C hrom S tar 7

Transcrição

C hrom S tar 7

ChromStar
Manual
7
ÿþýü
Software für Chromatographie
und Prozess-Analytik GmbH
Am Weidufer 32, D-28844 Weyhe
Tel.: 0421-802806, Fax: 0421-890346
E-mail: [email protected], Internet: www.scpa.de
Edition May 2012 for ChromStar 7
Version 7.0.12
ChromStar 7 - Contents
Page i-1
____________________________________________________________________
Contents
1.
Page
Introduction ......................................................1
2.
2.1
2.2
Installation ........................................................3
Setup .................................................................3
A/D-Converter ....................................................5
3.
3.1
3.2
3.3
3.4
3.5
3.6
3.7
Configurator .....................................................7
Creating a Configuration ....................................7
General Properties.............................................8
Documentation Setup.........................................8
Calculation Setup...............................................9
Plug In................................................................10
Data Sources .....................................................11
Device Control ...................................................14
4.
4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8
Method and Sequence .....................................18
Information/General ...........................................20
Information/Documentation ................................22
Channel1/Analysis .............................................23
Channel1/Analysis/Calculation...........................25
Channel1/Peak Table.........................................27
Time Table .........................................................31
Sequence...........................................................33
Calibrations ........................................................38
5.
5.1
5.2
5.3
5.4
Data Recording ................................................42
Chromatogram ...................................................45
Control Panel .....................................................47
DAD Data Recording..........................................49
Spectrum ...........................................................49
6.
6.1
6.2
6.3
6.4
6.5
6.6
Reprocess.........................................................51
Reintegration......................................................54
Manual Integration .............................................59
Reprocess - Calculation .....................................60
Reprocess – Method ..........................................60
Reprocess – Navigation Bar ..............................60
Reprocess - DAD ...............................................61
Page i-2
Contents – ChromStar 7
________________________________________________________________________________________________
6.7
6.8
Column Coefficients .......................................... 63
Chromatogram Transformation.......................... 65
7.
7.1
7.2
7.3
7.4
7.5
7.6
7.7
7.8
7.9
7.10
7.11
Batch Wizard.................................................... 67
Print ................................................................... 68
Reintegration ..................................................... 68
Recalculation ..................................................... 69
One-Point-Calibration ........................................ 70
Calibration with Calculation of the Average ....... 70
Multi Level Calibration ....................................... 71
Summary Table ................................................. 72
Compare Chromatograms ................................. 73
Convert Data ..................................................... 76
Scattering Calculation........................................ 76
Result Export ..................................................... 77
8.
8.1
8.2
8.3
8.4
8.5
8.6
8.7
8.8
8.9
8.10
Report-Editor ................................................... 78
Basic Items........................................................ 80
Chromatogram................................................... 80
Sample Information ........................................... 82
Method Values................................................... 82
Channel-Dependent Values............................... 83
Results .............................................................. 84
Page Status ....................................................... 85
DAD Items ......................................................... 85
Graph Items....................................................... 86
Runtime Parameters.......................................... 86
9.
Navigator .......................................................... 87
10.
User Administration ........................................ 89
11.
11.1
11.2
11.3
11.4
Working with ChromStar 7.............................. 91
How do I create a print template? ...................... 91
How do I record a Chromatogram?.................... 95
Methods for a Quantitave Evaluation ................. 99
Statistic Formulae.............................................. 104
ChromStar 7 - Introduction
Page 1
____________________________________________________________________
1. Introduction
The chromatography data and control system ChromStar 7 is used to
record and analyse chromatography data and – when linked to an
appropriate interface – to control chromatography equipment such as
solvent pumping systems, UV detectors and autosamplers. If a suitable
diode array detector is installed with the corresponding card, data from this
device can also be recorded and analysed.
The ChromStar data system is made up of several modules with the
following functions:
The Configurator is used to configure the devices in a
chromatography system.
The Navigator supports the graphic display mode used when
carrying out analyses and processing the results data.
The Sequence module determines how the samples will be
processed. A sequence and the methods named within it are an
absolute requirement for recording and analysing
chromatograms and DAD data.
The Method module is used to define or edit the method required
to record and analyse the chromatograms. The method contains
all the parameters needed to control the equipment.
The Data Recording module controls the devices and the
recording of chromatography data from a UV detector or similar
detectors, or from a DAD detector.
The Reprocess module allows you to present and process
chromatography data saved from DAD data and spectra in
different ways.
The Batch Wizard guides the user through calibrations,
presenting series in table form and making printouts, enables
the quantitative analysis of chromatograms, the presentation and
reprocessing of a group of chromatograms as well as many more
features.
Page 2
Introduction - ChromStar 7
________________________________________________________________________
The Report Editor creates printable versions of methods and
results data.
The User Administration tool determines access privileges. It
enables the administrator to register users and set passwords.
The Visualisation Editor generates an image of the
chromatography system that appears in the control window of the
runtime module and shows all the relevant device parameters.
The Skin Editor allows you to determine the graphic
presentation of the chromatograms.
ChromStar 7 - Installation
Page 3
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2. Installation of ChromStar 7
2.1 Setup
(applicable to version 7.0.2 and beyond)
Recommended operating system: Windows 2000, Windows XP or Windows
Vista. Windows Vista 64 is not yet supported as standard, although SCPA can
make the required files available on request. However, routine use is still a
little arduous at present, since the ChromStar A/D converter drivers are not
yet digitally signed and these drivers need to be loaded explicitly using the F8
key every time the system is started up.
Program installation: place CD in the CD drive and wait for the ChromStar
installation CD start-up screen to appear.
If the automatic start-up of programs from a CD is deactivated and the screen
does not appear, double-click on the content of the CD and open ‘Setup.htm’
by double-clicking on the file.
The CD’s start-up screen reads:
ChromStar 7.0
Chromatography Data System
Click once on this title to start the set-up program. If you are unable to do this
due to your Internet Explorer security settings, open the folder ‘ChromStar7’
on the CD and run the application ‘ChromStar 7.0.2 Setup.exe’ that you will
find there.
When using Windows 2000 or Windows XP as your operating system,
installation may be interrupted by an error message stating that the ‘.Net 2.0Framework’ is not available. If this happens, re-open or re-activate the set-up
screen and select ‘.Net Framework’. An installation program for the Microsoft
program environment will start up and install the Microsoft .Net Framework
2.0. Once this has been installed, you can recommence the installation of
ChromStar 7 (see above).
Preparation for opening the ChromStar 7 program for the first time:
If you have not yet done so, you should now install the A/D converter card(s)
and the required plug-n-play driver(s). The instructions can be found in the
ChromStar manual or at www.scpa.de/download.html, and the required drivers
are on the CD in the ‘Driver’ folder and on the same website as the
description.
Once ChromStar is equipped with A/D converter cards, the licence file needs
to be copied from the folder on the CD called ‘Licences’ into the licence file in
the ChromStar program folder. The licence file has the extension .lns, and the
Page 4
Installation - ChromStar 7
_____________________________________________________________________________________________________________
ChromStar program folder is called ‘C:\ChromStar7,’ unless another folder
name was entered during installation. If you intend the program to control a
diode array detector, you also need to copy the file called ‘SCPACFG.USR’
from the licence folder into the ChromStar program folder.
After installation, a shortcut to the ‘Chromstar7 Navigator’ will appear on the
desktop.
Double-clicking on this symbol will start the navigator. The first time you start
the program, you will receive a message saying that you have not yet
configured any devices, and you have the option to begin device
configuration. Select a name for the configuration that describes your analysis
device. The device configurator will start, allowing you to set the parameters
required for that device and register the components present such as pumps,,
autosamplers and detectors. Details of this process can be found in the online
help and the manual.
When configuration is complete and the configurator has been closed, the
navigator will appear. The navigator is a simple program that will help you to
start the different components of ChromStar.
Activating the licence: a licence file is required in order to use the data
recording module. This file includes both the user name and the serial number
of the A/D converter card. As a rule, ChromStar is supplied with an active
licence file. However, there are cases where this is not possible, for example
when updating an older version or when several systems are supplied as a
package. Non-activated licence files need to be activated using the internet. If
necessary, this should be done the first time you start up the data recording
module. This process is automatic: the serial number is entered in the licence
file and transferred to a database administrated by SCPA. If it is not possible
to connect your computer to the internet, you can activate your licence file by
informing SCPA of your serial number, for example by telephone. To read the
serial number, you can download the program ‘PCI Test’ from
www.scpa.de/download.html.
ChromStar is now ready for use!
ChromStar 7 - Installation
Page 5
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2.2 PCI A/D converter card and Ethernet box
2-channel PCI A/D converter card
Arrangement of plugs
.
ChromStar Integrator 1-channel PCI A/D card
.
Page 6
Installation - ChromStar 7
_____________________________________________________________________________________________________________
ChromStar Integrator 2-channel PCI A/D card
1
-- channel 1
2
+ channel 1
3
-- channel 2
4
+ channel 2
5
empty
6
empty
7
Start 2
8
GND
9
Start 1
10
GND
Ethernet box LC 421E
Back view with connections and arrangement of plugs for the A/D
converter card
ChromStar 7 - Configurator
Page 7
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3. Configurator
The Configurator module is used to configure the system. This process
includes selecting a data recording channel on one of the A/D converter
cards, setting up a diode array detector, interfaces to control pumps,
sample generators, detectors and additional devices and the configuration
needed for documentation in the method to be set up later.
As an
alternative to the A/D converter card, a two-channel A/D converter with an
RS232 interface for device control and its own IP address can be operated
using an Ethernet. Set values are determined for various parameters to be
used in data recording and analysis as well as integration, quantitative
calculations and the transmission of column coefficients. A configuration
can be given a name and linked to an image. This means it can be called
back up at any time.
When you click on the link to the Configurator module, a
window appears in which an existing system configuration
can be opened by clicking on the image in the menu bar on
the left and then editing as required. On the right-hand side of the same
window there are screen buttons to save, reload or delete the selected
configuration. A new device configuration can be created using Create
configuration. For graphic-driven system configurations, the buttons with
a green background each open the corresponding module. The Add Data
Sources button takes you to the Add Data Sources window, in which you
first select the data source and then set the accompanying parameters.
The Add Device button opens a window in which you can select devices.
The top left button leads to the General Properties, the next button to the
Documentation Setup for documentation in the method file and the right
button leads to the Calculation Setup. The Add a Plug-In button allows
you to link up additional programs. The Device Wizard button indicates
which device is linked to which interface.
3.1 Creating a Configuration
The screen button Create Configuration creates a new
system configuration. The configuration file contains
the extension *.ccf. The name of the new configuration
file is entered under Configuration File Name.
Above that, an image can be selected for the
configuration by clicking on the existing image and then
selecting an image from the list that appears. It is also
possible to select another image later for a configuration that has already
been saved, using the General Properties option.
In the new configuration data sources, devices and plug-ins can be added.
General properties, documentation setup and calculation properties can be
defined.
Page 8
Configurator - ChromStar 7
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3.2 General Properties
In the General Properties dialogue you can select the
file name of the device image and indicate the file path
(picture path). The image will appear above in the
Instrument picture box.
In the box below, you can select the directory that will
always appear as the default directory when creating a
method or recording data. This directory is the place where the results data
(chromatograms, *.cfd) will be saved.
If a qualification was carried out in the GLP mode the names of the
persons responsible for the IQ (Installation Qualification) and OQ
(Operational Qualification) can be entered. Only the administrator is
allowed do this.
Remote control of the data recording module can now be enabled.
3.3 Documentation Setup
Documentation Headings in the Method
This
dialogue
establishes
the
identifiers of the
individual phases
(groups) and rows
for the documentation in a method
(see page 24). When creating a new
method, you will first be asked
which configuration is to be used.
The documentation page for the
new method will be filled in with the
corresponding identifiers of groups
and rows entered there. Next, the
exact
specifications
of
the
chromatographic conditions will be
entered in the new method.
You can begin configuring the
documentation by clicking on Add
Group. The dialogue appears at the
top of the window to enter the group
name, with boxes below for the row
identifiers.
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Add Group can be repeated as many times as necessary until all the
specifications required to describe the analysis are present.
3.4 Calculation Setup
The Calculation Setup button allows you to pre-set various parameters for
different calculations in ChromStar.
Calibration mode
Exclusive, all standard substances are mixed in a solution
(default setting).
Additive: enables single point calibrations with standard
substances that must not be mixed and must therefore be
recorded on separate chromatograms.
Alternative peak table
parameter
Zero area (for the standard addition method) (default setting).
Recovery: takes into account a percentage recovery in the
peak table of the method file under Recovery (%). The
calculation method must be External Standard.
Calculate peaks in
window
All peaks in window: all the peaks are calculated in one peak
window (default setting).
Only highest peak: highest peak is calculated.
Only middle peak: the middle peak is calculated.
First peak in window: the first peak is calculated;
Last peak in window: the last peak is calculated.
List peaks
All peaks: all the peaks are listed in the list of results (default
setting).
Only peaks in windows: only the peaks indicated in the peak
table are listed.
Calculate column
coefficients
Yes: column coefficients are calculated (default setting). The
values can be entered as breaks in the results table.
No: no values are calculated.
Peak width calculation
Standard: the peak width at 10% of the peak height is used to
calculate asymmetry (default setting).
FDA compliant: peak width at 5% of peak height used.
Peak area Calculation
Slice-width-dependent, slice-width- independent or
ChromStar6-compatible.
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Resolution
determination Factor
2 (default setting). A different value can be entered here. This
value is used to calculate the resolution.
Original Method
Documentation
Default setting: None. Choosable: Only Method or Method and
Calibration. The original method appears in Reprocess in the
Navigation bar as additional submenu.
Close the Calculation Properties box by clicking on OK.
3.5 Plug-In
The "Add a Plug-In" button allows you to link up
additional programs. A licence file is required to use
most of the additional programs. Once you have selected
a plug-in, a new dialogue box will appear to set the
various program-specific parameters.
Plug-Ins are:
Alias Autosampler
Sykam Amino Acid
Reaction Module
Chromatography of amino acids with post-column
derivatisation
Conductivity Detector
controls a conductivity detector.
GamePort Start
starts a chromatogram via game port signal (no licence file
required)
Human Autosampler
Device
allows the autosampler table of the sequence to be used
without using an autosampler but injecting manually (no
licence file required)
Injection Cycle
starts data acquisition in time intervals.
Activity Correction
considers the radioactive decay during recording a
chromatogram.
Column Oven
controls a column oven.
Auxiliary Pump
controls an additional isocratic auxiliary pump.
Flow Monitoring
monitors the flow rate of the main pump and starts an
emergency sequence if the pump stops.
Server Transfer
transfers ChromStar data onto a server directory
The Server Transfer Plug-In is active in the background
once it is installed and inserted in a configuration. It moves
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or copies result files from the ChtomStar data directory into a
destination directory, i.e. into a server directory. Moreover
additional files can be copied (not moved) onto the server.
After Plug-In properties have been changed the computer
must be restarted.
In the PlugIn Properties box the following parameters are
defined.
Destination Directory. Name of the destination directory,
select by using the file select button to the right.
Move Result Files. Result files are moved.
Copy Result Files. Result files are copied.
Additional Files.
File Name. Entry of the name including path.
Destination Directory. Name of the destination directory,
select by using the file select button to the right.
Update. After each change in the selected file it is copied
into the destination directory.
Vali Signal Output
allows the validator LC120M with the program Validator 8 to
be used. A chromatogram is transformed via D/A-converter
into voltage signals which are given to the A/D-converter
board.
3.6 Data Sources
To define a data source, click on the green circle with a + sign in it and
check one of the following options in the Add Data Source dialogue box:
AD Board
Data is recorded from the A/D converter
card.
NetBox
The Ethernet Box is used to record data.
DAD
Data is recorded from a diode array detector.
You can check both DAD and either the A/D Board or NetBox so that data
from the diode array detector and the A/D converter card, or from the diode
array detector and the Ethernet box, can be recorded simultaneously.
Simultaneous data recording from an A/D converter card and the Ethernet
box is not possible. Once a data source has been selected, a green circle
will appear on this source and you can enter the A/D card properties, DAD
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properties or Ethernet box properties. In the case of the A/D converter
card, the green circle with the + sign indicates the first channel. Three
further channels can be selected (click on the green box with a + sign). If
the Ethernet box is selected as the data source, the first channel must be
selected with the green circle with the + sign, and one more channel can
be added (click on the green box with a + sign). For each channel,
channel-specific entries must be made in Channel Properties. The green
box with the red cross deletes the channel or data source.
A/D Board Properties / Default Settings
Slice width (ms)
Time in ms after which a new data point is registered
Slope Sensitivity
(µV/s)
Sensitivity in µV/s: maximum slope of the detector signal curve
that is still not considered as the beginning of a peak.
Baseline Max.
(mV)
Level of the detector signal in mV above which no new baseline
is recognised when a slope occurs that is greater than the slope
sensitivity.
Skim Ratio
Relationship between the heights of two neighbouring peaks
Filter Factor (data
points)
Number of points (5, 7, etc.), at which the slope of the detector
signal function is tested to determine the beginning and end of a
peak.
View Range (mV)
Area over which the y axis is displayed on the screen in mV
External stop
active
Allows an external stop when recording the chromatogram, e.g.
by turning the injector from the Load to the Inject position for a
second time .
Hardware serial
number
Serial numbers of the A/D convertor cards installed.
Channel Properties
Channel no.
Channel number (1, 2, etc.)
Name
Channel name
Dimension
Name of the dimension on the y axis, e.g. mV, µF, pH
etc.
Offset
Value of an offset
Factor
Factor by which the detector signal is multiplied
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DAD Properties / Presets
Integration time (ms)
Exposure time of the diode array in ms.
Wavelength
resolution (nm)
Wavelength resolution for saving data in nm
Wavelength range
(nm)
Wavelength range from... to ... in nm of the DAD
Intensity range (mV)
Display area from ... to ... of the intensity axis on the screen
in mAU.
Diodes
Number of diodes of the diode array in the diode array
detector.
Slice Width (Data
point distance) (ms)
Time in ms after which a new data point is registered.
Slope Sensitivity
(µV/s)
Sensitivity in µV/s, maximum slope of the detector signal
curve that is still not considered as the beginning of a
peak.
Max. Baseline Level
(mV)
Level of the detector signal in mV above which no new
baseline is recognised when a slope occurs that is greater
than the slope sensitivity.
Skim Ratio
Filter Factor (data
points)
Number of points (5, 7, etc.), at which the slope of the
detector signal function is tested to determine the beginning
and end of a peak.
Save DAD data
Indicates whether the DAD data set should be saved.
DAD Parameters
Shutter=0, Buffered=1
P0 to P4
Coefficients of the calibration function for the calculation of
wavelengths from diode numbers.
Disable/Enable write
protection
Wavelength range and diode numbers can be changed
when the write protection is disabled. Attention: this is only
necessary when the diode array in the spectrometer is
changed!
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Ethernet Box Properties / Channel Presets
Slice width (ms)
Time in ms, after which a new data point is registered
Slope Sensitivity
(µV/s)
Sensitivity in µV/s, maximum slope of the detector signal curve
that is still not considered as the beginning of a peak.
Baseline Max.
(mV)
Level of the detector signal in mV above which no new baseline is
recognised when a slope occurs that is greater than the slope
sensitivity.
Skim Ratio
Filter Factor (data
points)
Number of points (5, 7, etc.), at which the slope of the detector
signal function is tested to determine the beginning and end of a
peak.
View range (mV)
Area over which the y axis is displayed on the screen in mV
External stop
active
Allows an external stop when recording the chromatogram, e.g. by
turning the injector for a second time from Load to the Inject
position.
Hardware serial
number
-
NetBox 1 URL
Address of the Ethernet box, e.g. http://192.168.0.211/
3.7 Device Control
To register a device for control, click on the green circle with a + sign and
select one of the devices from the box entitled Add Device To Control.
Once a device has been selected, a line to the appropriate device will
appear, symbolising the interface. For controllable devices such as pumps,
autosamplers, detectors and valves, settings and parameters can be set by
clicking on the green box. The green box with a red cross deletes the link
to the device.
The second green circle by the control icon opens a
dialogue box in which the name of a visualisation file
(*.vis) can be selected. This file contains an image of the
system (visualisation) with all the devices and device
parameters. It appears in the data recording module in
the control station window, and shows the system in action with all the
changing device parameters. The file is created using the visualisation
editor.
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Pump Properties
Type
Type of Gradient
Port
Number of the RS232 interface
Address
Device number
NetBox Port
Use Ethernet box port
Driver
Name of a driver file if required (*.dll)
Presets
Default pump parameters: these can still be changed in the
method on the time table page.
Minimum pressure
Minimum pressure limit in bar
Maximum
pressure
Maximum pressure limit in bar
Flow rate
Flow rate in ml/min
Cycle time
Parameter that depends on the type of pump installed.
Additional
parameters
You can define extra optional parameters here. If they are
programmable, they will appear as extra functions in the time
table of the method file.
Isocratic Pump
The control panel in the runtime module only allows start and stop
of the solvent delivery.
Record pump
pressure
The pump pressure is saved in the *.cfd file as an extra data trail.
Confirm manual
solvent change
In the runtime module a question appears when the solvent is
changed.
Equilibration Stop
Time=0: no equilibration stop is carried out. If a time is entered
the pump stops after this time in case a sequence was loaded but
no analysis was started meanwhile.
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Autosampler Properties
Port
Address
Device number
NetBox Port
Driver
Presets
Depends on the type of autosampler installed
Loop volume
Injection volume
The injection volume can be changed later in the sequence
according to the type of autosampler.
Maximum volume
Maximum injection volume
Maximum injection
number
Maximum number of successive injections from one vial.
No vial action
Actions to choose from when a vial is missing: next vial or stop
the method.
Optional parameters
Detector Properties
Port
Address
Device number
NetBox Port
Driver
Presets
Default detector parameters: these can still be changed in the time
Wavelength
Range
Optional
parameters
programmable, they will appear as extra functions in the time control
file.
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Extra Device 1, 2, Valves
Addressing
Port
Address
Device number
Use Ethernet box
port
Driver
Name of a driver file (*.dll)
Driver parameters
Extra device
Device name
Name of the extra device
Extra parameters
Page 18
Method - ChromStar 7
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4. Method and Sequence
The method contains all the parameters required to record chromatography
data on a collection of tab windows. A sequence is also required to carry
out an analysis: this contains the order in which the vials will be processed
when using an autosampler or the order of injections when manual
injections are used, and the name of the method to be used for this
sequence (see section 4.7, page 32).
The Information/General tab contains general information; the results file
name, data recording parameters and the option to use a time table.
On the Information/Documentation tab, you can enter options for the
documentation of the chromatography method.
The Channel1/Analysis tab contains data recording parameters and
parameters for integration and analysis. When using more than one
channel to record data, further tabs will appear entitled Channel2 and so
on. The Calculation box contains options for the type of calculation, such
as percentage calculation or quantitative analysis with an external or
internal standard, options for calculating column coefficients and calibration
options.
The Channel1/Peak Table tab determines the peaks to be analysed.
The Time Table contains the sequence of functions for the HPLC pump or
other controlled devices in the HPLC system.
After you have clicked on the link to the Method module,
logged in using the user login screen and selected a
configuration from the icons representing the configurations
available, the Information tab of the method appears. An existing method
should only be loaded with the configuration that will be used with it in the
system. The suggested results file name (*.cfd) is the method name and a
sequential number.
The method is saved under File, Save As... A name should be
entered and the file will automatically be given the extension *.met.
This allows you to prepare the method for data recording with the
existing parameters (runtime 5 min, percentage calculation, no
autosampler, no device control) with the minimum of effort.
ChromStar 7 - Method
Page 19
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The menu points and screen buttons have the following functions:
File
New closes the method that has been loaded and resets all entries.
Open opens the window showing your saved files. You can
select a new method to load.
Import. The time table, the acquisition parameters, the evaluation
parameters or the documentation can be imported from an existing
method.
Save saves the entire method.
Save As... saves the entire method under a new name.
Save & Sign (only accessable in the GLP mode) saves and signs
the whole method (General Signature), the peak table (Peak
table signature) or the time table (Time table signature). The signature
is documented in the audit trail.
Audit trail Comment. This function key allows a comment to be
entered in the audit trail.
Export. The method can be saved as an *.html or *.xls file.
Print opens a print preview (*.cps) window from which you can
print the method file. Click on the downwards arrow behind
Printer to change the printer used. The default setting is your
computer’s default printer.
Recent files. The most recent methods are included at the bottom of
the list for faster access.
Close Editor closes the method window.
Extras
Method History opens the method history window. View
Options appear on the right of the window for filtering and
cleaning the transcript.
Actualize Documentation. When in the configuration the
documentation has been changed, the documentation of an existing
method can be actualized.
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Calibration
For the direct calibrations the calculation method must be external or
internal standard. The peak table must contain the peaks to be
evaluated (cp. page 35).
Direct One-Point Calibration carries out a one point calibration
immediately, as soon as the calibration chromatogram has been
selected.
Direct Average Calibration carries out an Average Calibration after
selecting the calibration chromatograms.
Direct Multi-Level Calibration carries out a Multi-Level Calibration
after selecting the calibration chromatograms.
Help
Contents opens the online help.
Show Help Question marks are included in the program windows to
provide extra information on the parameters concerned. This feature
can be turned on or off.
About ChromStar 7 Method opens an information box.
4.1 Information/General
This page of the method file contains the following
entries in the Information box:
Inserting the name of the author is optional.
When creating a new method, the current date is
entered automatically. When an older file is called up, its creation date
remains unchanged. The current date is only entered if a change is made
and the file is re-saved or saved under a new name using Save As....
Notes are also optional. They can be used to describe the
method in more detail.
The results file name (*.cfd) can be made up of the following elements:
User defined filename
current date
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current time
method filename
line number of the sequence
author name
sample identifier
auto-increment (serial injection number + vial
number)
Click on the individual elements you wish to include. The serial injection
number begins at 001 for the first injection, 002 for the second injection
and so on. When using an autosampler, the vial number is indicated,
followed by a hyphen and then the injection number. The results file is
automatically given the extension *.cfd.
Existing results data files will not be overwritten during data recording
under any circumstances. If no clear file name is entered when creating
the method (e.g. the same sample identifier for more than one injection), a
warning will appear when the method is called up in the data recording
module and a suitable message indicating the problem will be provided. If
the user clicks on the button to ignore the warning, an existing results file
will be moved to a folder with the same name as the results file, or under
certain circumstances folders will be given serial numbers in order to
effectively prevent results files from being overwritten. The Solve Problem
button opens the method in the method editor directly from the data
recording module. This can be used to change the requirements
appropriately and update the file with the corresponding button.
Acquisition Parameters
Runtime. This is the time during which the
chromatography data is recorded for a run.
(Enter a time between 0.01 and 10000 minutes, in steps of 0.001. The
default value is 5 at a data point distance of 500 ms.)
Slice width (ms) Data point distance. This
parameter indicates the interval in ms (from
10 to 9990, in steps of 10 ms) at which a new data point is to be
registered.
DAD Wavelength Range
If a DAD is selected in the configuration as
a data source, this box will appear. Enter
here the wavelength range in nm over
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which the DAD data is to be recorded and saved.
The time table can be activated.
Click on the time table for device control. An extra tab, Time Table, will
then appear with a table where you can fill in the sequence of functions
carried out by the chromatography devices (see page 30).
4.2 Information/Documentation
This page of the method file allows the user to include
documentation in the method. The names of the various
sections and the boxes they contain are determined in the
configuration. After a chromatogram has been recorded,
the documentation is saved along with the entire method in the results file
(*.cfd).
Example of documentation:
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4. 3 Channel1/Analysis
Data Acquisition and Integration
This page of the method file contains parameters for data
recording and integration. The parameters for the analysis
are explained in each section.
The following entries are made in the Acquisition box:
Further entries in this box are: Not Used
and DAD, i.e. data recording can be done
using one of the channels of the A/D converter card and/or a diode array
detector. For the latter, a DAD also needs to be set as a data source in the
configuration.
This parameter determines whether the
chromatogram should be standardised to the highest peak in the next
printout or not. No Standardisation means that the y-axis is allocated
according to the entries given for min. intensity and max. intensity. The
chromatogram is printed as it appears on screen. Click on this box to
switch between standardisation and no standardisation.
This is the time after the start of the analysis
(entries from 0 to 99.999 minutes, in steps of
0.001, default value 0) during which peaks are not used for normalisation.
Minimum mV value for the screen display of
the detector signal.
Maximum mV value for the screen display of
the detector signal.
When using a DAD, the wavelength at
which the chromatogram should be
recorded is entered here.
Wavelength resolution (nm) when saving the
DAD data.
The Integration box contains
integration parameters.
the
time-dependent,
programmable
Programming is done in a table. Entries
are made in chronological order. The list
of integration parameters is accessible using the downwards arrow. The
meaning of the parameters is as follows:
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Slope Sensitivity. This parameter controls the recognition of peak
beginnings and peak endings. The value (in microvolts/second) determines
the maximum amount by which the detector signal can increase without
being considered the beginning of a peak. Its value can be set between
0.001 and 10000 and changed over time.
Max. Baseline Level. This parameter determines the level of the detector
signal (in millivolts) above which an increase greater than the slope
sensitivity cannot be recognised as a new baseline. This parameter also
has the effect that a peak sliced off at the top (due to an overloaded
detector) is correctly integrated and not treated as a double baseline offset.
Its value can lie between 1 and 1000 and can be changed over time.
Skim Ratio. This parameter compares the relationship in height of two
neighbouring peaks with the height of the shared valley between them, to
determine whether a perpendicular should be dropped between the two
peaks when calculating surface areas, or whether the second peak is
merely a ‘bounce’ of the first whose baseline therefore runs exponentially
from the valley between the two peaks to the end of the peak. Its value can
be set between 0 and 20.
Valley Valley. This parameter sets a time window during data recording in
which a baseline is drawn between all neighbouring valleys, irrelevant of
the Max. Baseline Level and Skim Ratio values entered.
Horizontal. This parameter defines a time window in which the baseline is
plotted horizontally from the height of the signal at the beginning of the
time window until the end of the time window.
Integrate Inhibit. This parameter sets a time window during data recording
during which integration is inhibited.
Negative Peak. This parameter sets a time window during data recording
during which negative peaks are seen as positive and integrated
accordingly.
Filter Factor. This parameter indicates the number of points over which
the increase in the detector signal function is averaged. The minimum
value is 5 and the maximum is 15, odd numbers only. This increase is
tested over a certain number of consecutive points as a criterion for a peak
beginning and peak ending.
Zero Now (Baseline). This parameter allows a baseline for the integration
to be set manually at a defined time, e.g. after a disturbance of the
baseline caused by a change of solvent, of the flow rate or of the wave
length.
ChromStar 7- Method
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4.4 Channel1/Analysis/Calculation
The lower part of this page contains all the parameters you
need for a quantitative analysis in the Evaluation box.
Calculation Base. These parameters determine whether
peak areas or heights are used for calculation. Clicking on
one of the dots sets the basis for your calculation.
Calculation Method. These parameters determine the type of calculation
(for a description of the calculation methods and formulae, see page 89).
Percent. In this method, the results list indicates retention time, peak start
and end time, peak surface and height, and the surface and height
percentage for each peak. With the percentage method, the percentage
share of all peaks are calculated using entries in the peak table, but the
response factors and identifiers entered in the peak table are also taken
into account.
Normalisation. With standardisation, all the peaks, surface area
percentages and peak identifiers set in the peak table are shown.
External Standard. When using the external standard method, a surface
area and height calculation is made for all the peaks as well as a calculation
of the quantity of peaks found in the peak table.
Internal Standard. When using an internal standard, select this method for
your analysis. A calculation of the surface area and height of all the peaks
will be made, as well as a calculation of the quantity of all the peaks found
in the peak table. Additionally, one peak must be identified with the code IS.
Calculation Unit. This parameter sets the measurement unit for the results.
It is used as a column heading in the results table.
Minimum Area/Height. With this parameter (0 to 1000000, default value
100), a lower threshold is set for the value of a peak surface area or height
after integration or reintegration, beneath which the peak will be excluded
from the quantitative calculation. When making an analysis using peak
heights, a far lower value should be entered!
Number of Concentration Levels. Number of different standard solutions
(max. 15) that are used for a calibration with the calculation of an equalising
function (multi-level calibration).
Relative window update. This parameter determines whether the time
window with which peaks or peak groups are selected, should be brought to
its most recent position after a run, i.e. whether or not it should be shifted.
The peak that serves as a reference point is specified in the peak table with
the code RF. If the box is not checked or if no reference peak is specified,
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updating will not occur. Please note: do not use this function when
calibrating!
Column Parameters. To calculate certain column coefficients (see page
57), the following parameters need to be entered:
Column Length. Column length L in mm
Column Dead Time. Retention time of a non-retained substance T0 in min
(e.g. solvent peak) (dead time, RT-Solv)
The Show Calibration button displays
the results of a multi-level calibration
(calibration using standard solutions
with different weighed portions) and a
calibration with a calculation of the
average response factors.
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4.5 Channel1/Peak Table
The peak table contains all the peaks to be analysed with
their retention times, names, standard quantities for a
calibration and response factors (columns under
Concentration). Moreover deviations of the expected
results can be defined (columns under Limits) to generate displays of
validity ( not applicable with the percent method).
The peak table consists of the following columns under Concentration:
Start. This parameter (from 0.001 to 999.999, in min) indicates the start
time of the time window for a given peak.
End. This parameter (from 0.001 to 999.999, in min) determines the end
time of the time window for a given peak. This time must be a greater value
than the one entered under ‘Start.’
Amt. in Std. This column contains the known quantity (1.0000E-009 to
1.0000E+009, in the unit entered under calculation unit, with a default value
of 1) of the substance in a calibration mixture, so that the conversion factor
for this peak (Resp. Factor) can be calculated in a calibration run.
Resp.Factor. In this column, you will find the conversion factor (response
factor, 1.000E-009 to 1.0000E+009, default value = 1) for a given peak or
peak group. The default value of 1 is used in a calibration run. After the
calibration run, the conversion factor is automatically entered or updated in
the peak table.
Area0. The user can enter the surface area of a peak from a calibration run
here using the standard increase method. When calculating the unknown
quantity, this surface area is deducted from the total peak surface area. By
entering Recovery in the advanced settings of your configuration, the
column heading will change to Recovery(%). This makes it possible to take
a recovery rate into consideration when making a calculation. The default
value is 100 for 100% recovery.
Peak. In this column you can enter a peak name of up to 14 characters that
will be assigned to the retention time of this line. The name will appear in
the table of results after the corresponding retention time.
Code. In this column a code in the form of 1 or 2 letters can be allocated to
a specific peak. The following codes are possible:
IS – this code characterises the peak as a substance used as an internal
standard. Please note: the internal standard must also be indicated during
calibration. If more than one peak is coded IS, only the first one will be used
as the internal standard.
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RF – This code indicates a peak that should be used to correct the time
window after each run. For this to happen, the box needs to be checked
next to the parameter Rel. Window Update on the Analysis page.
G – a peak window is allocated the letter G for peak grouping. All the peaks
that occur within it will be taken as a whole by adding together their surface
areas. For their retention time, the average retention time of these peaks
will be calculated in the analysis. In the chromatogram, only the centre of
the peak group and the retention time or peak number will be shown. A
single line will appear in the report – marked as ‘G’ – for the peak group.
More than one peak window can be allocated the letter G in a single
chromatogram, and therefore several groups can be calculated.
S + figure – for peak sums, various peaks that are far apart from each other
are collated in the analysis. By allocating them the codes S1, S2, S3 etc.,
several peak sums can be created. The retention time and identifier of the
first peak in each sum is used. In the report, peak sums appear as a single
line, marked S + figure. The surface area is the sum of all the peak areas in
the peak window indicated by S + the same figure.
The peak table offers under Limits the following columns:
Set value is the expected value of the result.
N. Dev. is the negative deviation valid.
P. Dev. is the positive deviation valid.
The columns Peak start, Peak end and Peak(name) are the same as
above.
The waste bin on the right deletes a tagged line.
Graph will take you to the display of peaks in graph form.
Using a sub-menu, you can open a window under the table
(Under peak table) or outside it (In New Window) in which
the peak window can be drawn with the mouse after loading a
chromatogram (Load File To Graph or with the Import button).
chooses between the input columns Concentration
and Limits.
Copy To All Channels copies the peak table of the
first channel to the other peak tables when recording
data with more than one channel.
The number of the next standard solution
can be entered in this box by clicking on
the arrows or entering a figure. Afterwards
only the quantity of the standard solution can be entered under Quantity in
Std in the entry line. The user needs to do this for all the standard solution
peaks. If several peaks are selected, the quantity in standard can be
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entered for several peaks, as long as the peaks marked all have the same
standard quantity.
After clicking on the Graph button, a sub-menu will appear with the
following options:
No Graph. Default setting. If a graph window is open, you can use this
option to close it again.
Under Peak Table. The graph window opens under the peak table.
In New Window. The graph opens in a separate window.
Load File To Graph. You can choose one or more chromatograms from
the file selection window that will then appear in the graph window.
Reset Graph. The chromatogram shown in the graph will disappear from
the window.
To determine peak windows graphically, you first need to select one or
more chromatograms, By moving the mouse to the required place on the
chromatogram, you can draw a peak window by dragging out a coloured
box to cover the required area. The threshold values of the window will
immediately be entered in the first row of the peak table. You can now enter
the peak indicator under Peak. Then you can create another window by
moving your mouse, and enter the corresponding identifier in the peak
table. For calibrations, the standard quantity is set under Quantity in Std.
The peak windows determined can be changed later by moving the mouse.
This automatically updates the corresponding lines of the peak table.
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The window that opens when you click In New Window looks like this:
Use the Add option to select one or more chromatograms. The Reset
button makes all the chromatograms shown in the window disappear. More
data processing options will appear if you click the right mouse button.
The chromatogram window, which is being used here to determine peaks
graphically, also appears when you select Compare Chromatograms in the
Batch Wizard module. The meaning of the other function buttons is also
explained there (see page 74).
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4.6 Time Table
This page of the method file contains a table of the function
procedures of the chromatography devices being controlled. The
entries are in chronological order.
After clicking to add a new line the following functions can be accessed
using the downwards arrow:
Detector Attenuation. This function changes the attenuation (from 1 to
14) of the UV detector signal. The attenuation only affects the remote
control output of the detector.
Detector Wavelength. This function sets the wavelength (from 190 to 600
nm) of a UV detector.
Detector Zero. This function is used to carry out a zero point balance on a
detector signal.
Event. This function turns the outputs of the event box on and off at
indicated points in time. The numbers of the event outputs and their state
can be accessed using the downwards arrow.
Flow Rate. This function defines the flow rate (from 0.0 to 30.0 ml/min) at
a given time: the flow rate can be programmed independently of the value
set for time 0.0 or also set to any change required during an analysis. The
flow rate should not be changed during a solvent gradient.
Pump Maximum Pressure. This function programmes the pressure limit
(from 0 to 490 bar) above which the pump will switch itself off
automatically.
Pump Minimum Pressure. This function programmes the pressure limit
(from 0 to 490 bar) below which the pump will switch itself off
automatically.
Pump Start. This function starts an isocratic pump.
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Pump Stop. Depending on the pump to be controlled, this function can
stop the pump during a manual or semi-automatic injection. For example, a
stop can be programmed at a time when several injections are not reached
in a run because the time control file has previously been restarted at Start
at 0. Only when no further injections are made is this time point reached in
the time control file run and the pump will stop pumping solvents. The stop
function does not necessarily need to be programmed. The pump can also
be stopped after recording a chromatogram using the function button Stop
Pump in the control station of the data recording module.
Solvent Composition. This function defines the solvent composition at a
given time by entering the percentage share (from 0.0 to 100) of solvents
A, B and C. The percentage share of solvent D will be calculated
automatically. A time control file must have a solvent composition
programmed at time 0 if a pump is being controlled. The gradient
programming only requires the start and end conditions to be determined.
A linear solvent gradient will be calculated between two different solvent
compositions at two different times, whereby the percentage share of a
solution can either rise or fall.
Valve Position. This function allows the position of a valve to be switched
at programmed times. The number of the valve and its positions can be
accessed using the downwards arrow.
Next Valve Position. Using this function the valve can be switched one
position ahead. The number of the valve and the so called overflow
position can be accessed using the downwards arrow. The overflow
position is either the maximum number of valve positions or the maximum
valve position to be used.
The waste bin deletes a row.
ChromStar 7 - Sequence
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4.7 Sequence
The sequence contains the order in which vials are processed when using
an autosampler or the order of injections for manual injection, depending
on the selection made in the lower part of the window and the method to
be used in the analysis. When using an autosampler the sequence can
contain more than one
sequence line with tables, the
number of the sequence line
is on top to the left. With manual injection the sequence contains only
one table.
The data entry boxes in the upper part of the sequence row have the
following meaning:
Vial. Number of the vial to be processed; entries from 1 upwards, with the
maximum number dependent on the autosampler used.
to. Depending on the autosampler used, another vial number can be
entered here (1 or above). For each vial, a line will appear in the table
below. A consecutive series of vials will be processed, beginning with the
first vial number set and ending with the one entered here.
Analysis method. Here you must select the analysis method to be used.
If no path is entered the method must be in the same directory as the
sequence.
This button opens the chosen method in the method editor so that it
can be changed. Don’t forget to save it!
The red cross deletes the corresponding line of the sequence.
Calibration. If a calibration run of one or more samples needs to be done,
this column allows you to access and determine the type of calibration
using the downwards arrow (one-point, average, multi-level). For a single
point calibration, only one vial in the table below should be selected. For a
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calibration that gives the average value of the response factor, several
injections are carried out from one vial. For a multi-level calibration,
injections are carried out from a consecutive series of vials, where various
standard quantities are present in the vials. These are allocated to the
standard quantities entered in the peak table of the method using the extra
column Lvl. (Level). For further injections from other vials, a new sequence
row is created.
Result Report. Here you can select a print format to be used for the
printout of chromatograms for this sequence line. Use the downwards
arrow to access the list of print formats.
Additional. As an additional calculation for all chromatograms of this
sequence line a scattering calculation can be carried out. This sequence
line should not be changed and actualized while scattering calculation is
being carried out.
Summary Report. The result of the scattering calculation can be printed
out using an appropriate print report template.
The arrow closes or opens the table below.
The data entry boxes in the table have the following meaning:
Inj.. Number of injections (1 to 15, depending on the autosampler used)
that should be made from one vial. If a series of vials are selected for
processing, the number of injections entered here is first carried out from
one vial before the next vial is processed.
Vol.. The autosampler volume to be injected is entered in µl (depending on
the autosampler used).
Runtime. After this time (in minutes, accurate to a thousandth of a second)
has elapsed, the run will end. The runtime must not be equal to the runtime
in the method. If the runtime is shorter than in the method the
chromatogram is stopped at the time entered here. If the runtime is longer
than in the method the chromatogram is stopped at the method runtime.
The next injection is only made at the time mentioned in the sequence.
Sample identifier. Box to enter the sample identifier.
Factor. This factor is used as a dilution factor for a quantitative calculation
with an external or internal standard method. The result of the quantitative
analysis is multiplied by this factor.
Weight. Total quantity of the injected samples. In a quantitative analysis,
the quantities established for the peaks to be analysed are calculated
additionally as a percentage portion of this total quantity. The quantity must
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be stated in the measurement unit entered on the Analysis page in the
calculation box.
Int. Std. Quantity of the internal standard. If the quantity of the internal
standard differs in this injection from the quantity used for calibration, a
new value can be entered here. Entries must be made in the same
dimension as in the peak table.
Lvl (concentration level). Number of the standard solution with a given
standard concentration for multi-level calibrations. The level number
entered here must correspond to the concentrations entered under the
appropriate level number of the peak table.
Info. An extra entry with further information about the samples can be
added to every line. On clicking on the box under Info an entry box
appears to the right for additional sample information. The additional
information can only be copied by marking and Ctrl C and Ctrl V in the
desired box.
This button creates a new line of the sequence.
For quick changes in the table of a sequence line the following
means are available: The content of a marked box appearing in
blue can be copied into one or more table lines above (or below)
by dragging the black dot on bottom to the right with pressed
mouse button above (or below) into boxes which appear then
with a red frame. The content of a marked box is deleted with the
Delete key. When a cell contains a number at the end and after
the cell has been marked, this number can be increased for the cells below
using the Ctrl key and dragging with pressed mouse button.
On clicking with the right mouse key on one of the table lines a menu
appears allowing the following actions:
Paste clipboard
Copy selected cells into clipboard
Insert vial and move down
Delete selected vials and move up
The meaning of the menu points and screen buttons is as follows:
File
Open opens the file selection window where you can
load a new sequence.
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Import From CSV imports the data from a *.csv file. From
Old Method imports the autosampler table and sample
table of an old method into the new sequence.
Save saves the sequence.
Save As... saves the sequence under a new name.
Save & Sign (only accessable in the GLP mode)
saves and signs the sequence. The signature is
documented in the audit trail.
Audit trail Comment. This function key allows a
comment to be entered in the audit trail.
Export. The content of a sequence can be saved as .html
or .xls-file.
Print opens a window to print the sequence. The
printer selected can be changed in the Printer box.
The default printer is set as standard. OK starts
printing the document.
Recent Files. The most recently opened files are added to
the bottom of the menu for rapid access.
Exit closes the sequence window.
Edit
Undo deletes an entry.
Redo restores the deleted entry.
Sequence
Add Sequence Line adds a new line to the sequence.
Edit Progression opens a window in which the order of the
sequence rows can be changed using the upwards and
downwards arrows.
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Output
Save Chromatogram … into the method directory
Save Chromatogram … into the sequence
directory
Help
Contents leads to the online help.
About opens an information box.
With manual injection the sequence shows the injections to be carried
out. The menu procedure Add Sequence Line is dropped. The runtime
is only defined in the method .
On clicking with the right mouse key on one of the table lines a menu
appears allowing the following actions:
Copy selected cells into clipboard
Paste clipboard (only the first of the cells to be copied is klicked)
Insert new row behind selection
Remove selected rows
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4.8 Calibrations
The various calibration methods determine the conversion factor for
calculating an unknown quantity from a peak area or peak height: in other
words, the calibration function is established as the relationship between
quantities and peak areas.
The result of a one-point calibration appears under response factor in the
peak table. The Show Calibration button opens the calibration
chromatogram in Reprocess. The results of multiple calibration are shown
on screen when Show Calibration is pressed. When calibrating with an
average value, an average response factor is shown. Multi-level calibration
establishes a calibration function.
For one point calibration, a chromatogram is recorded using a calibration
mix containing known quantities of all the substances to be determined
later. Retention times, peak areas and peak heights are obtained by
integration. For analysis, the user should state on the Calculation page of
the method file, under Calculation Parameters, whether areas or heights
are to be used for analysis and the unit in which quantities are to be
shown. External Standard should be selected as the calculation method.
Entries are made in the peak table concerning the retention time window,
quantity of the substance in the calibration mixture and the peak identifiers.
The calibration is done using the Batch Wizard by clicking on one-point
calibration and entering the chromatogram name (*.cfd) and the method
file. One-point calibration can also be done directly in the method by
clicking on the corresponding menu option and entering the name of the
calibration chromatogram. The conversion factors obtained are saved in
the method file and appear under Resp.Factor in the peak table. A
conversion factor needs to be given for a substance to be used as an
internal standard as well. The substance needs to be identified as an
internal standard.
For a one point calibration in the current autosampler run, the injection
needs to be identified in the sequence under calibration as one-point.
If standard substances cannot be mixed and single point calibrations are
being done with the chromatograms of the individual substances, go to the
advanced settings in the configuration and select Additive as the
calibration type, so that the response factors of substances that are not
found in the chromatogram are not set to 0 each time. Additive calibration
only works for one-point calibration.
To do a multiple calibration with calculation of the average, begin by
recording several chromatograms from a standard solution. A standard
solution may contain several standards here. To do this calibration online,
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enter the vial with the standard solution and the desired number of
injections in one line of the sequence. Select Average under Calibration.
External Standard is used as the calculation method, and a calculation unit
should be entered. The peak table contains the standard quantities for all
the substances to be determined under Amt. in Std and the identifiers
under Peak. The subsequent analysis of the calibration chromatogram is
done using the option Average Calculation in the Batch Wizard. The
result in both cases is saved in the method on the Calculation page and
can be displayed on screen by clicking on Show Calibration.
The identifier of the analysed peak will appear at the top. The downwards
arrow gives access to the analysis of all the other peaks. In the graph
display, the peak area of individual chromatograms is shown in the upper
section (Zoom is selected); the red line shows the average, the blue line
shows the standard deviation and the green line shows the area of
reliability. These values appear on the right as percentages. The average
response factor is calculated from the quantity/average of the peak height.
This value also appears in the peak table under Resp.Factor.
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The table has the following columns:
No. Number of the standard chromatogram.
Ret. Time. Retention time of the selected peak.
Std. Amt. Amount of standard.
Area. Peak area.
Enab. Enabled: the chromatogram has been included in the calculation.
Disabled: the chromatogram has been excluded from re-processing in the
calculation batch.
File. Name of the chromatogram.
Multi-Level Calibration is used to establish a relationship between the
concentration of a substance and the peak area. This allows the
calculation of approximation functions. These are later used to calculate an
unknown quantity from a peak area. The multi-level calibration can be
done either in the current run or afterwards. To do it in the current run,
enter the series of vials with the calibration mixtures (from first vial to last
vial) in one line of the sequence. Several injections can also be made
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from a single vial. Multi-level is selected under Calibration. The allocation
of the vials to the various concentrations is done in the sequence under
Lvl. (Conc.Level). The level numbers entered here must correspond to the
appropriate level numbers in the peak table of the method file. On the
Cxhannel1/Analysis pagein the calculation box, External Standard must be
selected as the calculation method, the concentration unit is entered next
to Calculation unit and the number of different calibration mixtures is
entered next to Number of concentrations (this will correspond to the
number of vials). The various quantities in the calibration mixtures and the
peak identifiers are entered in the peak table. The retention times of the
substances to be determined must be known. Analysing the calibration
chromatogram afterwards is done using the Multi-calibration option in the
Batch Wizard. In both cases, the result is saved in the method on the and
can be displayed on screen by clicking on Show Calibration.
The identifier of the analysed peak will appear at the top. The downwards
arrow gives access to the analysis of all the other peaks. On the diagram,
the x-axis shows the peak area and the y-axis shows the quantity. An
approximation factor of the first order has appeared. The axes can be
swapped round using the option Invert Axes. The coefficients of the
regression function and the correlation coefficient are listed on the right.
The approximation function has the general form: y = K0 + K1*x + K2*x^2
+ K3*x^3.
The table has the following columns:
No. Number of the standard mixture chromatogram.
Ret. Time. Retention time of the selected peak.
Std. Amt. Amount of standard.
Area. Peak area.
Enab. Enabled: the chromatogram has been included in the calculation.
Disabled: the chromatogram has been excluded from re-processing in the
calculation batch.
File. Name of the chromatogram.
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Data Recording - ChromStar 7
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5. Data Recording - Runtime
In the data recording module, chromatography data is recorded and
displayed on screen. This module also allows you to control the devices in
an HPLC system.
Device control is done in the control panel.
The detector signal sent from an A/D convertor card is displayed in a
suitably titled chromatogram window (e.g. 'UV'). When recording data
with multiple channels, all the data traces will appear here, including the
one from the DAD for a previously set wavelength.
When using a diode array detector, the current spectrum is displayed in a
window. The two-dimensional data set from the diode array detector is
displayed in the DAD data recording window.
After clicking on the link to the Data Recording window, logging
into the user login dialogue and selecting devices from the icons
representing the devices available (configuration), the data
recording module window will appear with all the sub-windows appropriate
to the device configuration.
Now a sequence can be loaded and the recording of a
chromatogram can begin. The downwards arrow in the
button opens the list of recently loaded sequences.
The menu options and screen buttons have the following meanings:
File
Load Sequence ( also Ctrl O) opens a file selection window. In the
file selection window you can already choose on the right an
injection by dragging the start line with the mouse at the desired place (see
also below: File and Select next injection).
Recent Sequences. The most recently loaded sequences are included at
the bottom of the menu for rapid access (C:\....\*.csq).
Additional Exports. An additional export as CSV file can be chosen. The
available templates (*.cps) are stored in the ...\System\exporttemplates
directory. The CSV file is given the same name as the chromatogram and
is saved in the same directory as the chromatogram. Preset is None. The
export is independent of a print-out on a printer.
ChromStar 7 – Data Recording
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Set Next Injection. When using an
autosampler, a specific line of the
sequence, a vial number (followed by
the accompanying sample identifier)
and injection number can be selected
as the beginning of the analysis.
Printer. One of the installed printers can be selected for the printout.
The default printer is set as standard.
Exit closes the data recording module.
Analysis
Start (also with function key F5) starts recording the chromatogram
when a sequence is loaded.
Stop (also with function key F6) stops the chromatogram.
Abort (also with function key F7) aborts the chromatogram.
Window
Skin. The format in which the diagram appears can be changed by
selecting a different option (classic, high speed, matrix, simple, sky,
standard, timeless, void). The various types of display (skins) can be
changed in the skin editor.
Arrange Windows. If several windows are open in the runtime module,
they can be placed next to each other (Vertical), on top of each other
(Horizontal) or diagonally overlapping (Cascade).
View Mode. When recording data from multiple channels, the
chromatograms can be displayed on top of each other on a single pair of
axes (Stack) or in several diagrams (Split).
Device Control Panel. At the bottom of the menu you will see the open
windows. They can be activated or deactivated here. If the control panel is
deactivated, the information it contains will appear in the info bar at the
bottom edge of the runtime window.
UV. Chromatogram window.
Spectrum. This window displays the current DAD spectrum.
DAD. This window shows the two-dimensional data set from the DAD.
Process Overview. On loading a sequence this sub-menu will appear. The
Time Table card displays the time processing of the time table in the
method. The step in the table that is currently underway will appear with a
pink background.
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The Progress card shows the sequence table with the list of vials to be
processed and their actual status. The name of the result file (*.cfd)
appears when processing a vial is finished.
User
Change User takes you to the ChromStar user login dialogue.
Lock.
Help
Contents (also F1) opens the online help.
About opens the information window.
All devices are stopped.
Increase runtime (only possible without an autosampler).
Integrate on the fly. Evaluation of recorded data and representation in
Reprocess.
This button is only active when no chromatogram is currently being
recorded. It preserves baseline data by saving it as a chromatogram. After
clicking on the button, a window opens in which the time span can be set
using the double arrow in the baseline display. A method can also be
selected if none has
already been loaded.
The Rescue button
opens a dialogue box
in which the file name
can be entered (.cfd).
The chromatogram
obtained in this way
can be opened with
Reprocess and
integrated.
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5.1 Chromatogram
The chromatogram window is where the detector data trace is shown. The
name of the window, displayed in the title bar, can be set in the
configuration. In order to record a chromatogram, a sequence
needs to be loaded. To do this, either click on the appropriate
button or select the menu options File and Load Sequence, and then
choose the required sequence from the list of sequence files. In the file
selection window you can already choose on the right
an injection by dragging the start line with the mouse
at the desired place (see also below: File and Select
next injection). The name of the chosen sequence will
appear in the title bar of the runtime window. When
using a controlled pump, the initial solvent composition
determined in the time control file of the method is
pumped through the entire system. As soon as the pressure and detector
signals have stabilised and the column has been conditioned, the system
is ready to record a chromatogram. If an autosampler is being used, the
options File and
Select
Next
can
Injection
be
used
to
select a given
line
of
the
sequence
as
the beginning of
the
analysis.
When injecting
manually,
a
given injection
number can be
entered as the
starting point.
After the start button has been pressed, the autosampler will look
for the programmed vial and inject from it. A message will appear:
Wait for injection... from vial...
When injecting manually, the start button needs to be pressed
simultaneously with turning the injector to the 'Inject' position. The injection
and start can be synchronised using the start input of the A/D conversion
card.
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The information bar at the bottom of the Runtime window shows the time
that has passed since the start of the chromatogram, vial number, injection
number (sample identifier), name of the data file recorded (*.cfd) and the
user name.
The other screen buttons can only be selected during the recording of a
chromatogram and have the following meanings:
Stop. When you press the stop button, a window will appear that allows you to
select the type of stop you require.
Stop Autosampler. The autosampler is stopped, i.e. no further injections are
made. The chromatogram continues to be recorded until the end.
Stop Recording Data. Data recording is stopped, i.e. the chromatogram stops
immediately but the runtime continues until the next injection. This and all
subsequent injections are carried out.
Stop All. Data recording and autosampling are stopped. The chromatogram is
ended and no more injections are made.
Abort. Recording the chromatogram is broken off.
If the sequence or method running in the editor is changed, it can be updated
using this button. The things that cannot be changed are: options, results file
name, data point distance and data recording channels. None of the injections
that have already been made or sequence lines that have already been
completed can be updated.
This button at the bottom of the left-hand edge of the chromatogram window
opens a sub-menu with the items:
Set Reference Chromatogram with Select and Show: a reference chromatogram
can be selected and shown. It only appears when a chromatogram is being
recorded.
Set range opens a dialogue box in which the mV values to display the y-axis and
a time span for displaying the x-axis can be entered.
Display Peak Window allows peak windows to be shown for channel 1 to 4. The
peak windows only appear when a chromatogram is being recorded.
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By right-clicking on the chromatogram window, you can access another
menu with the following options:
Copy copies the chromatogram to the clipboard.
Save Image As saves the chromatogram as an image. The file type can be
selected.
Print prints the chromatogram without selecting a print layout, as it appears
on the screen, which in some circumstances means that only an extract is
printed.
Show Point Values. When this option is checked, moving the mouse over
the chromatogram will display a box containing the retention time and mV
value at the current cursor position.
Intensity Setup. Intensity values can be changed.
Undo Last Zoom undoes the last selection of an extract of the
chromatogram.
Undo All Zooms/Moves undoes all selections and movements to different
parts of the chromatogram.
Set Reference Chromatogram with Select and Show: a reference
chromatogram can be selected and shown. It only appears when a
chromatogram is being recorded.
By dragging the cursor with the left mouse button held down and the shift
key held down, you can select an extract from the chromatogram. The
cursor will appear as a cross and the extract will be indicated with a
dashed line. By dragging the cursor with the left mouse button held down
and the control key held down, you can move the chromatogram up, down,
left and right. The mouse cursor will appear as a hand, and the scale
values of the x and y axes will be moved along with the chromatogram.
5.2 Data Recording – Control Panel
The control panel shows the chromatography system with all the significant
measuring units such as flow rate, solvent composition, detector signal in
mV, time passed since starting the chromatogram, pump pressure etc. The
image of the chromatography system (showing current chromatography
values) can be set up by the user or a service technician in the
Visualisation Editor and saved as a visualisation file (*.vis). It is also stated
during configuration of the system (see page 14).
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A row of screen buttons operate the pump.
stops the pump.
opens a dialogue box in which the flow rate can be entered.
pumps 100% solvent A.
pumps 100% solvent B.
pumps 100% solvent C.
pumps 100% solvent D.
opens a dialogue box in which the solvent composition can be entered.
This button makes the display bigger or smaller.
The downwards arrow opens a window showing the device control RS232
dialogue with commands and responses.
When an isocratic pump is used this button starts the pump.
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5.3 DAD Data Recording
When recording data
with the diode array
detector, the DAD
data set can be
shown in a separate
window. Both a 3D
view and a density
plot are available as
visualizations
via
View Type.
The colour scale and
intensity can be set
under View Options.
5.4 Spectrum
When a configuration with a diode array detector is selected for data
recording,
the
spectrum window of
the DAD will appear
and
display
the
spectrum currently
being
measured.
With the shutter
closed,
the
first
elements
to
be
measured are the
dark flow and then
the
reference
spectrum.
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The menu options Spectrum and Reference (also function key F8) allow a
reference spectrum to be recorded at any time except during data
recording.
In the spectrum window, the various ways of measuring the DAD
are displayed. A selection can be made using the View Mode
button on the bottom left. The options are:
Reference. The reference spectrum is an emission spectrum saved in
order to calculate the absorption spectrum.
Absorption. The absorption spectrum is calculated from the appropriate
emission spectrum and the reference spectrum according to the LambertBeers law. Absorption spectrum = Log (Reference spectrum / Emission
spectrum).
Emission is the spectrum provided by the DAD.
The Dark Current is measured with the shutter closed and shut off from all
the other spectra, because it is a blind value generated by the electronics.
ChromStar 7 - Reprocess
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6. Reprocess
The Reprocess module displays the entire results file (*.cfd) containing the
chromatographic data. This includes chromatograms, the analysis of the
saved data, documentation of the methods used, the audit trail with all the
planned operations, and the two-dimensional data set recorded by the
diode array detector including all the spectra and chromatograms extracted
from it.
After clicking on the link to the Reprocess module, logging into
the user registration dialogue and selecting a results file, this file
will appear in the Reprocess window. The navigation bar on the
left enables access to further information. In the Reprocess module, it is
possible to carry out a reintegration with different integration parameters,
manual integration, a re-calculation of the column coefficients and a new
quantitative analysis. Various transformations can be carried out on a
chromatogram. Spectra can be selected and saved from the DAD data set.
Reintegration
Manual Integration
Calculation
Method
Audit Trail, Configuration and Sample Information
Reprocess - DAD
Column Coefficients
Chromatogram Transformation
The menu options and screen buttons have the following meanings:
File
Open opens the file selection window so that a new results file can be
loaded.
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Reprocess - ChromStar 7
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Recently opened chromatograms allow rapid access to the most recently
loaded results files.
Recently recorded chromatograms allow rapid access to the most
recently recorded chromatograms.
Save saves the entire data file. This operation is necessary when, for
example, a reintegration with different parameters is carried out,
spectra are extracted etc.
Save & Sign (only accessable in the GLP mode) saves and signs the
chromatogram file. The signature is documented in the audit trail. In
the Sample Info box appears a signature symbol.
Save As... saves the entire results file under a new name.
Close closes the results file. It leaves the Reprocess module open.
Import converts ChromStar 6 chromatograms and displays them in this
module.
Export allows you to export parts of the results file. The following exports
are possible:
Chromatogram as *.txt file, Results list as *.csv file, Chromatogram and
Results list as *.html file, Spectra as *.jdx file.
Moreover in the directory ..\system\exporttemplates report templates
created with the reporteditor for the csv-export are available.
Print opens a window in which a print layout (*.cps) can be selected
for printing the results file. The Settings option allows you to change
the printer. The default printer is selected as standard.
Exit closes the Reprocess module.
Opens the previous results file (chromatogram).
Opens the next results file (chromatogram).
List of chromatograms in the chosen directory. The order of
chromatograms corresponds to the setting in the navigator. The
buttons Previous and Next Chromatogram open the chromatograms
according to this list.
Audit trail Comment. This function key allows a comment to be
entered in the audit trail.
View
Skin with sub-options Classic, Highspeed, Matrix, Simple, Sky, Standard,
Timeless , Void etc. allows you to choose the window style for displaying
the chromatogram. The different skins can be changed in the skin editor.
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Reference Chromatogram with sub-options can Display the actual
reference chromatogram and allows to choose another chromatogram
using Browse. The name of the chosen reference chromatogram appears
in the chromatogram window.
Extra
Compare Chromatograms leads to the Compare Chromatograms window.
Compare Spectra opens the Compare Spectra window.
Summary diagram allows in a series of chromatograms an overview over
several parameters such as peak area, peak height, result etc. (Value) for a
given peak. All chromatograms of the used directory are shown. Using
Clear List and Add Chromatograms a defined series of chromatograms can
be chosen.
Batch Wizard leads to the Batch Wizard module.
Options
Export Configuration exports the configuration used to record the data file
loaded to a new configuration file (*.ccf).
Import Configuration imports a Configuration to a results file recorded
using a different configuration. This is necessary, for example, when
different parameters (set under Advanced Settings) need to be used to
calculate the column coefficient.
Load Evaluation Parameters… from another method
Save Method externally…
Save Integration Parameters externally…
Help
Show Online Help. Little light bulbs are incorporated into the program
window. They display extra information about the parameters concerned.
This option can be turned on or off.
About... opens a window with information about the version number and
copyright.
selected.
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printed.
Save Intensity the chosen intensity can be saved by File, Save.
chromatogram.
By dragging the cursor with the left mouse button held down and the shift
key held down, you can select an extract from the chromatogram. The
cursor will appear as a cross and the extract will be indicated with a
dashed line. By dragging the cursor with the left mouse button held down
and the control key held down, you can move the chromatogram up, down,
left and right. The mouse cursor will appear as a hand, and the scale
values of the x and y axes will be moved along with the chromatogram.
6.1 Reintegration
In the navigation bar on the left, select Chromatogram and the Channel 1
(for multiple channel data recording, you can also select 2 or 3 etc.). Then
the appropriate results list will appear under the chromatogram.
The menu options and buttons in the upper toolbar have the same
meaning as the ones in the chromatogram display (see above).
The data can be processed using the buttons in the extra toolbar. The
buttons have the following functions:
Set intensity and scaling. The meaning of the button changes
according to sub-menu option: Auto Scale or Custom Scale.
applies the current scaling to following chromatograms
chosen by means of previous or next chromatogram. Press
the button again to remove the lock of scaling.
Display chromatogram
Display results list
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Display: extrapolated
Display: data points
Display: histogram
Display solvent composition
Show/hide peak windows
Format results list (see below). The downwards arrow offers 3
standard results lists: Standard results list, Standard
calibration results list, and column coefficients list. Row
accent: A deviation can be accentuated by using a colour.
Annotation with peak numbers
Annotation with retention times
Annotation with peak names (only possible, when entries are
made in peak table of the method file)
Annotation with peak areas
Opens the Compare Chromatograms window (see page
74ff), the actual chromatogram is already loaded.
Show spectra for available peaks (see below)
Integration (see below)
Transformation Menu (see below)
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Format Results List
The button opens a
window in which the
results list can be formatted.
The following columns are available:
Amout
Quantity in standard solution
Area
Peak area
Asymmetry
Column coefficient
Bandwidth
Column coefficient: bandwidth at half peak height
Code
Code as entered in the peaktable of the method file
Deviation
valid or invalid, if defined in the peak table
Height
Peak height
HETP
Column coefficient: theoretical plate height
K'
Column coefficient: capacity
Name
Peak name
Peak End
Peak no.
Peak number
Peak Start
Plate number
Column coefficient: Plate number
Resolution
Column coefficient: resolution
Result
Depends on calculation method used
Ret. Time
Retention time
Selectivity
Column coefficient
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Window number
Row number in peak table (on the calculation page of the
method)
A column is chosen by using the downwards arrow under Column. You can
change the order of the columns using the red up- and downwards arrows.
The red cross removes a column from the table. Under Format the
number format is chosen and under Decimal Places the number of decimal
places. Using the button Right cell alignment the alignment in the columns
can be set to the left or to the right.
By holding down the shift key and dragging the mouse, rows and columns
of the results table can be selected. These can be copied to the clipboard
using Ctrl+C. From there the values can be pasted to an Excel document,
for example.
Show spectra for available peaks
If a DAD is used to record data, this option can be used to display all the
spectra in the peak table for peaks that have a peak name, and then to
transfer them using the Add Spectra button in the left-hand navigation bar
as a sub-option of Spectrum. The spectra can be saved to the *.cdf file
using the menu options File, Save.
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Integration
The button opens a window in which integration can then be carried
out manually or automatically. The meaning of the individual icons is
as follows:
opens the page of the method containing the integration parameters.
activates automatic integration, and the parameter buttons below become
active. The integration becomes easy by the grafic display of the parameter
values in the display of the chromatogram. The possible actions are
explained on the screen.
activates manual integration, and the 8 buttons for the manual integration
become active.
The Reintegrate button re-calculates the peaks.
Reintegration is done as follows: after opening the page with the
integration parameters, the parameters to be changed are selected from
the table and a value is entered. Click Reintegrate in the integration
window. The altered integration will appear in the results list. On the page
containing the integration parameters, the Load Evaluation Parameters
button can be used to load integration parameters from a different method.
The altered integration parameters can also be saved to another method
using the Save Externally button. The integration can be saved to the
results file using the menu option File, Save .
If you right-click on Channel1 (or a higher channel number for multiple
channel data recording) in the navigation bar, a menu will appear with the
options Change Integration Parameters, Reintegrate, Export As Text
File and Delete Chromatogram. This last option allows you to delete
chromatograms extracted from the DAD data set, for example. The
appropriate authorisation is required to delete chromatograms, as
determined in the user administration. The other options are also
accessible from other parts of the Reprocess module.
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6.2 Manual Integration
After activating manual integration in the Integration window the following
actievities are available:
Allows manual drawing of the baseline
Separates peaks with perpendiculars
Carries out exponential skimming
Carries out tangential skimming
Deletes all peak separations made
manually
Deletes marked peak separations
Undoes the last action
Repeats the last action
Manual integration is carried out as follows:
Firstly, select the option Draw Baseline. This makes a small blue
cross appear that can be moved to the starting point using the mouse
and fixed by left-clicking. Then drag the mouse to the end point and fix it by
left-clicking. This can be repeated for all the peaks to be integrated. After
pressing the Reintegrate button, the peak surfaces are calculated and
entered in the peak table.
An active operation can be deactivated by right-clicking the mouse in the
chromatogram field or by using the ESC key.
To carry out manual integration, it is recommended that you activate the
option Show Online Help.
Once all the planned manual operations have been carried out, the
results can be saved.
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6.3 Reprocess – Calculation
To make a calculation afterwards (quantitative analysis), the page in the
method containing the calculation parameters can be opened using the
menu option Method in the navigation bar and changed as required.
You can use the Load Evaluation Parameters button to load a method
containing the required response factors. It can be the same method that
was used to record the data; though in the meantime, however, it has been
used to do a one-point calibration after recording a calibration
chromatogram.
Next click the first sub-option (UV) for the chromatogram, then
press the Integration button followed by Reintegrate in the
integration window. The recalculated values will appear immediately in the
list of results under the chromatogram and can now be saved.
6.4 Reprocess - Method
The method used to record the results data can be viewed by clicking on
Method in the navigation bar. All the boxes and parameters that cannot be
changed are displayed in light grey. Certain parameters can be changed
afterwards, in order to carry out a different integration, calculation or
determination of a column coefficient.
The menu options and the buttons in the upper toolbar have the same
meaning as in the chromatogram display (see page 51 onwards).
For the meaning of the Load evaluation parameters menu point, see
section 6.3 above. Only the parameters that can be changed, such as
integration parameters, calculation parameters and the peak table are
loaded.
If a calibration was made using this method the calibration chromatograms
will appear as sub-menus in the context menu. After clicking with the right
mouse key on one of the chromatograms it can be saved externally.
The Save Externally menu point can be used to save the method under
another name.
6.5 Reprocess – Navigation Bar
All the information relevant to data recording is saved in the results file and
can be viewed by clicking on Sample Information, Method (see above),
Audit Trail and Configuration in the navigation bar.
The Sample Information contains all entries made in the sequence used
for data acquisition, such as sample identifier, autosampler vial number,
injection volume, factor, weight, actual value of the internal standard (if
applicable) and the entries in the sample information box. If in a
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recalculation in Batch the sample identifier was changed, the changed
name appears here, the original name is documented in the audit trail (see
below).
The upper part of the Audit Trail contains documentation with notes on
the computer used, user name, name of the configuration, the operating
system, date and time of data recording, ChromStar version, method,
license information, Sample ID and info, A/D converter cards, DAD
coefficients, and more. The lower part contains the run protocol.
The configuration used for data recording is shown under Configuration.
Under Sequence the sequence used for data acquisition (only in the GLP
mode, without sample information) is shown.
If data were recorded using a diode array detector, the submenu DAD
appears in the navigation bar. Extracted spectra and peak purity
calculations can be viewed using the corresponding submenus.
If a scattering calculation had been carried out, in all chromatograms used
for the calculation the additional Scattering point appears. The scattering
table is for all chromatograms used in this calculation the same. It contains
the names of the chromatograms, the results for all peaks, the average
value of the results, the standard deviation, and the own deviation in %.
The menu options and buttons in the upper toolbar have the same
meaning as in the chromatogram display (see page 51 onwards).
6.6 Reprocess - DAD
The data set recorded with a diode array detector can be displayed in a
two-dimensional field using the DAD option in the navigation bar, where the
x-axis shows time in minutes and the y-axis shows wavelength in nm. The
varying intensity of the detector signal is colour-coded (density plot).
The menu options and buttons in the upper toolbar have the same meaning
as in the chromatogram display (see page 51 onwards).
The buttons in the lower toolbar have the following meanings:
Density Plot
The split screen view shows a chromatogram in the upper part of the screen
and a spectrum in the lower part. When you move the cursor in one window,
the corresponding display appears in the other window.
3D view. The data points are displayed spatially. You can change the viewing
angle by moving the cursor.
Peak purity calculator. To calculate peak purity, begin by moving the cross
hairs to the required peak with the mouse and then press the button. A
window will appear with the peak purity calculation. In the upper part, you will
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see the three spectra used to make the calculation at the peak maximum, and
at half the peak height on the left and right of the maximum. The spectral
range can be limited by raising the start wavelength with the upwards arrow
and lowering the end wavelength with the downwards arrow. Extra information
can also be added to the peak. Save and Close will save the peak purity
calculation. An extra option will appear in the left-hand navigation bar.
Extract chromatogram opens a window showing the chromatogram at the
wavelength chosen by moving the cursor. Extract adds the chromatogram to
the other chromatograms in the navigation bar (Channel .., ... nm).
Extract spectrum opens a window in which the spectrum selected by moving
the cursor can be saved. A name can also be entered. When you press
Extract, the spectrum appears in the navigation bar under ‘Spectrum’ as
Spectrum 1 etc.
By right-clicking on the word ‘Spectrum’ in the navigation bar, you can delete
the spectrum.
This button opens a window in which the scaling of the x-axis (min), y-axis
(nm) and intensity (mV) of the spectrum and chromatogram can be changed.
This button opens a window in which colours can be allocated to maximum
intensity percentage values. The values can also be changed here.
This button opens a window to search for related spectra. First, you need to
set the folder or subfolder to be searched. If you check Search Window, you
can restrict the wavelength range for related spectra using the upwards and
downwards arrows. The standardization wavelength appears in the spectrum
display as a vertical red line. It can be moved using the upwards and
downwards arrows. The Search button starts the search. Search results are
shown in the table on the right. The first column indicates similarity in percent.
If you check a table entry, it will be added to the spectrum display. The name
of the *.cfd file containing the spectrum appears on the far right.
This button opens the Compare Spectra window. The window has the same
function buttons as the Compare Chromatogram window (see page 74
onwards).
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6.7 Column Coefficients
Various coefficients can be used to judge the efficiency of a column, such
as the theoretical plate number, height equivalent to one theoretical plate,
capacity coefficients and selectivity coefficients that should be tested from
time to time using the chromatogram from a test mixture.
The following can be calculated from: the chromatogram and the
parameters entered for column length and dead time: number of plates N
(Th Plate No), plate height H (HETP), capacity factor k', asymmetry factor
A, selectivity a, half value width W0.5 (width of the peak at half peak height)
and resolution (Resolution) R.
The parameters entered, column length L in mm and retention time of a
non-retained substance T0 in min (e.g. solvent peak) (dead time, RT-solv),
are used to calculate capacity, selectivity and plate height.
The calculation of the coefficient is done directly as the chromatograms are
being recorded if the option Calculate Column Coefficient is set to Yes in
the Advanced Settings of the Configuration If it is set to No, there is no
calculation. The values for column length and dead time are entered in the
method on the Calculation page and can be changed later using
Reprocess. If no values are entered for column length and dead time, the
values for plate height, capacity and selectivity are not calculated.
The calculation can be repeated later in Reprocess. The column
coefficients can be entered as columns in the results list in Reprocess
using Format Results List. To print this list, a print layout with a
correspondingly formatted table is required.
The column coefficients are calculated according to the following formulae:
Plate number
N = 5.545 * (T/W)2
Plate height HETP
H (µm) = L * 103 / N
Selectivity
a (i+1/1) = k'(i+1) / k'(i)
Capacity
k' = (T - T0) / T0
Resolution
Ri+1, i = ( Ti+1 - Ti) * F/ (W i + W i+1)
Asymmetry
A = wb / wf
FDA compliant:
A =( wb + wf )/ 2* wf
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Defined as follows: L = column length (in mm), T0 = dead time (in min), T =
retention time (in min), W = half value width (width of the peak at half peak
height), wf = peak width at 10% of peak height from the rising side of the
peak to the perpendicular, wb = peak width at 10% of peak height from the
perpendicular to the falling side of the peak. wf and wb are calculated at 5%
of the peak height if Peak Width Calculation=FDACompliant is entered in
the configuration. In that case, asymmetry is calculated according to the
formula A = (wb + wf )/ 2* wf. Ti+1, where Ti = retention time of two
neighbouring peaks. F= Factor, as determined in the configuration under
Factor for Calculating Resolution. The default setting is Factor=2.
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6.8 Chromatogram Transformation
The screen button Transformation Menu opens the Transformation
window in which various data transformations can be carried out. The
following functions are available:
Multiply chromatogram by a factor. The small black arrow allows you to
enter the factor.
Turn chromatogram round (invert).
Delete spikes. Pale red areas appear in the chromatogram. If you click on
one of these areas, all the peaks within it will disappear.
Change data point distance (slice width). The new data point distance in ms
can be entered in the box.
Smoothing chromatogram. The small black arrow allows you to enter the
number of data points over which an average should be found.
Baseline correction.
Enhance peaks. The small black arrow allows you to enter the gradient
width.
Frequency filter
Chromatogram arithmetic. The small black arrow leads to the operations
addition, subtraction, multiplication, division, link two chromatograms and
find the first and second derivative.
Allow for radioactive decay. The small black arrow allows you to enter the
half life time and a reference level.
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Undo the last action
Redo the last undone action.
Chromatogram Arithmetic
To carry out arithmetic operations on two chromatograms, click first on the
operation, then click again on Arithmetic and then select the second
chromatogram from the list of files. The result will be shown in the
Arithmetic window: the name of the operation carried out will be shown in
the title bar, with the two original chromatograms below and the result
(sum, difference, product, quotient, linked chromatogram, first or second
derivative) right at the bottom. The latter can be saved as the
chromatogram from an extra channel (Save As New Channel). Replace
replaces the original chromatogram with the sum, difference, product or
quotient, the linked chromatogram or the first or second derivative.
In the GLP mode Transform is not accessible, i.e. the Transform key is
grey.
ChromStar 7 – Batch Wizard
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7. Batch Wizard
This wizard takes the user through calibrations, analysing series, printing
series, comparing chromatograms and many more features. The choice of
folders, files, required parameters and method settings will be asked for
step by step, depending on the task in hand.
After clicking on the link to the Batch Wizard module, the module
window will appear and you can select one of the many functions
available. The box that is currently active at the time, in which you
need to enter data, is highlighted in blue. Click Continue to
activate the next box.
Operations:
Printjob
Reintegration
Integration With New Method
Recalculation
Recalculation With New Method
One Point Calibration
Average Calibration (Averaging response
factors)
Multi Level Calibration
Summary Table
Compare Chromatograms
Convert Data
Scattering Calculation
Result Export
The function keys have the following meaning:
Restart opens the start page. Back opens the previous page. Continue
activates the next page. Finish carries out the chosen action. Exit closes the
Batch Wizard.
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7.1 Print
You can print a series of chromatograms using the Batch Wizard. To do
this, select the function Print and click Continue.
After selecting the directory with the corresponding
button, all the
chromatograms in that directory will be displayed
below. You can
select one chromatogram (also by double-clicking) or several (using the
Shift or Ctrl key according to Windows convention) and add it to the list
below using Add. The Delete key removes chromatograms from the list.
Click Continue.
Answer the question ‘Do you want to print the results?’ as required. In
order to print, you need to select a report template from the list using the
downwards arrow, and a printer from the list of installed printers. Click
Continue.
The list that appears next contains all the print templates selected (under
‘Report’) and all the chromatograms selected (under ‘File Name’). The print
template for each chromatogram under ‘Report’ can still be changed using
the downwards arrow. A highlighted line can be deleted using Delete. Add
will take you back to the chromatogram selection screen, and the
chromatograms you have already selected will still appear in the list. You
can add more chromatograms.
Finish starts the print job.
7.2 Reintegration
You can use the Batch Wizard to reintegrate a series of chromatograms.
To do this, select the function Reintegration and click Continue.
After selecting the folder with the corresponding
button, all the
chromatograms in that folder will be displayed
below. You can
Click Continue.
Continue.
The list that appears next contains the function ‘Reintegration’ under the
heading Type, and all the chromatograms selected (under ‘File Name’). A
highlighted line can be deleted using Delete. Add will take you back to the
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chromatogram selection screen, and the chromatograms you have already
selected will still appear in the list. You can add more chromatograms.
Finish carries out the reintegration.
If you select the option Integration With New Method, you can select a
folder and chromatograms as above and then select a method. The
integration parameters of this method will be used for reintegration.
Options for printing the selection and editing the list are the same as
above. Finish carries out the reintegration.
7.3 Recalculation
The Batch Wizard allows you to recalculate one or more chromatograms.
To do this, select the function Recalculation and click Continue.
button, all the
below. You can
Click Continue.
Continue.
The list that appears next contains the function ‘Recalculation’ under the
selected will still appear in the list. You can add more chromatograms. The
calculation values Factor, Weight and Int.Std. can still be changed for each
chromatogram. The meaning of these parameters is given in the
Sequence (pages 34 and 35).
Finish carries out the recalculation.
If you select the option Recalculation With New Method, you can select a
folder and chromatograms as above and then select a method which
contains the desired parameters on the calculation page (calculation
method, response factors etc.). Options for printing the selection and
editing the list are the same as above. Finish carries out the recalculation.
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7.4 One point calibration
You can use the Batch Wizard to do one point calibrations at a later date.
To do this, select the operation One Point Calibration and click Continue.
button, all the
below. Click on
the chromatogram with the standard mixture and add it to the list below
using Add. Click Continue.
In the next box, you need to select a method. The list of methods can be
accessed using the downwards arrow. This method contains External
Standard as its calculation method on the Calculation page. It also
contains the dimension of the quantity under Calculation Unit and the
standard quantities of all the peaks to be calculated in the peak table.
Continue.
The list that appears next contains the function ‘One Point Calibration’
under the heading Type, and the chromatogram selected (under ‘File
Name’).
Finish carries out the one point calibration.
7.5 Calibration with calculation of the average
The calibration with calculation of average generates an average
response factor. To do a multiple calibration with calculation of the
average, several chromatograms are first recorded from a standard
solution. A standard solution can incorporate several standards here.
External Standard is selected as the calculation method and a calculation
unit is entered. The peak table contains the standard quantities for all the
substances to be determined under Amount in Std and the names under
Peak. Subsequent evaluation of the calibration chromatograms is done in
the Batch Wizard using the operation Calculate Average. After clicking
on this function, click Continue.
button, all the
below. You can
Click Continue.
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Standard as its calculation method on the Calculation page, the
dimensions of the quantity under Calculation Unit and the standard
quantities of all the peaks to be calculated in the peak table.
order to print, you need to select a report templates from the list using the
Continue.
The list that appears next contains the function ‘Average’ under the
Finish carries out the calibration.
The results of the calibration will be displayed in a window.
A chromatogram can be excluded from the calculation by clicking in the
Enabled column of the table (separately for each peak). The results of
calibration with calculation of the average are saved in the method by
clicking on Save.
7.6 Multi Level Calibration
For Multi level calibration, a calibration function is established as the
dependency of the peak area on the quantity of a peak. In the method,
select External Standard on the calculation page as the calculation
method, give the concentration unit under Calculation Unit and the number
of different calibration mixtures under Number of Concentration Levels.
The various quantities in the calibration mixtures and the peak identifiers of
all the peaks are entered in the peak table. Subsequent evaluation of the
calibration chromatograms is done in the Batch Wizard using the function
Multi Level Calibration. After clicking on this function, click Continue.
button, all the
below. You can
Click Continue.
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Standard as its calculation method on the Calculation page. It also
contains the dimension of the standard under Calculation Unit and the
standard quantities of all the peaks to be calculated in the peak table.
Continue.
The list that appears next contains the function ‘Multi’ under the heading
Type, and all the chromatograms selected (under ‘File Name’). A
selected will still appear in the list. You can add more chromatograms. If no
allocation of the standard concentration (using the level number) to the
injection number (or to the vial number when using an autosampler) was
made when recording the chromatograms in the sample table, you need to
enter the level number belonging to a chromatogram now in the table
under Level Number.
Finish carries out the calibration.
The results of the calibration will be displayed in a window.
A chromatogram can be excluded from the calculation by clicking in the
Enabled column of the table (separately for each peak). Clicking on Zero
will include the zero point in the regression. You can enter a blind value for
the zero point on the y axis. The results of calibration with calculation of
the regression functions are saved in the method by clicking on Save.
7.7 Summary Table
A series of chromatograms can be analysed and displayed in table form
using the Batch Wizard. To do this, select the function Summary Table
and then click Continue.
button, all the
below. You can
Click Continue.
The next window, entitled Chromatogram dependent Columns allows
you to create the first part of the table from the available table columns (file
name, vial number, injection number from vial, line number). After selecting
the columns with the red arrow click Continue.
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The second part of the table contains Peak dependent Columns such as
base height, quantity, peak identifier, relative quantity and retention time.
After selecting the columns with the red arrow click Continue.
The Peak identification list determines how a peak is to be identified: by
its peak identifier or retention time. This allows you to limit the number of
peaks found. The Import Peaks button allows you to add retention times
and peak identifiers from a selected method. Click Continue.
The Summary Table now contains the values relevant to the peaks for all
the chromatograms selected. Columns and rows of the summary table can
be selected, copied using Ctrl C and inserted using Ctrl V e.g. into Word or
Excel. Click Continue.
The Summary Export Options windows allows you to select where the
table should be exported to. The file format *.csv is available, in which the
columns can be separated with a semi-colon or tab. Enter the file name in
the Save As box.
Finish saves the file. A message will appear to tell you where the results
table has been successfully exported to. You can now open this file in a
spreadsheet program such as Excel.
7.8 Compare Chromatograms
A series of chromatograms can be presented in the Batch Wizard in order
to compare them. To do this, select the function Compare
Chromatograms and then click Continue.
button, all the
below. You can
Click Continue.
The list that appears next contains the function ‘Compare’ under the
Finish opens the comparison window. The selected chromatograms are
displayed in a window in cascade form.
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The meaning of the function buttons is as follows:
Add opens the file selection window. You can select one or more
chromatograms that you also wish to display.
Reset removes all the chromatograms displayed from the window.
Offset opens a window in which an offset between the chromatograms can
be selected on the x and y axes.
Colours opens the window in which colours are allocated to the individual
chromatograms. They can be changed by clicking on one of the coloured
boxes. Apply makes the new choice of colour take effect.
Print first opens a window in which you can change the pre-selected printer.
When you click OK, printing will begin.
Scaling opens a window in which you can change the scaling of the x and y
axes.
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selected.
printed.
chromatogram.
Display Peak Names displays peak names.
In the image below, the offset, colours and scaling have been changed from
the image above and another chromatogram has been added.
The ‘x’ button closes the comparison window.
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7.9 Convert Data
In the Batch Wizard you can convert chromatograms recorded with
ChromStar 6 into ChromStar 7 compatible chromatograms. To do this, select
the function Convert Data and click Continue.
The next input defines whether simple chromatograms or DAD data are to be
converted. Choose the directory into which the converted data are to be
saved. Click Continue!
After selecting the directory with the ChromStar 6 data with the corresponding
button, all the chromatograms in that directory will be displayed below. You
can select one chromatogram (also by double-clicking) or several (using the
Shift or Ctrl key according to Windows convention) and add it to the list below
using Add. The Delete key removes chromatograms from the list. Click
Continue!
Using Finish the data are converted. A list appears showing the successfully
converted data. Also the directory into which the converted data are saved can
be shown. The window is closed by OK.
7.10 Scattering Calculation
In the Batch Wizard a scattering calculation from a series of chromatograms
can be carried out. The chromatograms must be calculated with a method
other than percent. Select the function Scattering Calculation and click
Continue.
After selecting the directory with the corresponding button, all the
chromatograms in that directory will be displayed below. You can select one
chromatogram (also by double-clicking) or several (using the Shift or Ctrl key
according to Windows convention) and add it to the list below using Add. The
Delete key removes chromatograms from the list. Click Continue!
Answer the question 'Do you want to print the results?' as required. In order to
print, you need to select a report template from the list using the downwards
arrow, and a printer from the list of installed printers. Click Continue.
The list that appears next contains the function 'Scattering' under the heading
Type, and all the chromatograms selected (under 'File Name'). A highlighted
line can be deleted using Delete. Add will take you back to the chromatogram
selection screen, and the chromatograms you have already selected will still
appear in the list. You can add more chromatograms.
Finish carries out the scattering calculation. A message appears informing that
all tasks are successfully carried out. The result of the scattering calculation is
stored in each of the chromatograms involved and can be shown and be
printed in Reprocess.
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7.11 Result Export
In the Batch Wizard the results of a series of chromatograms can be
exported into a csv file. Choose the action Result Export and press Continue.
After selecting the directory with the corresponding button, all the
chromatograms in that directory will be displayed below. You can
select one chromatogram (also by double-clicking) or several (using
the Shift or Ctrl key according to Windows convention) and add it to the list
below using Add. The Delete key removes chromatograms from the list. Click
Continue!
Choose an export print template. Using the downwards arrow all templates in
the directory ..\system\exporttemplates are available.
Finish starts the export. The exported files have the same name as the
chromatograms and the file extension .csv. They are stored in the same
directory as the chromatograms.
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8. Report Editor
The Report Editor allows you to create individual report templates (*.cps).
The information to be printed can be generated from a range of items,
such as the chromatogram, results list, method, data recording and
analysis parameters, graphs, external files etc., with the current
parameters automatically being used. This means that the standard
templates provided can be modified and adapted.
After clicking on the link to the Report Editor module, you will
see a blank page which is the print template. On the right-hand
side, the available items are arranged under different headings.
Basic Items
Chromatogram
Sample Information
Method Values
Channel-Dependent Values
Results
Page Status
DAD Items
Graph Items
Runtime Parameters
The menu options in the Report Editor have the following meanings:
File
New closes the print template that was already loaded.
Open opens the file selection window where a new print template can be
loaded.
Save saves the entire print template.
Save As... saves the whole print template under a new name.
Print opens a window from which the print template (*.cps) can be printed
out. The standard printer is used.
Print preview opens the preview of the current print template.
ChromStar 7 - Report Editor
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Export Output saves the template with the example chromatogram as
*.csv-file.
Recent Reports allows quick access to recently opened files.
Exit closes the Report Editor.
Edit
Undo undoes the most recent change.
Redo repeats the change that has just been undone.
Paper
Page Layout opens a window in which the width of the page margins and
the header and footer can be set.
Background opens a window in which a background can be selected. This
will appear in the printout as the background on every page.
Items lists the elements included in the print template, classified under
header, page body and footer.
Zoom, Larger, Smaller, Standard increase or reduce the size of the
template on the screen, or return it to its original size.
Example File opens a file selection window in which you can select a
results file (*.cfd) or method file (*.met) and add it to the current print
template as an example.
Recent Example Files allows quick access to recently opened files.
Help
About opens a window containing information about the Report Editor.
To enter an item, click on it on the list on the right. Then move the cursor to
the place on the page on the left where you want the item to appear, hold
down the left mouse button and drag out an area of the size you want the
item to appear. The item will then appear with small crosses at the corners
and in the middle of the sides. When an item is indicated as active in this
way, it can be moved (when the cursor appears as two crossed arrows) or
enlarged/reduced (when the cursor appears as a double arrow). Clicking
on an active item with the right mouse button makes a menu appear
containing the options Properties, Delete and Duplicate. Clicking on
Properties takes you to a window that will differ for each item, containing
the properties that can be determined for that item. Clicking on Delete
deletes the item. Duplicate duplicates the item.
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8.1 Basic Items
The basic items are:
Text
Adds a box in which a text can be entered. The Properties option sets the
alignment of the text in the box, a border and the font.
User text
input
This box allows you to enter your own text directly before printing a results
file with a print template that contains this item. The text is entered in the
same window as the one where the print template is chosen. This item
should only be included in the print template once.
Group box
Adds a group box. When data is recorded from multiple channels, this box
only contains the values for added items belonging to one channel. The
Properties option allows you to add a border and the channel number. If
one of the elements in a group box has no value, no element at all is
displayed. In this case the elements should be placed in different group
boxes.
Item
Repeater
Adds a group box. The Properties option determines whether the items
should be repeated for a file or for several specific channel numbers.
CSVSeparator
A print report template which is to be used for CSV-export can start with
the element Separator. The template must be saved in the directory
...System\Exporttemplates, where it is available for the export in the
Runtime module and in Reprocess. Use the right mouse key to open the
property box. Now as separator are choosable: Semicolon, Comma,
Tabulator. If a template without separator is used for the export, the
semicolon is automatically used as default separator in the CSV file.
8.2 Chromatogram
The following items are located under the heading Chromatogram:
Chromatogram
Adds the chromatogram. Properties allows you to determine
various chromatogram properties to be displayed.
Version
Version number.
Point No.
Number of data points in a chromatogram.
Runtime
Runtime of the chromatogram in minutes.
Wavelength
When using a DAD: wavelength of the DAD in nm, at which the
chromatogram is registered.
Channel
Number of the recording channel.
Recording Offset
Offset when recording data. The detector signal value is shifted
by this offset amount.
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Recording Factor
Factor when recording data. The detector signal value is
multiplied by this factor.
Chromatogram Properties
The following chromatogram properties can be set:
General
General properties
Format: portrait or landscape format
Annotation: labels the peak number, peak name, retention time
and/or peak area
Scaling. Auto Scaling: Scaling as saved after recording the
chromatogram; Chromatogram Scaling: Scaling as entered in
Reprocess; Custom Scaling: Scaling as defined against MinX, Max
X, Min Y and Max Y
Legend
Axes
Composition and annotation of axes
Solvents
Plotting in the solvent run with different colours for different
solvents, e.g. when gradient programming
Peak windows
Shows the peak window
Chromatogram
Chromatogram display as curve and/or in high quality. Display of
baseline by clicking on Visible. Chromatogram and baseline display
in different colours and different line widths.
Data
Multiple channel display by adding the channel numbers; display of
the selected channel: channel number = 0.
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8.3 Sample Information
These items are entries made in the sequence used.
Injection/Vial
Number of the injection or vial.
Injections per Vial
Number of the injection from a vial in the case of successive
injections.
Sample identifier
Name of the sample entered in the sequence.
Sample factor
Factor by which the result of the quantitative analysis is
multiplied.
Sample weight
Total weight of the sample entered in the sequence.
Sample Int.Std.
Weight of the internal standard in a sample, insofar as this
weight differs from the quantity weighed in during calibration. It
is entered in the sequence.
Sample Conc. level
Level of calibration solution in a multi-level calibration, entered
in the sequence.
Sample information
Extra sample information, entered in the sequence.
Injection volume
Injection volume of the autosampler
8.4 Method Values
The items in this section are the same for all the chromatograms in one
method, independently of the channel number with which they were
recorded. The entries in ChromStar pertaining to the items are made in the
method file on the Information/General, Information/Documentation, and
Time Table pages. The following items are included here:
Author
Date
Date when the method was created.
Notes
Time table used
Devices were controlled with commands entered in the time
table.
Results file naming
Composite of the results file name (*.cfd, chromatogram).
Slice width
Data point distance in ms
Runtime
in min
Documentation
One of the groups in the documentation is printed. The choice
of group is made by right-clicking and selecting Properties.
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Complete
Documentation
The documentation page will be printed.
Time table
The time control table will be printed.
Method file name
Name of the method file without indicating the file path.
Method file name
(complete)
Adds the name of the method file ( *.met) and indicates the file
path .
Method History
The method history will be printed.
8.5 Channel-Dependent Values
The following items dependent on the channel can be incorporated into the
print template:
Channel type
UV or DAD
Cahnnel name
As defined in the configuration
Sensitivity
Integration parameter
Wavelength
DAD wavelength
Resolution
Wavelength resolution
Calculation Base
Entered in the method on the calculation page
Calculation Method
Concentration Level
Number of standard solutions (level, number of
concentrations) entered in the method on the calculation
page
Calculation Unit
Column Length
Column Deadtime
Peak Table
The peak table is printed.
Calibration Curve
After calculating the average response factor or doing a
multi-level-calibration, the graph or regression curve can be
printed.
Regression Table
The regression table is printed
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Integration Parameter
Table
The integration parameter table, entered in the method on
the data recording page, is printed.
Calibration Table
After doing a calibration, the calibration table can be printed.
8.6 Results
The following results items are available:
Results Table
The results table (for each channel) of a chromatogram is printed.
A group box needs to be added beforehand in which the table is
allocated to one of the data channels (Docked Channel)
by right-clicking on the table and selecting Properties. The item
‘results table’ is dragged into the group box, and then the table will
appear filled in. Properties allows you to change the table in
various ways, e.g. to highlight valid/invalid results.
Audit Trail
The Audit trail can be printed.
File
Documentation
Notes about the computer, operating system, device configuration
etc. can be printed in a list.
Deviation Display
If the result of a peak is out of the limit entered in the peak table, a
message is printed.
Scattering Table
can be printed.
Signature
In the print-out of a chromatogram signed in the GLP
Mode a picture will be inserted.The size of the picture can
be changed.
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8.7 Page Status
The following items are available here:
New Page
Starts a new page.
Page No.
Prints the current page number.
Number of Pages
Prints the total number of pages.
Filename (full)
Adds the name of the file to be printed (method file *.met or
results file *.cfd with file path ).
Filename
Name of the file to be printed without file path.
Template name (full)
Adds the name of the print template (*.cps with file path).
Template name
Name of the print template without file path.
Print Date
Current date.
Print Time
Current time.
Current User
Name of the current user.
The items Page Number and Number of Pages can be linked by the word
“of” and are located in the header or footer as preferred.
8.8 DAD Items
Items from a DAD data set are available for printing in this section.
Density Plot
Spectrum
Spectrum notes
Spectrum
parameters
Peak Purity
DAD
Parameters
Peak purity values can be printed in a list. If you right-click the
mouse in the table, a menu will appear with the options Properties
and Delete. Properties allows you to alter the table in various ways.
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8.9 Graph Items
Graph Items:
Picture
A picture is added to the print template. The name is selected from the file
selection window. The image must be available as a separate file at the
given location on the computer when printing.
Line
A line is added to the print template. Line thickness and colour can be
chosen freely.
8.10 Runtime Parameters
The following runtime parameters can be incorporated as elements in the
print template:
Pump pressure at
start
Counter-pressure of the system at the beginning of
chromatogram recording.
Injection time
Time of injecting in minutes and seconds.
Injection date
Date when the chromatogram was recorded.
ChromStar 7 – Navigator
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9. Navigator
The navigator makes it easier for users to work with ChromStar and will
take you quickly to the individual modules. First you need to enter your
user name and select the required device configuration from the list of
configurations available. Then the navigator box will appear with the most
recently used sequences on the left, an image of the system in the middle,
the individual program modules around it and the data folder determined in
the configuration containing the sub-folders and all the ChromStar files
they contain (*.csq, *.met and *.cfd) on the right.
In the GLP mode User name and password have to be entered. After
logging-in only instrument configurations appear for which the user has the
access right. Not-GLP-conform instrument configurations are marked.
When you click on one of the sequences on the left, all the working options
(start recording data, show or edit sequence) and all the file folders will
appear on the right with a black border. If you drag the file into one of the
boxes with a black border, first the border will turn yellow, and then when
you release the mouse button, either the appropriate operation will start
(the data recording module or sequence editor will open) or the file will be
copied to the selected folder. If you click on New Sequence, to begin with
only the folders where the sequence can be created will appear with a
black border. If you drag the mouse across one of these folders, a box will
open in which you can enter a new name. Click OK, and the sequence
editor will open. The new sequence can now be created and saved.
The double cross at the sequence editor can be moved either on the
Method editor or on the Reprocess module or on the Report editor. The
display on the left changes accordingly in “last methods”, “last
chromatograms” or “last report templates” and allows the corresponding
operations.
Clicking on a sub-folder on the right will reveal its contents. The file will
close if you click on it again. You can preview the chromatogram in a *.cfd
file by rolling over it with the mouse. If you click on one of the
chromatograms, a black border will appear around the operations that can
be carried out on it and the folders to which it can be copied. The possible
operations are reprocessing to do further work on the chromatogram or
processing the method it uses with the method editor.
Using these keys files can be sorted chronologically or
alphabetically, each in ascending or descending order.
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_____________________________________________________________________________________________________________
Clicking on a directory with the right mouse key opens a menu with the
sub menu Create sub directory, Explore directory and Delete and
allows these activities.
Clicking on a file with the right mouse key opens a menu with the sub
menu Open, Explore file and Delete and allows these activities.
A ChromStar7 data file (*.cfd, *.csq or *.met) can be opened or copied in
the appropriate module by drag and drop from an explorer directory onto
the Chromstar Navigator. The appropriate module appears with a black
border. On dropping the file the action is started.
The individual program modules can be opened with a single click.
ChromStar 7
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10. User Administration
A ChromStar user can enter his /her name and password for logging in.
Different access levels for using ChromStar program modules and
instruments can be assigned to the user. The list to the left shows all users
registered by User, Add User. The user's details such as name,
password (hidden) and permissions appear to the right on clicking on the
user’s name. In the box under Signum the user's name and function are
entered in the way they should appear as signature.
The following menu options and screen buttons are available:
File
Security Level – With Authorization. The user must log into the User
Registration box with their name and the corresponding password. The
password must have at least 7 characters and must contain a number or
another sign.
Security Level – No Authorization. The name of the most recent user
appears in the User Registration box and can log in immediately, as well as
another registered user.
Security Level - GLP. In the GLP mode a user can only open a ChromStar
module with his name and password as entered in the administration
module. The GLP mode can only be quitted by the administrator. In the GLP
mode the system can be closed manually in the navigator by using the
appropriate button or automatically after a certain time (Inactivity time,
s.below). All ChromStar modules are closed except the data acquisition
module. This one can be opened only by a user with access rights. With
the automatic closing a warning appears some time previously .
Synchronisation. A path name belonging to a network can be defined for
the user's administration. In case the entry box for the name of the directory
is empty no synchronisation occurs.
General Audit Trail. The general Audit Trail (gat.bin file, in the
...\ChromStar\system directory) saves in a list all actions carried out with
ChromStar together with date, time and user's name. Documented actions
are: Opening and closing a module, saving a file and much more. The
general audit trail can be opened and - also in parts - be printed-out.
Exit closes the module and saves all changes.
Setup
Lock-Time. Here the administrator enters a time for which a user is not
allowed to open a ChromStar module, if the user has tried three times in
vain to open ChromStar (e.g.by using a wrong name or password). A
locked user can only be de-locked by the administrator.
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Inactivity Time. Here the administrator enters a time of inactivity, after
which all ChromStar modules are closed - except data acquisition module.
Time of inactivity means no entry in ChromStar were made.
Service
This menu procedure can only be used by the administrator to re-establish
GLP mode conformity
User
Add User opens a box in which the new user’s name is entered. In the
next box, user data such as the password and various authorisations
are determined and limited as appropriate. In the Device Authorisations box,
one or more device configurations can be chosen and excluded from using.
Delete User deletes the selected user from the list.
Help
About opens an information box.
ChromStar 7 – Working with ChromStar
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11. Working with ChromStar 7
11.1 How do I create a print template?
To print a chromatogram in the way it is displayed above, you need to create a
print template (*.cps) with the report editor. The name of this template can
then be entered in the sequence under Report. This means that the
chromatogram will be printed on the printer installed as the standard printer as
soon as the chromatogram has been recorded. If you already have the
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chromatogram, open it in Reprocess and select File, Print to print it. You can
select the template and, if required, a different printer.
Below you will find step-by-step instructions on how to create a print template.
Double click on the Report Editor to open it.
The menu options Paper and Layout set the margins and page format (DIN
A4):
Create the header:
Click on Basic Elements, followed by Text. The cursor appears as a + and
can be moved to the required position in the header. Drag out a box by
holding down the left mouse button; double-click on the box to highlight the
word Text, replace it with the word Chromatogram, then repeat the whole
process to add the words Date: and Time:.
Click on Page Status, then Print File Name (full). Move the cursor to the
required position (after the word ‘Chromatogram’) and drag out a box. Repeat
the whole process for the items Print Date and Print Time.
To layout and format the items, hold down the Shift key and click on all the
items in the header consecutively. Then right-click with the mouse and select
the following from the menu: Layout and then same y position, Size and then
same height, Font and then Times New Roman 10.
Create the page body:
Click on Method Values. Select the items Author and Notes one after the
other and add them at the required position on the page. To edit a single box:
highlight the box by clicking on it, then double click so that the sub-boxes can
be altered independently of the others.
Click on Sample Information. Select Sample Identifier and add it at the
required position.
Add the basic element Text and replace the word Text with Data Acquisition
Parameters.
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Click on Method Values, select the item Slice Width and add it at the
appropriate position.
Click on Channel-Dependent Values, select Sensitivity and add it at the
appropriate position.
Click on Chromatogram, select Chromatogram and add it at the appropriate
position.
Right-click, then click Properties to open a dialogue in which you can set
various chromatogram properties.
Under Axes you can set the font in which the chromatogram scales will be
shown (Scale font) and the axis labels (Label font) (here: Times New Roman
12). The Baseline Visible option has been checked under Chromatogram.
To add the results table, you first need to add the basic element Group Box.
Right-click, open Properties, and in the dialogue that appears you can remove
the caption (Caption style is set to No Text).
Click on Results, select the item Results Table and add it to this group box.
Highlight the results table by double-clicking. Now right-click and select
Properties. In the dialogue, you can determine which columns will appear in
the results table under Column. The table shown here contains the columns
Peak number, Peak Start (min), Peak End (min), Ret. Time (min), Area,
Height (mV), Percent and Name. All the other columns are clicked away to the
left using the << arrow. For each individual column, set a heading under
Name, the number of decimal places under Accuracy and the column width
under Width. Under Column Heading Font, Column Font, Results Header
Font, Description Font and Value Font, select Times New Roman each time.
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The page numbering is located in the footer. To add numbering, add the
basic element Text twice and change the text to Page: and of: respectively.
Under Page Status, select the items Page Number and Number of Pages
which are added and formatted in the same way as described for creating the
header.
Use the menu options Paper, Example File to load a chromatogram into the
newly created print template so that you can test the individual elements and
parameters.
The menu options File, Print Preview will open the page view as shown
above.
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11.2 How do I record a chromatogram?
These instructions assume that you have already installed ChromStar 7 and
that you have configured a system consisting of a pump, autosampler and UV
detector.
Start ChromStar 7 using the Start Menu (Start, Programs, ChromStar7,
Navigator).
First you use the Navigator to select your system configuration.
The image of the selected system – entered in the configuration under
General Properties – will appear at the centre of the Navigator. In this case
the UV system is selected. Both the configuration file (Anlage-uv.ccf) and the
image of the system (Helgas Anlage.jpg) are in the System sub-directory of
the current Chromstar 7 directory.
The Navigator shows the most recently used sequences on the left. On the
right you will see the data directory specified in the configuration with all its
sub-directories and ChromStar files such as sequences, methods and
chromatograms.
Now you need to switch on the chromatography devices belonging to the
system (pump, autosampler and UV detector).
To create a new sequence, click on New
Sequence, hold down the mouse button and
drag it to the directory where the sequence
is to be saved. The folder will be highlighted
with a yellow border.
Enter the file name in the dialogue box and
click OK. The new sequence will be saved at once and opened in the
Sequence Editor. Now you can enter the required data.
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Enter the desired vial number or vial series in the first line of the sequence.
On the right you can select the method to be used from the methods
available. The table below contains pre-set values for the number of
injections, injection volumes and runtimes. You can change these values as
required.
The button to the right of the method name is used to open the method
selected in the Method Editor. Now it can be changed as required. The
results file name (chromatogram) on the Information/General page will
be generated automatically from the method name and a serial number
(autosampler vial and injection number), unless you state otherwise. Entries
for Author and Notes are optional. The data recording parameter Runtime has
been set to 2 min and the data point distance is 250 ms. If Time control table
is checked, a separate page will appear where entries can be made which
solvent is to be pumped and at which flow rate.
Under Information/Documentation you can enter the chromatography
conditions (optional):
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The Time Table determines what the pump does:
Now all the important entries in the method have been made, and the method
is saved.
The samples are put into the autosampler at the pre-determined positions.
The RP18 column is installed between the autosampler and detector and the
solvent mixture of 70% methanol and 30% water is connected to the pump as
Solvent A.
In the Navigator, drag the sequence into the circle Start Acquisition. This
automatically opens the Data Acquisition Window and the pump will start
pumping the solvent mixture at the pre-set flow rate.
The following display is shown at the control panel:
As soon as the pressure and detector signal are constant and the
column is adequately conditioned, the sequence can be started by
pressing Start.
While the autosampler is preparing the injection the following message will
appear: Wait for injection, Vial: 1 Injection: 1.
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After the injection, information will appear in the information bar at the bottom
edge of the data recording window to show the time elapsed since the
injection, the vial processed (number, injection number and sample identifier)
and the name of the chromatogram.
The detector signal of the channel identified as UV appears in the
chromatogram window.
After recording both chromatograms – as determined in the autosampler table
– the pump can be stopped and the data recording window closed. The two
chromatograms, new demo-001-1.cfd and new demo-002-1.cfd, will appear in
the Navigator in the file list on the right and can, for example, be displayed
and further processed in Reprocess.
ChromStar 7 - Calculation Methods
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11.3 Methods for a Quantitative Evaluation
ChromStar offers six different methods for making a quantitative calculation:
1) Percent method
2) Standardisation method (Normalisation
3) External standard method
4) Internal standard method
5) External standard method with calibration function
6) Internal standard method with calibration functions .
These methods were determined on the Calculation page of the method file by
making the appropriate entries under Calculation Method. An internal
standard is defined in the peak table with the code IS.
For the first three calculations, the following universal formula is used to
calculate quantities:
RESi =
(PKSi * RFi ) * MULT/ DIV (1)
Where:
RESi
=
Result of the quantitative calculation for peak i
PKSi
=
Peak area or peak height of peak i
RFi
=
Response factor of component i (RF value as
determined by the type of calibration)
MULT and DIV depend on the method used and will be explained in the
following paragraphs.
11.3.1 Percent Method
In the percent method, a percentage analysis of all the peaks found is carried
out. The factors in formula (1) have the following values:
RFi
=
1
MULT
=
100
DIV
=
sum of PKSi
and (1) therefore takes the form:
RESi =
PKSi * 1* 100 / (sum PKSi)
No entries in the peak table are required for this calculation method. All the
peaks whose area or height is greater than the value entered as the minimum
area or height are used in the calculation.
This calculation is automatically made for every chromatogram if no instruction
to the contrary is given. The calculation can be repeated with saved data in
the Reprocess or Batch modules.
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11.3.2 Normalisation
In Normalisation, only peaks that are included in the peak table and fall within
a defined time window are brought into the calculation. The RF value
(response factor) has the value given in the peak table for each peak. The
pre-set value is 1. It can be changed manually for each peak or determined in
a calibration run. The other factors have the following values:
DIV
=
Sum of (PKSi * RFi)
MULT
=
100
RFi
=
Quantity/area of peak i, determined by calibration run
and formula (1) is therefore as follows:
RESi
=
PKSi * RFi * 100 / sum of (PKSi * RFi)
11.3.3 External Standard Method
To calculate the quantity by this method, the RF values of a sample with
known quantities of the substance to be investigated (standard) is determined
with the help of a calibration run. Only peaks that fall within the time window
determined in the peak table are included in the quantity calculation. The
factors in formula (1) have the following values:
RFi
=
Quantity/area of component i, determined in the
calibration run for each peak
MULT
=
1
DIV
=
1
and (1) is therefore as follows:
RESi =
PKSi * RFi
In the run that is underway, the entry under Factor in the sample table of the
method file is equal to the multiplier MULT in (1). If no entry is made, this
multiplier will have the value 1. If a value other than 0 is entered in the sample
table of the method file under weighed portion, the percentage weight
proportion of each component in the total quantity of the sample in the current
run will be calculated additionally to this according to formula (2):
RESi2
=
RESi * 100/ Wspl
(2)
where Wspl is the total weight of the sample entered under weight.
If the advanced settings of the extra peak table parameters in the
configuration are set to Recovery, it will be possible to take a recovery rate
into consideration in the quantitative analysis. The calculation method should
be External Standard. The recovery rate is entered as a percentage in the
peak table under Recovery (%). Different values can be entered for the
individual substances to be determined. The default setting is the value 100
for 100% recovery.
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In the calculation, the quantity is determined according to the following
formula:
RESi = PKSi * RFi * 100 / RCi
Where:
RESi = Result of the quantitative calculation for component i
PKSi = Peak area or peak height of peak i (of component i)
RFi = Response factor of component i
RCi = Recovery rate in % for component i
11.3.4 Internal Standard Method
In this method, which is not dependent on an exact sample quantity injection,
a substance (the internal standard) is added to both the calibration standard
with known quantities and the sample to be investigated. For this method, it is
necessary to define the standard peak as the internal standard in the peak
table by entering IS under ‘Code’. If no such identifier is found, the error
message
"No internal standard peak defined"
will appear. The standard peak is defined as the largest peak found in the time
window identified as IS. If no peak is found within this time window, the error
message
"No internal standard peak found"
will appear. Both error messages will be followed by an analysis according to
the percentage method.
In the internal standard method, the response factors are determined
according to the following formulae:
Resp. Fact. i
=
Amount i Std. * PKS IS Std. / PKS i Std.
Resp. Fact. IS
=
Amount IS Std. / PKS IS Std.
where
Amount i Std.
=
Amount of substance i in the standard solution
Amount IS Std.
=
Amount of the internal standard in the standard
solution
PKS IS Std.
=
Peak area or height of the internal standard in
the calibration run
PKS i Std.
=
Peak area or height of substance i in the
calibration run
The quantity calculations are made according to the following formulae:
Amount i
=
Resp. Fact i * PKS i / PKS IS
If another amount of internal standard than in the standard solution is injected,
the PKS IS is multiplied by the ratio amount IS Std / amount IS Inj.
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Amount IS
where
PKS i
PKS IS
Amount IS Inj.
= Resp. Fact. IS * PKS IS * Amount IS Std./ Amount IS Inj.
=
=
=
Peak area or height of substance i
Peak area or height of the internal standard
Amount of the standard in the current run
(entered in the peak table under Amt. in Std.
or for the calculation in the Batch Module under
Int. Std., if a quantity other than the calibration
mixture is being used in the current run)
In the current run, the value Amt. in Std. in the peak table for the internal
standard is copied from the value entered in the sample table under Int. Std.,
as long as a value other than 0 has been entered here.
The results list of the quantitative analysis with the internal standard method
still also contains the calculation of the quantity of the substance found as a
proportion of the amount of the internal standard (Rel.).
For the internal standard,
Rel =
Amount IS / Amount IS Inj.
11.3.5 External Standard Method with Regression Function
The calculation of the unknown quantity is made with the equation:
RESi
=
K0i + K1i*PKSi + K2i*PKSi2 + K3i*PKSi3 (4)
where
RESi
=
Result of the quantitative analysis of peak i
K0i, K1i, K2i, K3i = Regression coefficients of the 1st, 2nd or 3rd order for
component i
PKSi
=
Area or height of peak i
The coefficients are generated by multi-level calibration.
In the Batch Module you can choose which order will be used for the analysis.
K0 to K3 will change according to the order chosen. The coefficients and
graph display of the regression can be accessed with the Show Calibration
button on the Calculation page of the method file. Those peaks in the peak
table for which the multi-level calibration was carried out are the ones to be
calculated.
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11.3.6 Internal Standard Method with Regression Function
In multi-level calibration with an internal standard, a calibration relationship is
created between the quantity in the standard solution and the relationship of
the peak area of the substance to the peak area of the internal standard.
The calculation of the unknown quantity is made with the equation:
Amount i =
K0 i + K1 i * (PKS i / PKS IS) + K2 i * (PKS i /PKS IS)2
+ K3 i * (PKS i / PKS IS)3
where
K0i, K1i, K2i, K3i = Regression coefficients of the 1st, 2nd or 3rd order for
component i
PKSi
=
Area or height of peak i
PKS IS
=
Area or height of the internal standard in the current
run
If the quantity of the internal standard differs between the individual solutions,
the vial is standardised to the quantity of the internal standard in the first
calibration solution.
PKS IS Norm. = PKS IS * Amount IS 1 / Amount IS
The coefficients are generated by multi-level calibration. Here the internal
standard in the calibration mixtures needs to be present in the same quantity
(otherwise standardisation occurs, see above). During calibration it is also
decided which order is to be used for analysis. Depending on the order
chosen, K0 to K3 will change. The coefficients and graph display of the
regression can be accessed with the Show Calibration button on the
Calculation page of the method file. Select the method Internal Standard on
the Calculation Parameters page. In the peak table, the standard peak must
be characterised as the internal standard in the peak table by entering IS
under ‘Code’. It must be present in a known quantity in the mixture to be
investigated. This value is entered in the Batch Module under Int. Std. at the
beginning of the calculation if the amount of internal standard in the solution to
be investigated is different from the amount in the first calibration solution.
Those peaks in the peak table for which the multi-level calibration was carried
out are the ones to be calculated.
In the current run, the value Amt. in Std. in the peak table for the internal
standard is copied from the value entered in the sample table under Int. Std.,
as long as a value other than 0 has been entered here.
Page 104
Statistic Formulae - ChromStar 7
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11.4 Statistic Formulae
In the calibration calculations Averaging the response factor and using a
Calibration function and in the scattering value calculations the following
formulae for the statistic evalution are used:
Average value
(xi = measuring value, n = number of measures)
Standard deviation
Relative deviation
(s = sigma)
Confidence region
Own deviation in %

C hrom S tar 7

Transcrição

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