Agilent EZChrom Elite User’s Guide
Notices Copyright © Scientific Software, Inc 1997-2005 © Agilent Technologies, Inc. 2006-2008. No part of this manual may be reproduced in any form or by any means (including electronic storage and retrieval or translation into a foreign language) without prior agreement and written consent from Agilent Technologies, Inc. as governed by United States and international copyright laws. Manual Part Number G4650-90008 Edition January, 2008 Document Revision 3.3 Printed in USA Agilent Technologies, Inc.
Contents 1 Using This Guide ................................................................................. 8 Introduction ............................................................................................................8 Who Should Read This Guide? ...........................................................................8 2 Basics of Operation ............................................................................ 9 3 Instrument Wizard ..............................................
Open Data File Options.............................................................................. 20 Open Method and Sequence Files................................................................... 22 Save Data Files............................................................................................ 23 10 Reading CDF Files ............................................................................. 25 11 The Instrument Window...................................................................
Set up a trace.......................................................................................................50 Remove a Trace ...................................................................................................54 Set Limits for X-Axis and Y-Axis.......................................................................54 13 Chromatogram Operations............................................................... 57 About Chromatogram Operations .............................................
15 Data Acquisition and Control........................................................... 74 Data Acquisition and Control............................................................................ 74 Single Run Acquisition....................................................................................... 74 Run a Sequence .................................................................................................. 79 Stop a Run in Progress .........................................................
Examine a Custom Report ...............................................................................109 Changing Integration Parameters ..................................................................
1 Using This Guide Introduction This User’s Guide provides a basic operating overview and Tutorial for the Agilent EZChrom Elite data system Who Should Read This Guide? This document is designed for new users who will be doing acquisition of data and processing of results.
2 Basics of Operation This section describes the basic operation of EZChrom Elite, its file structure, features of the application windows and chromatogram windows. 3 Instrument Wizard Each time you start an instrument application (by doubleclicking the instrument icon from the Main window), an Instrument Wizard will appear. This wizard is designed to direct you to the basic functions of the instrument window.
Create a sequence This button starts the Sequence Wizard that steps you through creation of an acquisition or reprocessing sequence. Run one sample This button opens a dialog where you can use a stored method to run a single sample. Run sequence of samples This button opens the Run Sequence dialog where you can start data acquisition using a stored sequence. Show at instrument startup If this box is selected, the Instrument Wizard will appear each time this instrument is started.
4 Client/Server Operation When operating in a Client/Server mode, you will have one or more Client Workstations along with one or more Agilent Instrument Controllers, configured on a network. All instruments are physically attached to the Agilent Instrument Controllers (AICs), and the Agilent Instrument Controllers are the machines where the actual data acquisition and control of instruments occur.
User’s Guide
To turn the Navigation pane on 1. Select View followed by the Navigation pane command. To turn the Navigation pane off 1. Click the x button at the top of the Navigation pane. You can also "park" the Navigation pane at the left of the Instrument window, which provides additional work space. Once the Navigation pane is parked, you can view it again by simply moving your mouse over the Navigation tab that appears at the left. The pane will disappear when you move your mouse back into the work space.
Views Data Display options, Manual Integration Fixes, Baseline Check Status 7 Data Acquisition Using Agilent Instrument Controllers When you start a run or a sequence from a client workstation, you are actually submitting that run or sequence to the Agilent Instrument Controller (AIC) or EZServer where the instrument is attached. Once you have submitted a run or sequence, the AIC assumes control over the acquisition and control functions.
workstation (if the user has appropriate privilege assignment). In order for sequence changes to take effect, the sequence must be saved (File>Sequence>Save). When the sequence is saved, all other clients will be notified of the change, and the next run of the sequence will be executed from the modified sequence. Closing a Server Instrument Once an instrument application is started on an Agilent Instrument Controller, it will remain open after all runs are completed.
About Data File Structure A data file is created on the designated drive whenever you acquire a sample, or when you save a data file using the Save As 32-bit… command. The file contains the following information: File Information Header. This contains information such as the date and time of acquisition. Complete method parameters used to acquire and process the data (this is the "original" method saved only when the data is acquired).
first. If the check fails, the file cannot be opened and an error message will appear in the instrument activity log. Checksum verification, when enabled, is enterprise-wide. The checksum feature is enabled from the Enterprise Options dialog in the Main menu, and is labeled Extended Security. GLP and Extended Security Turn on Extended Security In order to adhere to good laboratory practices, the software does not let you over-write a data file.
and cannot be removed or overwritten. To view the changes logged in the Audit Trail for a method, 1. From the File menu, select Method followed by Audit Trail... This will display the Audit Trail listing for the current method. If the audit trail option is turned on for the current method, this box will display the logged changes to the method. User The user who was logged into the system at time of the change. Logged The time the change was logged into the system.
9 Opening and Saving Files Open Data Files Whenever you open a file using the data system, you will be presented with a dialog box that allows you to not only open the file, but specify parameters for searching, as well as on the previewing file contents. The Open File button command ribbon gives you access to the Open File menu. When you select one of the types of files to open, a dialog will appear where you can select the file from those on your hard disk.
systems with advanced file security enabled. Users will be limited to storing files within the current project folder and the Enterprise Common folder. A folder bar will be included in the dialog to facilitate navigation. Open Data File Options The Options box allows you to save time by loading additional information at the time the data file is opened. Method If you select Current, the current method will not change when you open the data file.
Results When one of the Results options is selected, the data file will be opened along with the selected results. When a data file is opened with results, the integration and baselines that generated those results will be displayed automatically when the chromatogram is drawn on the screen. If Most Recent is selected, the data file will be opened with the results from the last time the chromatogram was analyzed.
Note: When using the Search feature, make sure the Windows Hide File Extensions for Known File Types option is turned OFF. To turn this off, from My Computer, click Tools followed by Folder Options... and then click the View tab. Open Method and Sequence Files To open a method or sequence file, 1. From the File menu, select Method or Sequence followed by Open. 2. 22 The Open dialog boxes for Method and Sequence files are identical.
Searching for Method and Sequence Files The criteria you can use to search for specific method and sequence files include selection of specific text found in the file description (Text in Desc.), Analyst name, and date Created or last Modified. Note: When using the Search feature, make sure the Windows Hide File Extensions for Known File Types option is turned OFF. To turn this off, from My Computer, click Tools followed by Folder Options... and then click the View tab.
To save the current data in a new data file, type the name of the new data file in the File name field, then click Save. Use the buttons at the upper right of the dialog box to view details of a highlighted file, or to view the description of a highlighted file. An entry "Saved from " will be logged into the saved file as the first entry. If the Compress Data box is selected, the file will be saved in a compressed format.
10 Reading CDF Files When opening a CDF file, the software looks for one of the following Y-axis labels: "microvolts", "uvolts", "uv", "uau} or "millivolts", "mvolts", "mv", "mau" If one of these labels is not found, the software will try to read it from an AIA.ini file, which is used to get multipliers for non-standard file types. If there is no AIA.ini file available, the software will try to make an estimate based on the range of values.
Enter the information as prompted, and then click the Login button. User name: Enter the user name which has been assigned to you on the network. Password: Enter your assigned network password. Save Password: If the "save password" option is enabled by your system administrator, a Save password checkbox will appear.
If you select this box, the password you type at login will be saved when you click the Login button. Once the password has been saved, whenever you login to the instrument, the saved password will be used. Note: The password will be saved when the Login button is clicked, whether or not the login is successful. Domain: Select the domain you have privileges on from the drop-down list, or type in the correct domain. (Not shown unless you are using a domain controller.) Project: Select a project to log into.
To view details of any line in the instrument activity log, click on the line to highlight it, then do a right mouse click within the spreadsheet. From this pop-up menu you can view details of the highlighted line, print it, or print the entire activity log. In addition, you can export, archive, or purge the activity log from this menu. Note: Over time, the instrument activity log file may become large, so periodically you should archive the file to a floppy or another location and then purge it.
• Instrument control and data acquisition • Analysis and review of data • Reporting • Data export You can customize the appearance of the application window if you choose, adding or removing the Toolbars and Status information. However, these features are designed to make the system easier to use, and most users will prefer to have these turned ON. Change View Preferences To change the appearance of the instrument window, User’s Guide 1. From the View menu, click Preferences. 2.
Preferences - General This tab is used to set up general preferences in the instrument window. Toolbar options For each area of the window listed, you can turn on or off the Toolbar and Tooltips if available. Click on the toolbar area, then check the Show toolbar and Tooltips boxes to enable your choices for that area. Status bar options Select the checkbox to turn on the status bar. The status bar provides brief information at the bottom of the instrument window, if enabled.
2. 3. 4. 5. Click Files to change the file view preferences for the instrument window. Select the file type, then enter the number of files you wish to display in the Max files box. This determines the number of recent files displayed in the File menu. Click Clear Files to clear the current recent files list for the selected file type. Click Clear All Files to clear the recent files for all the file types. Locking your screen To lock the window from unauthorized use, from the Window menu choose Lock.
12 The Chromatogram Window About The Chromatogram Window Whenever there is data to be viewed, it is shown in a chromatogram window. Normally, one chromatogram will appear in each window, and multiple channel data files will display multiple chromatogram windows - one for each channel. However, it is possible to add multiple traces to a single chromatogram window and perform comparison and mathematical operations on them.
As data is being acquired, it is also displayed in a chromatogram window. At the end of a run the data becomes the "current data". You can change the appearance of the chromatogram and select annotations, fonts, and labeling. Utilities are available to print the current window view, copy it to a clipboard, or save it in a file.
Zooming You may want to examine a chromatogram in more detail, or zoom in on a portion of the chromatogram. To do this, drag a box around the area of interest by holding down the left mouse button and dragging the box until it highlights the section of interest. Then release the mouse button. To move quickly to the previous level of zoom, double-click on the chromatogram.
Add a Trace (Viewing Multiple Chromatograms) The chromatogram window is used to view data, either current data (real-time) in the instrument window, or data recalled from the disk. You can view multiple chromatograms in a single chromatogram window if you wish. This is convenient if, for example, you want to compare a past run with your current data or overlay an oven or pump profile. To add a new trace to the chromatogram window, User’s Guide 1. Click the right mouse anywhere in the chromatogram window.
3. Select the New Trace tab. Fill in the fields to add a trace to the chromatogram window and set its properties. These properties apply only to the trace selected and are not saved as part of the method. When a new trace is opened, the properties will be set to default values. Added traces are normalized by default. Data Source Enter the name of the file from which to get the trace. You can also click the File button adjacent to the field and select a data source.
Current Data This selection allows you to select a trace from the current chromatography data. Current Method This selection enables you to select a trace from your current method (if available). For example, you could load an oven temperature program from an HP5890 instrument method. Open Data This allows you to select a stored data file from which you can select a trace for display. Trace Select the trace to be displayed. Click the button to display available traces.
Y min If you have selected a User Defined scale, enter a minimum value for the Y-axis. Y max If you have selected a User Defined scale, enter a maximum value for the Y-axis. Units Select the units for display. X Offset Enter a value in units for offset of the X-axis. Y Offset Enter a value in units for offset of the Y-axis. Y Scale If desired, enter a multiplier that will be applied to the entire trace here.
3. 4. 5. User’s Guide Once you have added a data file to the list, you can select the channel by clicking on the Trace field, then click the down-arrow button. If multiple channels for that file are available, select the desired channel by clicking on it with the mouse. To delete a trace from the display list, click on its name or on its number, then click the Delete button. When you are ready to open the multiple traces, click Open. The selected files/channels will appear in your chromatogram window.
Annotate a Chromatogram To change the annotations on the chromatogram, 40 1. From the chromatogram window, do a right mouse click and then select Annotations. 2. Click the Annotation tab in the Properties box. This brings forward a dialog where you can designate how you want the trace you are adding to be annotated.
3. 4. 5. 6. User’s Guide Select the trace from the drop-down list. Then select what features you wish to annotate. Normal choice is Peaks. However, if you have the SEC option installed, you can select SEC to annotate specific SEC features on your chromatogram. For the selected trace, click Peaks or Groups to select what kind of annotation to use. Click on an Available Annotation.
7. Note: Click the check box(s) to display Baseline, USP Width, or Retention Time Windows, Show undetected named peaks, and Group ranges on the trace. With the SEC option installed, you will have access to additional SEC annotation features. The Reference Peak window annotation displays the window set in the Peak Table. This window is not adjusted for relative retention time. 8. Continue to select as many annotations as you wish for this trace. When you have finished, click OK. 9.
Scheme If you have previously saved an appearance scheme on disk, you can select it from this box. The Save As… button allows you to save the existing appearance scheme on disk by giving it a name. The Delete button allows you to delete a scheme and start again. Item This drop-down list lets you select which part of the chromatogram window for which you wish to change the appearance. The choices will include the graph itself (including background and legends), and the available traces.
Graph, you will have access to setting up appearances of sub-items including the background, axes and labels for the graph. If the item selected is a chromatogram data channel, you will have access to setting appearances of sub-items such as baselines, start and stop tic marks, and annotation. If the item selected is text, you will have access to the Font formatting commands as well. When a sub-item is selected, you will have access to fields appropriate to that item.
Sub-items available in the Appearance tab are as follows. Item Sub-Item Description Graph Background Select the color of the graph background. Default is black. Graph Title Select a color and font for the Title of the graph. There must be a Graph Title defined in the Axis Setup tab in order for it to appear in the window. Graph Left Y-Axis Select a color for the left Y-Axis of the graph. Graph Left Y-Axis Major Ticks Select a color for display of major unit marks on the Left Y-Axis.
Graph Legend Select a color and/or font for display of the graph legend. The legend indicates what traces are currently displayed in the window. The Legend is turned On or Off from the Axis Setup tab. Graph Grid Select a color for display of the grid lines. Grid lines are turned On and Off from the Axis Setup tab. Data Trace Select a color and/or line type for display of the selected trace. Data Annotation Select a color and font for display of the trace Annotation(s).
Change the Axis Properties The Axis Setup tab allows you to configure the appearance of the axis on your chromatogram. These settings apply to active traces. To change the Axis properties, 1. In the Chromatogram Window, do a right mouse click and then select Axis Setup. Graph Title Enter a title for the graph, if desired. This appears at the top of the graph.
Axis Using the drop-down list, select the axis of interest: Left Y-Axis, Right Y-Axis, or X-Axis. Then for your selection, you can choose the limits for the axis. For Y-Axis selections, you may choose Use limits of trace to get the limits from one of the traces in the window, or you can select the Manually set trace's limits to box and set the Y-Axis limits to your desired range. If you choose None, no Y-Axis values will be displayed.
Chromatogram Window with Legend and Grid displayed Chromatogram Window with no Legend or Grid displayed Orientation Select portrait or landscape orientation for your graph by clicking the appropriate button.
in the Data Graph Properties. To change the Data Graph Properties, 1. In the Chromatogram Window, do a right mouse click and select Properties. 2. Select the tab for the properties you wish to view or change, as shown below Properties tab Used to Trace Setup Add or remove traces, set legends, set scaling. Axis Setup Add a graph title, change data range, set margins and orientation, turn on and off legends. Appearance Set color schemes, line styles, and fonts.
Show Click this box to show the trace in the chromatogram window. De-select this box to remove the trace from the display (but leaving it open). This is a convenient way to temporarily remove a trace from the viewing window. Lgnd Click this box to show the legend for the trace. The legend appears in the upper right corner of the window and displays the name of the trace. De-select this box to remove the legend for this trace from the chromatogram window.
the legend (color, etc.) is done in the Appearance tab for the Graph item. Note: If you have not turned on the Legend in the Axis Setup dialog, this box will have no effect. Data Source Enter the name of the file from which to get the trace. You can also click the File button adjacent to the field and select a data source. The data source can be a chromatogram or it can be a stored profile such as temperature or flow program.
Autoscale to 3rd largest peak Scales such that the 3rd largest peak is on scale. User Defined Allows you to enter a value for Y max and min. Normalized Allows you to normalize one trace to fit on the graph. Y min If you have selected a User Defined scale, enter a minimum value for the Y-axis. Y max If you have selected a User Defined scale, enter a maximum value for the Y-axis. Units Select the units for display. X Offset Enter a value in units for offset of the X-axis.
Hide Details Click this button to hide the current trace details and display only the spreadsheet. Reset Scaling Click this button to reset the scaling values to their original values. Remove a Trace If you have multiple traces in your chromatogram window, and you want to remove one or more of them from the chromatogram window, click the right-hand mouse button anywhere within the window, and select the Properties… command. A spreadsheet will appear where the currently displayed traces are listed.
3. Click the Axis Setup tab to set absolute ranges for the trace. Select X-Axis, to set the range for the X-Axis. Click Autoscale to set the X-Axis range automatically to the range of the longest chromatogram (the default selection), or click Use this range to enter an absolute range in minutes. The Get Current Limits button brings in the X-Axis range from the current chromatogram window.
5. 56 always be displayed in the chromatogram window using these ranges until you change or reset them. To reset the scaling of all chromatograms to default values, click the Reset Scaling button.
13 Chromatogram Operations About Chromatogram Operations There are a number of chromatogram comparison and mathematical operations that are available from the chromatogram window. These are accessed by doing a right mouse click in the chromatogram window and then selecting Operations.
Operation Action Move Trace Lets you "grab" and move a trace within the chromatogram window. Stack Traces Positions multiple traces with an offset. Align Adjusts a second chromatogram such that a peak (or point) on one chromatogram will be aligned with a peak (or point) on the first chromatogram. Stretch Performs a two-point contraction or expansion of chromatogram relative to another.
Move a Trace To "grab" a trace and move it with your mouse 1. In the chromatogram window, do a right mouse click and select Operations followed by Move Trace. 2. Move your cursor over a trace until the cursor changes to a "move" icon. "Grab" the trace by clicking the left mouse button and dragging the trace to a new location. When you release the mouse button, the trace will be placed where your cursor was located when you released the button.
Chromatograms before stacking Chromatograms after stacking To remove these offsets, 1. In the chromatogram window, click the right mouse button and then select Properties. 2. Click the Trace Setup tab, then scroll to the right to the X-axis and Y-axis offset columns where you can delete or change these settings. Click the Reset Scaling button to restore ALL settings to their original values. Or, you can use the Stack command again, entering "0" for both stack parameters. 3.
Align two traces To Align one chromatogram to another, 1. 2. In the chromatogram window, do a right mouse click, and then select Operations followed by Align. Click first on the point of the first chromatogram to which you wish to align, then click on the peak (or point) of the second chromatogram which you wish to align to the first point. The second chromatogram will be adjusted such that the peak (or point) you clicked second will be aligned with the first point you clicked.
First peak of top chromatogram aligned to first peak on bottom chromatogram. Stretch a chromatogram The stretch function allows you to perform a two-point contraction or expansion of chromatograms relative to another. To stretch a chromatogram, 1. In the chromatogram window, do a right mouse click and then select Operations followed by Stretch. 2. 3. Select points (or peaks) on the first chromatogram to which the second will be stretched (or contracted). Select two points on the second chromatogram.
Chromatograms before stretching. Bottom chromatogram stretched relative to top chromatogram. Normalize Traces This function allows you to normalize one or more chromatograms to the first chromatogram, adjusting the heights such that the apex height of a selected peak matches that of the peak selected on the first trace. Once you have selected this command, you will be prompted to select the start and then the apex of a peak in the first trace.
To un-do the normalization, use the right-hand mouse button/ Properties command to view the trace spreadsheet. Click the Trace Setup tab, then scroll to the right to the X-axis and Yaxis offset columns where you can delete or change these settings. Click the Reset Scaling button to restore ALL settings to their original values. Chromatograms before normalization. After Normalization.
Perform Mathematical Operations on Traces Performing mathematical operations on traces can be done from within the chromatogram window. To perform a mathematical operation on a trace, 1. In the Chromatogram Window, do a right mouse click and select Operations, then select the operation you wish to perform. 2. Follow the instructions displayed to perform the operation. The result of the operation will appear in the window. For a list of available operations, see Chromatogram Operations.
Smoothed result trace is displayed with original trace. Calculate Derivatives To calculate and display the 1st or 2nd derivative of a chromatogram, 66 1. Do a right mouse click on the chromatogram, and then select Operations followed by 1st Derivative or 2nd Derivative. A prompt will appear in the window Click on trace. 2. Click on the chromatogram for which you wish to perform the operation. The result trace will appear in the window.
Trace before 1st derivative. 1st derivative trace displayed with original trace.
2nd Derivative displayed with original trace Add two traces To add two traces to a chromatogram window, 1. In the chromatogram window, do a right mouse click, and select Operations followed by Add. 2. Click on 1st trace to select the first file by clicking the mouse on the chromatogram. Click on the 2nd trace to select the trace to be added to the first by clicking on the trace with the mouse. The result trace will appear in the window.
Note: In order for this operation to be valid, both traces must have the same sampling frequency. Multiply Traces To multiply two traces, 1. In the Chromatogram Window, do a right mouse click and select Operations followed by Multiply. 2. Select the first trace by clicking the mouse on the chromatogram or trace. Select the trace to be multiplied by the first by clicking on the 2nd trace with the mouse. The result trace will appear in the window.
ymult = the y multiplier for the trace that converts it from microvolts to the trace's displayed units Utilities About Chromatogram Utilities The Utilities menu in the chromatogram window gives you access to commands for saving, copying, or printing the current chromatogram window. To access the Utilities, in the Chromatogram Window do a right mouse click and then select Utilities. 70 Utility Action Print Sends the current chromatogram view to the printer.
Print a trace This command sends the current chromatogram window view to the printer. Copy to clipboard To copy the contents of the window to the clipboard, 1. From the chromatogram window, do a right mouse click and select Utilities followed by Copy to clipboard. This command copies the current chromatogram window to the clipboard as a metafile. From here, you can paste the view into a word processing document or other application that supports the clipboard.
Parameters that can be set using graphical programming include: 72 Command Action Width Inserts a Width event at the point on the chromatogram. Threshold Inserts a Threshold event at the point on the chromatogram. Shoulder Sensitivity Inserts a Shoulder Sensitivity event at the point on the chromatogram. Integration Off Turns off integration at the point on the chromatogram. Valley to Valley Turns on valley to valley baseline detection.
Force Peak Stop Force the end of a peak. Move Baseline Manually move a baseline. Reset Baseline Force a baseline to the point. Reset Baseline at Valley Reset the baseline to the next valley. Adjust Retention Time Window Adjusts the retention time window. Adjust Group Range Adjust the group range. Define Single Peak Define a single peak and add it to the peak calibration table. Define Peaks Define multiple peaks and add them to the peak calibration table.
15 Data Acquisition and Control Data Acquisition and Control Commands that are available from the Control menu are related to data acquisition and control of the instrument. In general, there are two ways to acquire data: 1) single run acquisition, where you acquire data for a single injection and 2) sequence acquisition, where you acquire data automatically for a series of runs using a pre-programmed sequence that defines the number of injections, methods, file names, and calibration.
Run Information This section allows you to specify files for the run. Sample ID Enter a Sample ID for the run. This can contain text and numbers, and is saved with the data file. You can also click the arrow and select from a number of predefined ID’s. Method Enter the name of the method to be used for data acquisition and processing. Include the entire path name if the method is not in your default method directory.
field. It is not possible to use an existing file name, unless the file exists in located in a directory whose path contains the term "public". For example, if you data files are saved in a directory entitled "C:\Public\Data", the files saved in this directory can be overwritten. The software automatically appends a .dat file extension. Number of runs Enter the number of runs you wish to make. The runs will automatically proceed without review until completed, incrementing each file name as designated.
Internal Standard Amount For calibration runs, the Internal Standard Amount is taken from the method Peak Table. For unknown runs, enter the amount of the Internal Standard in your unknown sample. Multiplication Factors Enter one to three multiplication factors to be used for this run. All quantitated peaks will be multiplied by these factors. Dilution factors Enter one to three dilution factors to be used for this run. All quantitated peaks will be divided by these factors.
Clear replicates Click this box if you want to clear all existing replicates from the existing calibration level before running the sample. Average replicates Click this box if you want to average the replicates for this calibration level. Baseline Check This box will appear if you have the Baseline Check option implemented in the instrument configuration. When this box is checked, it will trigger a baseline check prior to the start of the run. Begin run By default, run will start immediately.
Run a Sequence Once you have created and saved a sequence, you can use it to acquire and process data. To start a sequence acquisition, 1. From the Instrument Window, click the Sequence , or from the Control menu, click Run button Sequence Run.... 2. Enter or select a sequence to use, set a run range, mode, printing options and review options, then click Start.
Range Enter a range of runs to be executed. For example, an entry of 4 - 6 will execute runs 4, 5, and 6 of the sequence. An entry of 4- designates the 4th run through the end of the sequence. Mode Select the manner by which you want to handle autosampler dual towers (if any), processing mode, and bracketed calibration (if used). Tower If your instrument is configured for Dual Tower, you can select the tower mode to be used for the sequence run. Selections include Dual, Front, and Rear.
Seq. w/ Back Calc Select this if you want to perform the sequence mode of bracketing calibrations and back-calculate calibration runs. Review Results Review Click this box if you want the sequence to pause between runs for you to review results. Calibration Review Click this box if you want the sequence to pause after each calibration set, where a calibration set is defined as one or more calibration runs that occur in a sequence.
Select the On Stop or Error box to send email notification when the sequence stops or if an error occurs. When you have completed the dialog box, click Start to initiate the sequence acquisition. You may see the data displayed in real time in the chromatogram window(s), if the "current data" is selected for viewing. Stop a Run in Progress When you want to stop data acquisition during a run, 1.
Stop current run and sequence run This selection stops the run currently in progress, and terminates the sequence it is a part of. Other queued items will proceed. Stop sequence after current run completes This selection will abort the sequence after the current run in progress is completed. Stop all run queue items you submitted This selection stops the run currently in progress, and terminates all the items in the queue that were submitted by you. Queue items submitted by other users will be unaffected.
Note: If you are not the user that submitted the run or sequence or are using an instrument offline, you do not have access to the Stop or Extend Run command. 16 Turn Off GPIB Instruments If you are using two instruments attached to a GPIB board, it is important to close the instrument application (or close the instrument window and then close the server) prior to turning off the power to the instrument or instrument modules, otherwise the other instrument attached to the GPIB board may freeze.
17 Tutorial This section walks you through the basics of using EZChrom Elite. Follow the steps to set up a method and acquire a data file, then optimize the method for integration and set up calibration. Use the Tutorial files provided with the software to "play" with the software and become comfortable with its use. Details on data file structure, application window features, how to open and save files are located in Basics of Operation.
Step 4: Using Tutorial Files • Review a Multi-level Calibration • Explore a Peak Table • Examine a Custom Report • Change integration parameters 18 Tutorial - Acquiring and Analyzing Data In this section of the Tutorial you will use the data system to acquire data and optimize the integration, set up and run a calibration standard, and create and run a sample sequence. Details for advanced operation are located in later sections of this manual.
When you click this button, the Method Wizard will appear, allowing you to select how you want to use the wizard. Select the Create a new method button to start creating your tutorial method.
When you select this button, the Method Wizard sets up a bank of buttons in your application window that allow you to "step" through all dialogs of method generation. A save button is also provided. Create a Data Acquisition Method The first step toward acquiring a data file is to create a data acquisition method.
The default Sampling Frequency should be adequate for most types of chromatography. For Frequency data sampling, the data rate is selected in Hz (samples per second). For Period sampling, you can select the number of seconds (or milliseconds) between data points. The default is Frequency and this is how most chromatography applications are set up. Make sure that the Run Time is long enough for your last expected peak to elute.
None Sampling of data starts immediately when Start is clicked. Manual User must start the data sampling. External Data sampling starts when externally triggered. When you have completed the acquisition setup information, click the X box in the upper right hand corner of the tab window to exit the dialog. Save Your Method Once you have set the acquisition parameters, save your method using a name you will recognize. Click on File>Method/Save As… A dialog box will appear.
Select the folder in which you want to save your file. In the Filename field, type Test.met as the filename for saving the method. Run a Preliminary Sample You will now use the data acquisition method you just saved to make your first data acquisition run. To aid in later steps of the Tutorial, it is best to run a standard sample for your first acquisition run. 1. User’s Guide To start the run, select Control>Single Run or click the Single Run button.
2. At this point, enter Test in the Sample ID field. 3. In the Method field, either type your method name, along with its path, or select the Test.met file from a file list on your disk by clicking on the open file button adjacent to the field. Enter a path for storage of data files in the Data Path field, or select a path from a list presented by clicking the open file button adjacent to the field. Enter Test.dat as the name for storing your data in the Data File field.
into your method as Integration Timed Events. These events can be placed at the beginning of the run to apply to all peaks, or they can be inserted at a certain place in the chromatogram such that only some peaks are affected. Follow the steps below to add the timed event to turn off integration to your method. Note: You can perform this step using one of the multi calibration level.dat files provided with the software. 1. 2.
The points where you clicked your mouse are shown as Start Time and Stop Time. The integration will be turned off between these points on your chromatogram. The Value is set at zero, as no numeric value is required for this event. Click Analyze Now to add the event to the method and re-integrate the chromatogram. (Add to Table will simply add the event to the integration timed events table without re-integration.
the row number, then press the Delete key on your keyboard. You can also delete the event using the Edit/Cut command. Using this command, you could re-insert the event using the Edit/Paste command if you wished. To temporarily view the effect of removing an event without actually removing it from the table, click the check box adjacent to the event to de-select it. To re-select the event, click the check box once again. 6.
2. integration the chromatogram and show the baselines. Click the Define Single Peak button on the Toolbar. A dialog box will appear for the first detected peak in the chromatogram. The retention time of the first detected peak will appear. If you want to add this peak to the peak table, complete the dialog for this peak. If you do not wish to add this peak to the peak table, click the Next button. If you want to move to a specific peak in the chromatogram, click on that peak with your mouse.
would enter Concentration Level 2 and the amount for that level. Continue to enter level concentrations until you have completed the number of calibration levels desired.) Units Enter the units to be used for display of results. ISTD ID # If you are doing internal standard calibration, enter the ID # for the internal standard peak for this compound. This is the peak ID number from the peak table. If you don’t know it, you can add it in the peak table later.
the chromatogram is displayed on the right of the dialog box. When you are finished adding peaks to your peak table, click Done. Each peak you defined will become a row in your peak table. Note that if you already had peaks in your peak table, the peaks you just defined will be added to those already present. To view the peak table, click the Peak/Group Tables button from the command ribbon. 4. Once you have defined your peaks, click the Peak Table button and the calibration peak table will appear.
Calibrate Using a Stored Data File In order for the software to calculate amounts for unknown samples, your method must contain the areas generated for each component in your standard sample. In order to get these areas into your method, you can either run the standard sample again, designated as a calibration run, or you can calibrate the method using the stored run from before, using the Analysis>Single Level Calibration command.
6. Leave the Amount Values set to "1". For details on how these values are used, see Method Development. 7. Click on the Calibration checkbox, then enter a "1" for Calibration Level. Since this method is currently uncalibrated, it is unnecessary to select any of the boxes dealing with calibrations or replicates. However, if you are unsure of the method contents, click the Clear all calibration box before starting. 8. When you have completed the dialog, click Start.
2. Type the method to be used for the acquisition, or select the name from a list of available methods by clicking the File Open button. 3. For Method, enter the method name (including path) for the method you have been using for this Tutorial. If you don’t know the entire path name, you can select it by clicking the file button next to the field, and selecting your method name from the list displayed. If you are following the Tutorial, enter Test.met as your method name.
5. 6. 7. 8. 9. 102 The Sample ID field is for an identification for the samples. Click the blue right arrow and select the Line number and Method name. This will cause each sample to be identified with the sequence line number and current method name. In the Data Path field, enter the path where you want the data to be stored, or select an existing path by clicking the File Open button. For Data File, click the blue right arrow, and select Sample ID.
10. In this dialog, the calibration ID and calibration file names are automatically set to the identifications from the previous dialog. Set the number of calibration levels to "1", and leave the calibration repetitions per level at "1" also. Leave all other boxes unchecked, then click Next.
11. Select the check boxes to cause the samples to be included in a summary report and calibration summary report. Do not select the other boxes. 12. Click Finish. A sequence spreadsheet will appear, with the file and method names you specified shown. 13. At this point, the sequence is set up to run 1 calibration sample and 3 unknown runs.
Sample ID’s and Data File names are numbered automatically to prevent duplication. In order to run a calibration standard as the first run, you must designate that run to be a calibration run. This has been done automatically by the Sequence Wizard. Unknown runs always have a Level of "0". The information in the Run Type field may be abbreviated if there is more than one run type designation. To view the possible Run Types, click the arrow next to the run type.
Enter the name of your sequence file by typing the name, along with path, in the Sequence Name field. You can also select it from a list of sequence files on your disk by clicking the file button next to the field. Leave the other parameters as their defaults. Prepare your autosampler to inject your standard sample, followed by 3 unknown samples. When you are ready to inject your first sample, click Start.
level calibration, use the multilevel calibration.met file provided with the data system. 1. 2. Open the multilevel calibration.met method file by clicking the Open button followed by Open Method. Select the multilevel calibration.met file from your disk. It will be located in the \datasystem\Methods folder. (Where datasystem = your installation program folder.) Once you have opened the multilevel calibration.met method, click the Review Calibration button, or select the Method/Review Calibration command.
for the different fit types displayed are shown, along with the goodness of fit calculation, r2, which is not calculated for the Point-to-Point curve since it is by definition a perfect fit to the data. For additional details on using Review Calibration, see Method Development. To close the window, click the X box at the upper right corner of the Review Calibration window. Explore a Peak Table The Peak Table is where method calibration information is located.
including the Levels, which contain the calibration amounts for each compound at each level of calibration. Note that it is possible to customize the Peak Table such that only parameters needed for a given calibration are displayed. Details on what each column represents, along with how to customize the Peak Table, are given in the Method Development chapter of this manual. Examine a Custom Report A complete suite of report templates are provided that can be used without modification to generate reports.
If you wish, you can modify the standard report templates, or create entirely new reports using the Custom Report feature. You can create custom method reports and / or custom Sequence reports. These are described in detail in the Custom Reports section. To view the custom report in the multilevel calibration.met file, first open the file if it is not already open. (Use the File Open button, followed by Method, then select it from the file list.
Changing Integration Parameters Another important aspect of using a computerized data system is the ability to customize the integration using Integration Timed Events. In this part of the Tutorial, you will use the multi calibration level 3.dat data file provided to become familiar with how to enter integration timed events into your method, and to view the effects of some of these events. Complete details on how each integration timed event works are given in the Integration section. 1. 2.
3. Add the Valley-to-Valley timed event to integrate the cluster of 4 large peaks with valley-to-valley baselines. To do this, click the Valley button on the integration toolbar. Then, click the mouse once before the first large peak, then again just after the last peak. When the dialog box appears displaying the start and stop points for the event, click the Analyze Now button and view the chromatogram.
4. Click the Integration Events Table button from the command ribbon. Note the addition of the Valley to Valley event in the table. 5. Remove the Valley to Valley event by clicking its number with the mouse, then select Edit/Delete from the menu bar. You can also test integration without the event, yet leave it in the timed event table, by deselecting the check box next to the Valley to Valley event and then re-integrating the chromatogram.
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