitative mode”, “quantitative method (single point internal standardizationization)”,


“standard (select ‘1 point’), “power” , “ unit of standard sample concentration” and “unit of
sample result”.
b Fill in component table
Click “component table” icon, appear the method edition window, fill in name of sample
constituent analyzed, RT(min) and standard concentration.

54
Chromatography Data Handling System

Respectively fill in the component and corresponding RT (min) and standard concentration at
each point, etc. During the analysis of the internal standardization the internal standardization
can not input the compound name while input “internal standardization” so that the system
can identify the internal standardization. See the below figure when the cursor moves to
“internal standardization”. If the concentration of the internal standardization in the standard
sample is equal to the concentration of internal standardization in the unknown sample as
shown in the above figure, Input “1” for the standard concentration of the internal
standardization, also input “1” for the internal standardization quantity of the sample
(describe it later).

Explanation:
In the operation procedures of single point internal standardization, “internal standardization
quantity, sample quantity” are available for filling in, this method can be only provided with our
instrument as show in the above figure.
Pay attention to the unit.
a Assume x to be some of constituent to be analyzed, add the internal standardization to
be ‘1’, so the concentration in the standard sample and the sample to be analyzed are all
0.01mg/ml。

55
Chromatography Data Handling System

Therefore, the standard concentration of the internal standardization and the internal
standardization quantity of the sample all input 0.01, or both input 1 as shown in figure:

b If the concentration of the internal standardization is not as same as the concentration in


the standard sample and the sample to be analyzed as shown in the following figure, the
concentration of the internal standardization in the sample is 0.01mg/ml while the
concentration in the sample is 0.015mg/ml.

Therefore, input 0.01 to the standard concentration of the internal standardization, input
0.01 into the standard quantity in the sample as shown in the following figure:

NOTE: The internal standardization quantity parameter for the specific concentration is only
required to be input only when the concentration of the internal standardization are the same
for all of the sample and not the same as the internal standardization concentration used in
the standard sample. When the internal standardization concentration are as same as the
concentration in the standard sample and the sample to be analyzed, it is OK only for
inputting 1. During the internal standardization quantity, it is used as the divisor for
calculating the constituent content.
a Definite calibration curve

56
Chromatography Data Handling System

h) Look over the sample result


Enter into “Re-analyze” page, click “open data file” icon, appear “Select Data File” window
to select the sample spectrogram file. After that, click once “re-calculation” button, again click
“screen report” icon and see the screen report table:
9) Printing report

5.4Normalization method
The operation method is basically as same as the external standardization without making the
calibration curve. After finishing the experiment during the normalization method, directly click
“report” button to observe the experimental result.
The procedures are as follows:
1) Start the chromatograph and software of work station.
2) Edit the data and record the parameters
Click once” data recording parameter” icon, appear the method edition window, set the
position for saving the file.
3) Edit integral parameter
Click once “integral parameter” icon, appear the method edition window, here set the integral
parameter value and edit “time program table”. Press the acquiesce value if it is not set.
4) Edit “ sample information” table
Click “sample information” icon, edit “sample information” table, definite ‘sample name’ for
the experimental data file.
5) Sample analysis
After running the chromatograph table, input the standard sample. Click once “Start “button, the
program automatically proceeds to the data acquisition on the spectrogram. And mark the RT(min)
of the peak. When all of the peaks are appeared, manually stop running the program or set “stop
time’ in the “integral parameter” table to auto-stop. The data file (spectrogram) acquired is
automatically saved by the system. The data file will be auto-continuously be named, such as:
Test001.dat, Test002.dat, Test003.dat…….
6) Look over the sample result
a Edit “Quantitate parameter”
Click “Quantitate parameter” icon, appear the method edition window:

Select the selecting items of “quantitative method” to be used, “quantitative method


(normalization method”, “standard” and “power”, it is not necessary to make any of the

57
Chromatography Data Handling System

selection in this method.


b Fill in component table
Click “component table” icon, appear the method edition window, fill in name of sample
constituent analyzed, RT(min) and standard concentration. See the below figure:

i. After filling in the component table, click “screen report” button, so observe the
normalization analytical result.
5.5Calibrating normalization method

The other operation procedures are basically as the same with the external standard
without making the calibration curve. In the normalization method, correction factor
for each constituent should be filled in the component table, and then directly click
“report” to observe the experimental result after completing the experiment. See the
following figure:

58
Chromatography Data Handling System

Appendix Ⅰ Technical terms


Integration
In the chromatography program of work station, use “integral parameter” to decision-making
integral event at some of peak or some of zone in the work station of the chromatograph. The
following instance demonstrates in details for every integral parameter how to influence the
integration of displayed spectrogram. Use “manual integration treatment” to make the
re-integration of some zone or some peak in the spectrogram.
When re-integration is made for every time, both peak width and slop are very important
parameters. These parameters are used to determine starting, ending and changing of the peak and
identify the real peak from the noise.
Peak width
When the work station is running, a data point will be acquired for the analysis at a interval of a
certain period. In general speaking, 20 more acquired points should be secured on a whole
chromatograph peak in order to obtain the measuring accurate of the peak. Set “peak width”
parameter to control the time space of data acquisition point. When “peak width” value is equal
or less real half peak width, 20 more acquisition points on a whole peak can be secured. Identify
if the setting of “peak width” value is correct according to the following example.

Decrease peak width Peak width Enlarge peak width

Too small peak width Proper peak width Too big peak width
Slop
It is first derivative. This makes the integral calculation to identify starting and ending of the peak
from the baseline noise and drift. The following example shows how to influence the starting and
ending points if the peak width and slop are displayed incorrectly.
Decrease slop slop Increase slop

Too

59
Chromatography Data Handling System

small slop Proper slop Too large slop

Partition
The unit of the partition value isμv/s in the work station of chromatograph.

As shown in the above figure, θ is an angle value of initial point, assume “α” to be the
partition value to be set in the integral parameter.
When:θ>α, A and B is treated as non-separated peak, so the non-separated peak is
vertically separated.

When:θ<α, both peaks of A and B are fully separated.

(baseline)drift
In normal operation condition, The following figure shows the response signal curve only
produced by fluid phase slowly orients to change conditions with the time.

1) The baseline drifts upward, the initial point a and landing point b are not symmetry, in this
case, set the drift parameter ( positive value) to make the adjustment until the initial point and
landing point is symmetry.

60
Chromatography Data Handling System

2) When the baseline drops down, this similar situation is also occurred.

The moment, set the drift parameter(negative value) to adjust the initial point and the landing
point of the peak.

Time program
In the LC analysis of equal concentration as shown in the following:

When begging the analysis, appear a sharp peak, there is a width peak appearing for a long time
interval, afterwards again appear a sharp peak. On this occasion, use the time program to design a
time table, set the integral parameter at the different time section. Use the time program set to
repeatedly execute the same treatment. Therefore this peak treatment method is suitable for
cycling analysis.
NOTE:“The definition and setting of parameter and integral parameter in “time program
table” are all the same.
Peak operation
All of the peak treatment, including peak determination, baseline drift correction, the separation

61
Chromatography Data Handling System

for not fully separated peak and measurement of peak area are all finished in the peak treatment
parameter.
This section describes peak treatment parameter and using these parameters to make the peak
treatment.
Peak width
The peak width parameter is very important parameter in the peak treatment parameter. During the
integration, the program will be proceeding under the optimal condition according to the peak
width value. The setting of the half peak width can be little bit narrower than the minimum narrow
width of the peak in the spectrogram. The unit is S.

Peak width and deleting the peak undetermined


The noise peak width is normally less than the peak, therefore, if the peak width is set to the
minimum peak width (at half peak height), so the peak width set less peak is deleted as the noise.
Slop
The parameter used to set the peak slop is called “slop” (also called sensitivity of peak
determination). When the slop value is big, the sensitivity of peak determination is decreased;
When the slop is small, the sensitivity of peak determination is high and determine the peak width.
As shown in the following, use the signal slop to determine the peak. When the slop exceeds the
slop value pre-set,the initial point of the peak is determined. When the slop is less than the slop
value pre-set, the end point is arrived in consideration.

Minimum peak area(height)


This parameter can not be directly influence on the peak treatment. After executing an ordinary
peak treatment according to peak treatment parameter, if the peak area (or peak height) is less than
this value, eliminate this peak from the treatment by the following situation:
1) No display for the RT(min) in the spectrogram treatment.
2) Screen report
3) Quantitative determination (or correction calculation).

62
Chromatography Data Handling System

APPENDIX Ⅱ Usage of multiplier and divisor


During running work station program of chromatograph, in the re-analysis of single sample,
“multiplier” and “divisor” should be available for filling in. The parameters of RT(min), area and
peak height of the chromatography peak are all saved during the acquisition data of the work
station. After the integral calculation for these data, the report result will be displayed or output.
When the sample is analyzed by an analyst, in the most cases, the unit of the analytical sample
result is normally as same as the unit of the concentration. Such as: In the section of 4.1---external
standard operation procedures, the unit of standard sample concentration is mg/ml. If
“multiplier ”,”divisor” are not used any more (or multiplier and divisor values are all ‘1’), the
result unit should be also :mg/ml. But sometimes the analyst requires the unit of the report result
not to be the same as the unit of the analytical sample. If “multiple” and “divisor” are not set, the
conversion should be again made manually after the screen result is displayed, such as: Assume
that the result unit is g/ml,proceed the calculation manually:
mg
× 1000 = g/ml
ml
If set “multiplier=1000” in the program, directly obtain the report result to be g/ml, so it saves the
time of obtaining report result and directly print out the result by the printer.
As example for determining the content of tanshinone II in drug of “composite salvia miltiorrhizae
tablet ” as follows:
1) Preparation of working standard sample (single point):
Weigh 9.8mg working standard sample, dilute it to 50ml; again take 5ml from it and dilute it
to 25ml (equal to dilute it to 250ml), the calculation is : 9.8mg/250 ml = 0.0392mg/ml 。
2) Sample preparation:
Weigh analytical sample to be analyzed (tablet) of 1.0007g and dilute it to 25ml. The single
drug weight has been know: 0.24g.
The single point external standardization is used for the following description.
Operation procedures are as follows:
1) Start the chromatograph and software of work station.
2) Edit data, record parameter.
Click once “data recording parameter” icon, appear the method edition window, set the
position of file storage.
3) Edit integral parameter
Click once “ integral parameter” icon, appear the method edition window, here set the
integral parameter value and edit “time program table”. Press the acquiesce value if it is not set.
4) Edit “ sample information” table
Click “ sample information” icon, edit “sample information” table, definite ‘sample name’ for
the experimental data file.
5) Standard sample analysis
After running the chromatograph table, input the standard sample. Click once “Start “ button, the
program automatically proceeds to the data acquisition on the spectrogram. Stored file name is
Test026.dat. the RT(min) of the peak obtained is 14.34min.. When all of the peaks are appeared,
manually stop running the program or set “stop time’ in the “integral parameter” table to auto-stop.
6) Sample analysis

63
Chromatography Data Handling System

Input known sample, the program automatically save the file name: Test027.dat. the RT(min) of
the peak obtained is 14.44min.
7) Draw calibration curve
a Edit Quantitate parameter
Click “Quantitate parameter” icon, appear the method edition window:

Set “quantitative mode”, “quantitative method (external standardization)”, “standard “(8 pieces of
concentration standard sample, select ‘8 point’), “power” , “ unit of standard sample
concentration” and “unit of sample result”.
b Fill in component table
Click “component table” icon, appear the method edition window, fill in name of sample
constituent analyzed, RT(min) and standard concentration.

b Definite calibration curve


Click ”calibration curve” icon, appear the method edition window, definite well the
standard point file, and then click “ re-correction” button to obtain the calibration curve.

64
Chromatography Data Handling System

8) Call the sample file required on the “re-treatment” page:

9) Click “report” button to observe the report result.


As shown from the screen report, the concentration of the tanshinone II in the sample
is:0.0637 mg/ml. But it is not the content of the tanshinone II in a single tablet, therefore, calculate
it manually at this step.
Mass of the tanshinone II:0.0637 mg/ml ×25 ml = 1.5925 mg (or content of tanshinone II
in the drug sample weighed 1.0007g )
The content of tanshinone II in a single tablet:(1.5925 mg×0.24g/片)/ 1.0007 g = 0.382
mg/tablet.
But, if a big amount of sample are analyzed, the manual calculation is required for each
sample at this step. So it is not very convenient for the operator. Therefore, “multiplier” , “divisor”
are set in the program to solve this problem. During inputting “component table”, only to input
sample weight of control sample is required, while the analytical result obtained is still mg/tablet.
In this case, the report result can be obtained only by one step analysis without manual calculation.
10) Using “multiplier” and “divisor” as follows:
a Firstly determine the values of “multiplier” and “divisor”(its function is to converse
the result unit).
All calculating procedures mentioned above are described like:
1. Calculate the concentration of control sample:9.8mg/250 ml = 0.0392 mg/ml.
2. Mass of tanshinone II : 0.0637 mg/ml×25 ml = 1.5925 mg

65
Chromatography Data Handling System

3. Content of tanshinone II in the single tablet:(1.5925 mg×0.24g/片)/ 1.0007 g =


0.382 mg/tablet.
NOTE: At the step 3, the concentration of standard sample in the component table is:
0.0392 ,unit: mg/ml; unit of sample result: mg/ml. Now it only require to input weighting sample
of the control sample while obtaining the unit of the sample result is mg/tablet, or the standard
concentration: 0.0098 (9.8 mg) unit: g ; unit of sample result: mg/tablet.
“unit of standard sample concentration “ in the step 1 aforementioned hs been changed, from
mg/ml to g. The multiplier during the conversion is 1000,divisor is 250.
The result unit in aforesaid step 2 and 3 have been also changed from mg/ml to g/tablet. The
multiplier during the conversion is 25 X 0.24, divisor is 1.0007.
Multiplication by all of the multipliers and divisors to obtain:
1000 × 25 × 0.24 240
=
250 × 1.0007 1.0007
So that, in the re-analysis window of single sample, respectively set the multipliers and
divisors to be:240,1.0007.
b Definite “Quantitate parameter”
c Click “edit current method” button, appear the method edition window, and then
repetitively edit ““Quantitate parameter”, “component table”.
1. Edit ““Quantitate parameter”. Among them, “ unit of standard sample
concentration is changed from “mg/ml”to“g”, so the data value on the standard
concentration bar in the “component table” should be in the unit of “g”. “unit of
the sample result is“mg/tablet”.

2. Fill in “component table”. The value on the standard concentration bar should fill
in “g”, or 0.0098 (unit is g).

66
Chromatography Data Handling System

3. Edit “current sample information table”. Click this icon, appear the edition window,
and then here input the values of the multiplier and the divisor. The multiplier is
240, and the divisor is 1.0007. “Enter”.

4. Click “re-calculation” button, the system proceeds re-calculation to the values (note:
Click “re-calculation” button after changing the Parameters of method file and
information file for each time, so the changed command will transfer to the internal
memory, in this case, the system can be able to perform the re-treatment to the
data.).

5. Click once “screen report” button, obtain the content of the sample result,
or Content of tanshinone II in the single tablet is 0.3821 mg tablet,directly obtain
the result unit is mg/ tablet. The obtained result is in accord with the result obtained
by the manual calculation.

67
Chromatography Data Handling System

APPENDIX III Flow chart of data processing

Explanation:
1) The contents in method file:
Integral parameter, time program table, Quantitate parameter, component table, calibration
curve, report format, method information, data recording parameter and screen plotting
parameter.
2) There are four modes for calling the method file (as shown in dotted line frame).
① Load Method
Select the method file from the saved method files.
② Open the method from the data.
Select the same method file used foe some sample analysis to make the experiment.
③ Open the data file
Use this function to open the spectrogram file on ‘re-analysis’ page. It only calls the data
information out of the method file from the data file, or there is no method file, so the user
can again select the method file of the analysis result of treating sample. NOTE: The
acquiesce method file on the ‘reanalysis’ page is still the current method file of the main page
of the work station.
④ Open the data file at the same time open the method.
Use this function to open the spectrogram file on ‘re-analysis’ page, it is able to call it
with original method file.
3) Data processing
A、If the original method file used before is required to make the experiment, click “file” menu on
the main page before the experiment, here select opening the method file mode(‘Load Method’

68
Chromatography Data Handling System

or ‘Load Method from data’), and select the method file.


B. If the original method file used before is not required to make the experiment, while using
self-definition method file to make the experiment. Open the method edition window, set ready
parameters for each method file (if the same item is made for a long time, that means the method
used is as same as the method used last time, no need to to set the parameters of the method file
again since we have already set ‘working parameter last time’ item on “parameter calling mode”
window. Afterwards, again open ‘sample information’ window to make the edition. The analysis
can be started after completing these operations.
C. After finishing the experiment, the experimental result is saved as a data file, this data file
includes method file, sample information, spectrogram and result data.
D. If the user wants to make “re-treatment” for the sample analysis result, firstly enter into
“re-treatment” page, then call the spectrogram, here select the original method file, also available
to select again the other method file to make re-treatment (“open data file” under the acquiesce
situation).
E. After determining to make the method file of the re-analysis, click “re-calculation” button to
make the selected method file used by the spectrogram the re-treatment (No matter the calling
mode of the method file is selected for any mode, this operation procedures are required).
F. The sample analysis after the treatment can obtain a new data file.

69
Chromatography Data Handling System

APPENDIX Ⅳ Each component in paste report to word/wps file


For example:
Step 1: Enter (custom report) the page of editor as follows:

Step 2: Click once “ open data file” icon, appear the window below:

Select the data file in the path frame, then “Enter” to call the data file.
Step 3: Click once “open template” icon, appear the following window:

Select the template file, press “ Enter”, the report format following assigned template output
can be observed on the edition window. Each component here can be still edited until the
appearance is satisfied by yourself.

- 70 -
Chromatography Data Handling System

Step 4: If the spectrogram is required to be pasted, use the right key of the mouse to click the
spectrogram, appear the menu:

Step 5: Select “copy to cut and paste plate”.


Step 6: Enter word/wps file required to be edited, move the mouse to the position of the
spectrogram, see the below:

Step 7: At last, click once “ paste “item on the “edition” menu, so the spectrogram is paste to
word/wps file. As shown in the following:

- 71 -
Chromatography Data Handling System

Step 8: In word/wps file, the edge frame of the spectrogram can be enlarged or shortened
until the appearance is satisfied. See the below:

- 72 -
Chromatography Data Handling System

《Content determination of tanshinone IIA》——Only for reference


1、Linear relation
Accurately weigh15.omg control sample of tanshinone IIA and put into 100ml brown measuring
flask. Dissolve it with methyl alcohol and dilute it to the scale mark. Accurately suck 0.5, 1.0, 1.5,
2.0, 2.5 and 3.0ml into 50ml brown measuring flask and then dilute it to the scale mark with the
same solvent above-mentioned. According to the aforementioned chromatography condition,
sampling 20μl,make the determination, the regression analysis is made by the integral value A
of the peak area to sampling quantity C of tanshinone IIA. The regression equation:A =4.329×
106 C–3.61×102,r=0.9998, C(μg). That shows tanshinone II A has very good linear relation in
the range of 0.03~0.18μg,and the straight line is passed through the initial point. The single
point external standardization is used to proceed the determination and calculation. The data of
the linear relation and standard curve and spectrogram are as shown in the following figure:

Fig.6 HPLC chromatogram of control sample of tanshinone IIA

Fig 7 Standard curve diagram of tanshinone IIA


Table 3 Data of linear relation
Sampling Mean integral
Integral value of area peak
quantity(μg) value
0.03 129014 129664 129454 129377
0.06 256231 256096 255333 255887
0.09 385688 384743 419272 396568
0.12 515600 513454 516965 515339
0.15 646779 646757 645911 646482
0.18 779687 779828 778720 779412
2. Testing stability
Take sample control solution and sample solution aforementioned for each, respectively, put aside
for 0, 1, 2, 4 and 6 hours,then start the operation. sampling 20μl,determine the peak area,

- 73 -
Chromatography Data Handling System

calculate the relative standard deviation of the peak area of tanshinone IIA. See table 5. That
shows the stability is good. The control sample and the sample to be determined are shown in
fig.8 and 9.
Table 5:Test stability Integral value of average peak area at different time (n=3)
Mean
Sample 0h 1h 2h 4h 6h RSD(%)
value
Control
416150 417508 419094 418871 417723 417869 0.28
sample
Tested
375217 375327 374026 367780 372439 372958 0.83
sample

Fig. 8 HPLC chromatogram of control sample Fig.9 HPLC chromatogram of tested sample
of tanshinone IIA of returning tanshinone medicine
which is to be taken after being
mixed with boiling water.
3. Test for recovery ratio of adding sample
Accurately weigh known content sample properly, again respectively add a certain amount of
control sample of tanshinone IIA, the operation is made by the determination items of the tested
sample. Calculate the recovery ratio, see the table 7. That shows the recovery ratio is better.
Table7:Test for recovery ratio of adding sample
Average
Sampling Original Adding Determined Recovery
recovery RSD
№ amount amount amount amount ratio
ratio (%)
(g) (μg) (μg) (μg) (n%)
(%)
1 5.1009 52.0 50.0 100.32 98.32
2 5.3822 54.8 50.0 103.67 98.83
3 5.5118 56.2 50.0 105.33 99.17 98.53 0.73
4 5.3229 54.2 50.0 101.53 97.35
5 5.4565 55.6 50.0 104.56 98.97
4、Confirmation of minimum determination
Suck the control sample solution, dilute it to 0.1μg/ml. Make the determination as the operating
method mentioned above, determine the peak area, calculate the minimum determining amount
to be 2ng (signal to noise: 5:1), see figure 7.

- 74 -
Chromatography Data Handling System

- 75 -