Professional Documents
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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
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
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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
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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.
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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.
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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
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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 “
P D
On this page, click once “P” or “D” button to appear the following page:
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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
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D. “ ”,is column.
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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:
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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:
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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:
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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.
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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:
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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:
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Peak icon at the right down angle of the window is pre-observation button of the spectrogram file,
see the following figure.
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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.
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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.:
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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:
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:
After clicking with the left key of the mouse, the starting point is changed as shown in
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figure below:
d. And then again click “adjust landing point” as shown in figure below:
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:
f. After clicking it with the left key of the mouse, the landing point is changed, the
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spectrogram of the re-integration is again appeared on the page as shown in figure below:
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:
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.
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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.
Quantitate parameter(Ctrl+D)
Display the dialogue frame of the Quantitate parameter.
Integral parameter(Ctrl+J)
Display the dialogue frame of integral parameter.
Constitute table(Ctrl+I)
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Display the dialogue frame of constitute table edition. Edit compound name required to be
analyzed, etc.
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
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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.
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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:
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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.
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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:
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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:
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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::
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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.
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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
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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:
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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.
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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:
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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:
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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:
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”.
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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:
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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:
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Chromatography Data Handling System
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
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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.)
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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. :
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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. :
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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.
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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.
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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.
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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:
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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.
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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
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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:
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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
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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:
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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。
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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:
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
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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:
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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:
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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
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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.
(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.
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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
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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.
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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.
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2. Fill in “component table”. The value on the standard concentration bar should fill
in “g”, or 0.0098 (unit is g).
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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.
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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’
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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.
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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 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:
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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:
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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.
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