C h r o m S t a r 7. M a n u a l

Transcription

1 C h r o m S t a r 7 M a n u a l

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3 ÿþýü Software für Chromatographie und Prozess-Analytik GmbH Am Weidufer 32, D Weyhe Tel.: , Fax: Internet: Edition May 2012 for ChromStar 7 Version

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5 ChromStar 7 - Contents Page i-1 Contents Page 1. Introduction Installation Setup A/D-Converter Configurator Creating a Configuration General Properties Documentation Setup Calculation Setup Plug In Data Sources Device Control Method and Sequence Information/General Information/Documentation Channel1/Analysis Channel1/Analysis/Calculation Channel1/Peak Table Time Table Sequence Calibrations Data Recording Chromatogram Control Panel DAD Data Recording Spectrum Reprocess Reintegration Manual Integration Reprocess - Calculation Reprocess Method Reprocess Navigation Bar Reprocess - DAD...61

6 Page i-2 Contents ChromStar Column Coefficients Chromatogram Transformation Batch Wizard Print Reintegration Recalculation One-Point-Calibration Calibration with Calculation of the Average Multi Level Calibration Summary Table Compare Chromatograms Convert Data Scattering Calculation Result Export Report-Editor Basic Items Chromatogram Sample Information Method Values Channel-Dependent Values Results Page Status DAD Items Graph Items Runtime Parameters Navigator User Administration Working with ChromStar How do I create a print template? How do I record a Chromatogram? Methods for a Quantitave Evaluation Statistic Formulae...104

7 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. features. 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

8 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.

9 ChromStar 7 - Installation Page 3 2. Installation of ChromStar Setup (applicable to version 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 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 2.0- Framework 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 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

10 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 ChromStar is now ready for use!

11 ChromStar 7 - Installation Page 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 Arrangement of plugs.

12 Page 6 Installation - ChromStar 7 ChromStar Integrator 2-channel PCI A/D card Arrangement of plugs 1 -- channel channel channel 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

13 ChromStar 7 - Configurator Page 7 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.

14 Page 8 Configurator - ChromStar 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.

15 ChromStar 7 - Configurator Page 9 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 Alternative peak table parameter 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. 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 List peaks Calculate column coefficients Peak width calculation 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. 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. Yes: column coefficients are calculated (default setting). The values can be entered as breaks in the results table. No: no values are calculated. 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.

16 Page 10 Configurator - ChromStar 7 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 Plug-Ins are: Alias Autosampler 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. Sykam Amino Acid Reaction Module Conductivity Detector GamePort Start Human Autosampler Device Injection Cycle Activity Correction Column Oven Auxiliary Pump Flow Monitoring Server Transfer Chromatography of amino acids with post-column derivatisation controls a conductivity detector. starts a chromatogram via game port signal (no licence file required) allows the autosampler table of the sequence to be used without using an autosampler but injecting manually (no licence file required) starts data acquisition in time intervals. considers the radioactive decay during recording a chromatogram. controls a column oven. controls an additional isocratic auxiliary pump. monitors the flow rate of the main pump and starts an emergency sequence if the pump stops. 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

17 ChromStar 7 - Configurator Page 11 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 NetBox DAD Data is recorded from the A/D converter card. The Ethernet Box is used to record data. 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

18 Page 12 Configurator - ChromStar 7 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) Slope Sensitivity (µv/s) Baseline Max. (mv) Skim Ratio Filter Factor (data points) View Range (mv) Time in ms after which a new data point is registered Sensitivity in µv/s: maximum slope of the detector signal curve that is still not considered as the beginning of a peak. 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. Relationship between the heights of two neighbouring peaks 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. Area over which the y axis is displayed on the screen in mv External stop active Hardware serial number 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. Serial numbers of the A/D convertor cards installed. Channel Properties Channel no. Name Dimension Offset Factor Channel number (1, 2, etc.) Channel name Name of the dimension on the y axis, e.g. mv, µf, ph etc. Value of an offset Factor by which the detector signal is multiplied

19 ChromStar 7 - Configurator Page 13 DAD Properties / Presets Integration time (ms) Exposure time of the diode array in ms. Wavelength resolution (nm) Wavelength range (nm) Intensity range (mv) Diodes Slice Width (Data point distance) (ms) Slope Sensitivity (µv/s) Max. Baseline Level (mv) Skim Ratio Filter Factor (data points) Save DAD data DAD Parameters P0 to P4 Disable/Enable write protection Wavelength resolution for saving data in nm Wavelength range from... to... in nm of the DAD Display area from... to... of the intensity axis on the screen in mau. Number of diodes of the diode array in the diode array detector. Time in ms after which a new data point is registered. Sensitivity in µv/s, maximum slope of the detector signal curve that is still not considered as the beginning of a peak. 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. Relationship between the heights of two neighbouring peaks 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. Indicates whether the DAD data set should be saved. Shutter=0, Buffered=1 Coefficients of the calibration function for the calculation of wavelengths from diode numbers. 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!

20 Page 14 Configurator - ChromStar 7 Ethernet Box Properties / Channel Presets Slice width (ms) Time in ms, after which a new data point is registered Slope Sensitivity (µv/s) Baseline Max. (mv) Skim Ratio Filter Factor (data points) View range (mv) Sensitivity in µv/s, maximum slope of the detector signal curve that is still not considered as the beginning of a peak. 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. Relationship between the heights of two neighbouring peaks 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. 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 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.

21 ChromStar 7 - Configurator Page 15 Pump Properties Type Port Address Type of Gradient Number of the RS232 interface 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 Flow rate Maximum pressure limit in bar 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 Record pump pressure Confirm manual solvent change Equilibration Stop The control panel in the runtime module only allows start and stop of the solvent delivery. The pump pressure is saved in the *.cfd file as an extra data trail. In the runtime module a question appears when the solvent is changed. 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.

22 Page 16 Configurator - ChromStar 7 Autosampler Properties Port Address NetBox Port Driver Presets Number of the RS232 interface Device number Use Ethernet box port Name of a driver file if required (*.dll) Depends on the type of autosampler installed Loop volume Injection volume Maximum volume The injection volume can be changed later in the sequence according to the type of autosampler. Maximum injection volume Maximum injection number No vial action Maximum number of successive injections from one vial. Actions to choose from when a vial is missing: next vial or stop the method. Optional 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. Detector Properties Port Address NetBox Port Driver Presets Number of the RS232 interface Device number Use Ethernet box port Name of a driver file if required (*.dll) Default detector parameters: these can still be changed in the time table of the method file. Wavelength Range Optional parameters You can define extra optional parameters here. If they are programmable, they will appear as extra functions in the time control file.

23 ChromStar 7 - Configurator Page 17 Extra Device 1, 2, Valves Addressing Port Address Number of the RS232 interface Device number Use Ethernet box port Driver Name of a driver file (*.dll) Driver parameters Extra device Device name Extra parameters Name of the extra device 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.

24 Page 18 Method - ChromStar 7 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.

25 ChromStar 7 - Method Page 19 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.

26 Page 20 Method - ChromStar 7 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

27 ChromStar 7 - Method Page 21 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 minutes, in steps of 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

28 Page 22 Method - ChromStar 7 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:

29 ChromStar 7 - Method Page 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 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 the time-dependent, programmable integration parameters. 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:

30 Page 24 Method - ChromStar 7 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 and 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.

31 ChromStar 7- Method Page 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 , 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,

32 Page 26 Method - ChromStar 7 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) average response factors. 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

33 ChromStar 7 - Method Page 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 to , in min) indicates the start time of the time window for a given peak. End. This parameter (from to , 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 E+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 E+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.

34 Page 28 Method - ChromStar 7 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

35 ChromStar 7 - Method Page 29 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.

36 Page 30 Method - ChromStar 7 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).

37 ChromStar 7 - Method Page 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.

38 Page 32 Method - ChromStar 7 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.

39 ChromStar 7 - Sequence Page 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

40 Page 34 Sequence - ChromStar 7 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

41 ChromStar 7 - Sequence Page 35 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.

42 Page 36 Sequence - ChromStar 7 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.

43 ChromStar 7 - Sequence Page 37 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

44 Page 38 Method - ChromStar 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,

45 ChromStar 7 - Method Page 39 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.

46 Page 40 Method - ChromStar 7 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

47 ChromStar 7 - Method Page 41 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.

48 Page 42 Data Recording - ChromStar 7 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.

49 ChromStar 7 Data Recording Page 43 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.

50 Page 44 Data Recording - ChromStar 7 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.

51 ChromStar 7 Data Recording Page 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 Injection can 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.

52 Page 46 Data Recording - ChromStar 7 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.

53 ChromStar 7 Data Recording Page 47 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).

54 Page 48 Data Recording - ChromStar 7 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.

55 ChromStar 7 Data Recording Page 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.

56 Page 50 Data Recording - ChromStar 7 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 Lambert- Beers 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.

57 ChromStar 7 - Reprocess Page 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.

58 Page 52 Reprocess - ChromStar 7 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.

59 ChromStar 7 - Reprocess Page 53 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 Contents opens the online 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. 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.

60 Page 54 Reprocess - ChromStar 7 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. Save Intensity the chosen intensity can be saved by File, Save. 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. 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

61 ChromStar 7 - Reprocess Page 55 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)

62 Page 56 Reprocess - ChromStar 7 Format Results List The button opens a window in which the results list can be formatted. The following columns are available: Amout Area Asymmetry Bandwidth Code Deviation Height HETP K' Name Quantity in standard solution Peak area Column coefficient Column coefficient: bandwidth at half peak height Code as entered in the peaktable of the method file valid or invalid, if defined in the peak table Peak height Column coefficient: theoretical plate height Column coefficient: capacity Peak name Peak End Peak no. Peak number Peak Start Plate number Resolution Result Ret. Time Selectivity Column coefficient: Plate number Column coefficient: resolution Depends on calculation method used Retention time Column coefficient

63 ChromStar 7 - Reprocess Page 57 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.

64 Page 58 Reprocess - ChromStar 7 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.

65 ChromStar 7 - Reprocess Page 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.

66 Page 60 Reprocess - ChromStar 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

67 ChromStar 7 - Reprocess Page 61 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

68 Page 62 Reprocess - ChromStar 7 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).

69 ChromStar 7 - Reprocess Page 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 W 0.5 (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 T 0 in min (e.g. solvent peak) (dead time, R T-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 = * (T/W) 2 Plate height HETP Selectivity H (µm) = L * 10 3 / N a ( i+1/1 ) = k' (i+1) / k' (i) Capacity k' = (T - T 0 ) / T 0 Resolution Ri+1, i = ( T i+1 - T i ) * F/ (W i + W i+1 ) Asymmetry FDA compliant: A = w b / w f A =( w b + w f )/ 2* w f

70 Page 64 Reprocess - ChromStar 7 Defined as follows: L = column length (in mm), T 0 = dead time (in min), T = retention time (in min), W = half value width (width of the peak at half peak height), w f = peak width at 10% of peak height from the rising side of the peak to the perpendicular, w b = peak width at 10% of peak height from the perpendicular to the falling side of the peak. w f and w b 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 = (w b + w f )/ 2* w f. T i+1, where T i = retention time of two neighbouring peaks. F= Factor, as determined in the configuration under Factor for Calculating Resolution. The default setting is Factor=2.

71 ChromStar 7 - Reprocess Page 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.

72 Page 66 Reprocess - ChromStar 7 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.

73 ChromStar 7 Batch Wizard Page 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.

74 Page 68 Batch Wizard - ChromStar 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 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 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

75 ChromStar 7 Batch Wizard Page 69 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. After selecting the folder with the corresponding button, all the chromatograms in that folder 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 Recalculation 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. 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.

76 Page 70 Batch Wizard - ChromStar 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. After selecting the folder with the corresponding button, all the chromatograms in that folder will be displayed 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. 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 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. After selecting the folder with the corresponding button, all the chromatograms in that folder 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.

77 ChromStar 7 Batch Wizard Page 71 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, the dimensions of the quantity under Calculation Unit and the standard quantities of all the peaks to be calculated in the peak table. Answer the question Do you want to print the results? as required. In order to print, you need to select a report templates 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 Average 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 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. After selecting the folder with the corresponding button, all the chromatograms in that folder 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. 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

78 Page 72 Batch Wizard - ChromStar 7 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. 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 Multi 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. 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. After selecting the folder with the corresponding button, all the chromatograms in that folder 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. 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.

79 ChromStar 7 Batch Wizard Page 73 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. After selecting the folder with the corresponding button, all the chromatograms in that folder 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. The list that appears next contains the function Compare 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 opens the comparison window. The selected chromatograms are displayed in a window in cascade form.

80 Page 74 Batch Wizard - ChromStar 7 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. By right-clicking on the chromatogram window, you can access another menu with the following options:

81 ChromStar 7 Batch Wizard Page 75 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. 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.

82 Page 76 Batch Wizard - ChromStar 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 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.

83 ChromStar 7 Batch Wizard Page 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.

84 Page 78 Report Editor - ChromStar 7 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.

85 ChromStar 7 - Report Editor Page 79 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 Contents opens the online 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.

86 Page 80 Report Editor - ChromStar Basic Items The basic items are: Text User text input Group box Item Repeater 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. 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. 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. Adds a group box. The Properties option determines whether the items should be repeated for a file or for several specific channel numbers. CSV- Separator 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 Point No. Runtime Wavelength Channel Recording Offset Version number. Number of data points in a chromatogram. Runtime of the chromatogram in minutes. When using a DAD: wavelength of the DAD in nm, at which the chromatogram is registered. Number of the recording channel. Offset when recording data. The detector signal value is shifted by this offset amount.

87 ChromStar 7 - Report Editor Page 81 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 Solvents Peak windows Chromatogram Data Composition and annotation of axes Plotting in the solvent run with different colours for different solvents, e.g. when gradient programming Shows the peak window 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. Multiple channel display by adding the channel numbers; display of the selected channel: channel number = 0.

88 Page 82 Report Editor - ChromStar Sample Information These items are entries made in the sequence used. Injection/Vial Injections per Vial Sample identifier Sample factor Sample weight Sample Int.Std. Sample Conc. level Sample information Injection volume Number of the injection or vial. Number of the injection from a vial in the case of successive injections. Name of the sample entered in the sequence. Factor by which the result of the quantitative analysis is multiplied. Total weight of the sample entered in the sequence. 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. Level of calibration solution in a multi-level calibration, entered in the sequence. Extra sample information, entered in the sequence. 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 Results file naming Slice width Runtime Documentation Devices were controlled with commands entered in the time table. Composite of the results file name (*.cfd, chromatogram). Data point distance in ms in min One of the groups in the documentation is printed. The choice of group is made by right-clicking and selecting Properties.

89 ChromStar 7 - Report Editor Page 83 Complete Documentation Time table Method file name Method file name (complete) Method History The documentation page will be printed. The time control table will be printed. Name of the method file without indicating the file path. Adds the name of the method file ( *.met) and indicates the file path. 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 Cahnnel name Sensitivity Wavelength Resolution Calculation Base Calculation Method Concentration Level Calculation Unit Column Length Column Deadtime Peak Table Calibration Curve Regression Table UV or DAD As defined in the configuration Integration parameter DAD wavelength Wavelength resolution Entered in the method on the calculation page Entered in the method on the calculation page Number of standard solutions (level, number of concentrations) entered in the method on the calculation page Entered in the method on the calculation page Entered in the method on the calculation page Entered in the method on the calculation page The peak table is printed. After calculating the average response factor or doing a multi-level-calibration, the graph or regression curve can be printed. The regression table is printed

90 Page 84 Report Editor - ChromStar 7 Integration Parameter Table Calibration Table The integration parameter table, entered in the method on the data recording page, is printed. 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 File Documentation The Audit trail can be printed. Notes about the computer, operating system, device configuration etc. can be printed in a list. Deviation Display Scattering Table Signature If the result of a peak is out of the limit entered in the peak table, a message is printed. can be printed. 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.

91 ChromStar 7 - Report Editor Page Page Status The following items are available here: New Page Page No. Number of Pages Starts a new page. Prints the current page number. 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 Template name (full) Name of the file to be printed without file path. Adds the name of the print template (*.cps with file path). Template name Print Date Print Time Current User Name of the print template without file path. Current date. Current time. 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 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. DAD Parameters

92 Page 86 Report Editor - ChromStar Graph Items Graph Items: Picture Line 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. A line is added to the print template. Line thickness and colour can be chosen freely Runtime Parameters The following runtime parameters can be incorporated as elements in the print template: Pump pressure at start Injection time Injection date Counter-pressure of the system at the beginning of chromatogram recording. Time of injecting in minutes and seconds. Date when the chromatogram was recorded.

93 ChromStar 7 Navigator Page 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.

94 Seite 88 ChromStar 7 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.

95 ChromStar 7 Seite 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.

96 Page 90 User Administration - ChromStar 7 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 User This menu procedure can only be used by the administrator to re-establish GLP mode conformity 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.

97 ChromStar 7 Working with ChromStar Page Working with ChromStar 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

98 Page 92 Working with ChromStar - ChromStar 7 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.

99 ChromStar 7 Working with ChromStar Page 93 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.

100 Page 94 Working with ChromStar - ChromStar 7 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.

101 ChromStar 7 Working with ChromStar Page 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.

102 Page 96 Working with ChromStar - ChromStar 7 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):

103 ChromStar 7 Working with ChromStar Page 97 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.

104 Page 98 Working with ChromStar - ChromStar 7 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 cfd and new demo cfd, will appear in the Navigator in the file list on the right and can, for example, be displayed and further processed in Reprocess.