Chromatography Software Training Materials. Contents

Transcription

1 Chromatography Software Training Materials This document contains information on how to build a method, start the instrument to acquire data, and then process the data using the Galaxie Program. You will need to turn in the answers to several questions (see other handout). Please ask questions if you are uncertain as to what the instructions are asking. For Method creation and Sequences you need to use a computer connected to an instrument. Contents Reprocessing a Chromatogram Overview... 3 Initial Demonstration... 3 Figure 1 Pull-down Menu to open Chromatogram for Display and Reprocessing... 3 Figure 2 Window Used to Preview and Open Chromatograms... 4 Figure 3 Galaxie Main Screen Window... 4 Figure 4 Menu Generated by Right-clicking on the Chromatogram Name... 5 Figure 5 Zooming the Chromatogram Screen... 5 Figure 6 Zoomed Chromatogram... 6 Figure 7 Unzooming the Chromatogram... 6 Figure 8 The Chromatogram Properties Button... 7 Figure 9 Chromatogram properties Main Information... 7 Figure 10 Chromatogram with Hand Displayed... 8 Figure 11 How to Move the Boundary between the Windows... 8 Figure 12 Integration Events Selected... 9 Figure 13 Two Events Added to the Timed Events Table Figure 14 Reintegrated Chromatogram with Integration Off From 0 to 2 Minutes Figure 15 Initializing the Peak Table from the Chromatogram Figure 16 Peak Names Added to the Chromatogram Figure 17 The Report Style Dialog Box Figure 18 Saving the Method Figure 19 Final Report Questions to Answer Reprocessing Data Building a Method and Running an Acquisition Figure 20 Screen for Method Selection Figure 21 Method Information Screen Figure 22 Display of Method Sections with Preset Method Sections Figure 23 Control Screen with Control of the Signal in the ProStar 310 Detector... 16

2 Figure 24 Signal Part of Varian ProStar 325 Instrument Control Method Section Figure 25 Pump Control Page for the ProStar Figure 26 Acquisition Screen Figure 27 Integration Events Screen Figure 28 Peak Table Figure 30 Calibration Screen Figure 31 Formats Screen Figure 32 Step 5 of the Suitability Wizard Making a Single Injection Figure 36 Quick Start and Quick Start Icon Figure 37 Quick Start Menu Figure 38 Acquisition parameters screen Figure 39 Galaxie Run Screen Questions to Answer Using a Sequence - Overview Building a New Sequence Figure 40 New Sequence Wizard Selecting the System Figure 41 Entering a Name and Description for the Sequence Figure 42 Main Sequence Editing Screen Figure 43 Manual entry of data or selecting data from the drop down menus Figure 44 Entering the First Line of Data Figure 45 Selecting the Boxes Where Information Will be Copied Figure 46 Filling the Block of Entries Figure 47 Special Editing Icons for a Sequence Figure 48 Auto Fill Block Window Figure 49 Method Options Screen Figure 50 Bracket Building Figure 51 Creating Two Non-overlapping Brackets Figure 52 Setting Bracket Figure 53 Selecting B2 as the bracket for the Last 2 Unknowns and Standards Questions to Answer Page 2 of 37

3 Reprocessing a Chromatogram Overview The following is covered in this section: Opening a chromatogram Manipulating the main Galaxie window Changing data handling, peak identification, and report parameters associated with the chromatogram Manually reintegrating the chromatogram Saving the chromatogram and method Viewing the report. Initial Demonstration Open the chromatogram Std1a1.DATA by clicking on File, Open, Open Chromatogram. Figure 1 Pull-down Menu to open Chromatogram for Display and Reprocessing When you click on Open Chromatogram, you will see the following window. Each of the data files is listed in the left-hand window, and when one is selected, the chromatogram is displayed in the right hand-window. Select a few different files to view the chromatograms, and then select Std1a1.DATA and click on the Open button. Page 3 of 37

4 Figure 2 Window Used to Preview and Open Chromatograms When you open the chromatogram you should see the main chromatogram screen below. Figure 3 Galaxie Main Screen Window Page 4 of 37

5 The Galaxie Main Screen Window is divided into three main parts: The Browser Window is used to select the activity that you want to perform. The Processing parameters and results window is used to specify how you want to perform the activity and to view the numeric results of the activity. The Chromatogram Window is used to view the results of the activity graphically. The Galaxie Main Screen Window also contains status information (on the bottom), pull down menus, and icons. Right-click on the data filename to see additional menu items as shown below. Figure 4 Menu Generated by Right-clicking on the Chromatogram Name When you have finished reviewing the drop down menus; practice zooming the chromatogram to expand the scale. Hold the left mouse button while moving the cursor from the upper left to the lower right of the area in which you wish to zoom. Figure 5 Zooming the Chromatogram Screen Page 5 of 37

6 The screen will look like the figure below. Figure 6 Zoomed Chromatogram To unzoom the screen, you may either double click anywhere on the screen, or hold down the cursor and pull it in a different direction. Figure 7 Unzooming the Chromatogram Practice zooming and unzooming different parts of the screen. To find more information about the chromatogram, click on the Chromatogram Properties button. Page 6 of 37

7 Figure 8 The Chromatogram Properties Button Figure 9 Chromatogram properties Main Information The Main Information screen displays the basic information about the sample and injection. The Signal Information screen displays the chromatogram. The Variables screen displays that information about this chromatogram which is stored in each variable. Page 7 of 37

8 Look through these screens for the present chromatogram. You can move the entire chromatogram by clicking the right mouse button until a hand appears and then moving the chromatogram. Next, move the chromatogram to several different positions in the chromatogram window. Figure 10 Chromatogram with Hand Displayed You can also adjust the amount of space allocated to each window by moving the cursor over the bar separating the Chromatogram and the Processing Parameters and Results Window. This is useful when you need extra room in the Processing Parameters and Results Window. Move the boundary between the windows to increase the amount of space in the Processing Window but keep the chromatogram fully displayed. Figure 11 How to Move the Boundary between the Windows Page 8 of 37

9 When a chromatogram is collected, default peak width and threshold values are used to automatically process the chromatogram. This default processing is displayed on the chromatogram screen. Additional integration events can be added and processed. Select the integration events in the lower Browser Window as shown in Figure 12. Figure 12 Integration Events Selected With the Integration events selected, right click on the lower screen, select add event, select forced peaks and select Turn Integration. You can select this entry with either a right or left mouse click. The Turn Integration event controls whether integration is on or off. When you have selected the Turn Integration event, it will be displayed in the timetable with a default time of 0 minute and with a Red On/Off designator. This will tell the system to stop performing integration. No peaks will be detected. Repeat this process and select this entry again. Adjust the time for the second entry to two minutes and change the marker to green by clicking on it. This turns integration back on. Your display should look like Figure 13. Page 9 of 37

10 Figure 13 Two Events Added to the Timed Events Table To apply these new events to the chromatogram, you will need to reintegrate the chromatogram by either clicking on the integrate icon or by pressing the F5 button. Figure 14 Reintegrated Chromatogram with Integration Off From 0 to 2 Minutes Your chromatogram should look like Figure 14. There should be no baseline below the first peak. This is due to turning integration off for the first two minutes. Also, there should be a label on the chromatogram when integration was turned off and turned back on. Page 10 of 37

11 The next step is to identify the peaks. Click on the peak identification section of the menu tree. Right click on the table and select Initialize from chromatogram. This is displayed in Figure 15. Figure 15 Initializing the Peak Table from the Chromatogram The peaks are now entered into the peak table as shown in Figure 16. Now change the name of the peaks by typing in new names. To validate an entry, you must leave the line. Next, click on the Integrate icon. The peak names will be added to the chromatogram. Note that the peak names are not stored with the chromatogram until the chromatogram is reintegrated. Page 11 of 37

12 Figure 16 Peak Names Added to the Chromatogram To print the results, select the report style from the lower browser window. When the report screen is displayed, click on the yellow folder icon. The Open file dialog is displayed. Click on the LC-Data directory and select the Varian Standard Report Style. Figure 17 The Report Style Dialog Box Now that you have formatted the report, you will save the method and save the chromatogram. Right-click on the method in the upper browser window and on SaveChromato method as shown in Figure 18. Page 12 of 37

13 Figure 18 Saving the Method Right-click on the chromatogram name and save the chromatogram. Now preview the report it by clicking on the File menu and selecting Print Preview. Your report should look like the Report in Figure 19 but without the Demo software watermarking in the background. Figure 19 Final Report Questions to Answer Reprocessing Data Using the same chromatogram as you used in the tutorial, build a new method with the following characteristics: 1. The five peaks initially detected in the chromatogram will have the following peak names: a. Peak 1 = Uracil Page 13 of 37

14 b. Peak 2 = Acetophenone c. Peak 3 = Benzene d. Peak 4 = Toluene e. Peak 5 = Naphthalene 2. Reject from the report all peaks between 3 and 4 minutes 3. Reject from the report all peaks which have a height of less the 14,000 counts. 4. Print out your report. It should look like the report below. Page 14 of 37

15 Building a Method and Running an Acquisition For this section, the computer needs to be connected to an instrument (the LC or GC). To build a new method, you will need to select File, New, New Method. The system screen will appear as in Figure 20. Figure 20 Screen for Method Selection After you have selected the instrument you will have to give a name to the method as shown in Figure 21. Figure 21 Method Information Screen On the Method Information Screen you will enter a name for the method and a description of the method. Page 15 of 37

16 Once the method is built, the main Galaxie Screen will appear with the presets in the method sections. This is shown in Figure 22 below. Figure 22 Display of Method Sections with Preset Method Sections The browser window will have the method displayed in the upper window and the individual preset sections of the window in the lower browser window. The workspace will have the details of the method section highlighted in the browser section. Now we will go through each section of the method and highlight what is in the section and what must be filled out. Click on the Control item in the lower browser menu. A Window similar to will be displayed (the screens will change depending on what instrument is available on that computer). The following figures are specific for an HPLC instrument. Page 16 of 37

17 Figure 23 will be displayed (the screens will change depending on what instrument is available on that computer). The following figures are specific for an HPLC instrument. Page 17 of 37

18 Figure 23 Control Screen with Control of the Signal in the ProStar 310 Detector If an instrument is comprised of multiple modules, each of these modules is listed on the left hand side of the screen. Click on the modules to see the various parts to the instrument. The 325 Module Control consists of several parts: the Signal, Peak sensor, Relays and Miscellaneous parts. Click on the Signal part of the module control. This will display entries that can be made into the signal part of the instrument control section of the method. See Figure 24. Figure 24 Signal Part of Varian ProStar 325 Instrument Control Method Section You will always have to make appropriate entries in to the control sections of a method. For example, on the screen above, you must enter the appropriate wavelength at which you wish to monitor the detector signal. There are several conventions that occur throughout Galaxie. The Add Line, Delete Line, and Return to Presets button are common throughout the method sections. Try adding several lines to this table, deleting them and then returning the table to its presets. Besides the appropriate wavelength, you must also enter the appropriate Run (end) Time in the instrument control method. Many instruments have their own time control. Also, you may want to run the pumps longer than the detector to clean the column before the next run without saving data from that part of the run. The 210 pump icon will look like the display shown in Figure 25 showing the pump solvent control page. Page 18 of 37

19 Figure 25 Pump Control Page for the ProStar 210 This is an example of controlling the pump gradient and flow. Note on the graphical display on the bottom of the screen that the pump composition is represented on the left side of the plot and the flow is on the right side of the plot. Also, the plot ends at the last timed event (see the table directly above the plot) while the Run Time, displayed at the top of the screen, is longer (20 minutes here). Depending on the type of instrumentation that you are using, you may have to set the Run Time on each of the modules independently. Now take a look at the entries for each of the modules that comprise the instrument with which you are working. Next, click on the Acquisition entry in the lower browser window and the screen shown in Figure 26 will be shown. Page 19 of 37

20 Figure 26 Acquisition Screen The Acquisition Screen allows you to enter a variety of information about the sample and how to acquire data about it. The most important entries are the File prefix and File Identifier and the Acquisition length. The file prefix specifies the first part of the file name in which the chromatogram will be stored. The File identifier specifies the second part of the name of the data file. The file identifier must be numerical. In the example above, the data file name will be Test Run_55.DATA. This will be stored in the directory specified by the login Project. The other required entry is the Acquisition length. This specifies the total acquisition time. It can be different than the detector run time but should not be longer. If you are using an autosampler to make an injection, you will need to specify the Vial # and Rack # from which the sample will be injected. If appropriate, the Injection Volume should also be specified. No other entries need to be made on this page. The preprocessing screen only specifies whether you want to subtract a blank baseline from the run before you perform any peak processing. This is not used except in special situations. Page 20 of 37

21 Click on the Integration Events entry in the lower browser window. The Integration Events screen as shown in Figure 27 will be displayed. Figure 27 Integration Events Screen Right click on the space below the table and the first of the three menus shown above will be displayed. Move the cursor over the entry in the menu to get the next menu. Do this for the Add Event and Detection menu entries. The Integration Events Screen is a time-based table of events that controls the detection and processing of peaks. Two events are preset, Peak Width and Threshold. The Peak Width parameter is used in peak detection to distinguish between spikes on the baseline (the set peak width is much larger than the actual peak width) or baseline drift (if set peak width is much smaller than the actual peak width) and the peaks. It bunches points together so that peaks of different width look the same to peak processing. The default value of the Set Peak Width is 0.2 minute. This is fine for most peaks. However, if the peak width varies greatly during the run, you may have to program this using either the Set, Double, or Half peak width parameters. The Threshold Parameter is used to distinguish between baseline noise and the peaks of interest. The parameter operates by taking the highest peak in the chromatogram and setting its value to It then adjusts all other points in the chromatogram relative to this value. When the signal exceeds the set threshold above baseline, it will be detected. In the preset case, the software will detect a peak when it is 10/ or 0.01% of the biggest peak in the chromatogram. There are many other Timed Events that can be applied. Look at them using the procedure above. None of these events need to be set before a chromatogram is collected. They can be used to reprocess the chromatogram later. Click on the Peak Identification entry in the lower browser window. A blank screen will appear. Right click anywhere on the screen and click on Add. The first line of the table shown in Figure 28 will appear. Page 21 of 37

22 Figure 28 Peak Table The Peak table has information about the peak name, retention time, and how to distinguish between peaks if they are close together. Add a line to this table with a peak name of peak1 and a Retention time of 5 minutes as shown in the figure above. Although the peak table can be built here, it is easier to build it by using a standard chromatogram that has already been collected. This is detailed in Lesson 3. Click on the Calibration entry in the lower browser window. When the screen appears, click on the Internal Standard entry in the section labeled Type. The button in the lower left of the display labeled Initialize from ID tables will become active. Set the level number to 3 and click on the Initialize from ID tables button. The screen should now look like Figure 29. Figure 29 Calibration Screen The parameters for calculating the results are set on this screen. Galaxie can use many different variations for result calculation. Click on the Formats entry in the lower browser window. Three buttons will be displayed, Peak report, Group report, and Chromatogram format. Click on Chromatogram format. You should see a display similar to Page 22 of 37

23 Figure 30. Figure 30 Formats Screen The Formats table allows you to specify how the standard report will be displayed. You can specify everything about what will be on the chromatogram and what will be in the report tables. Look through the entries in Chromatogram format as well as in Peak report and Group Report. Click on the Suitability tests in the lower browser window. Click on Add and step through the wizard, selecting Peak on the first page, all of the second page, and As. 10% on the third page. Your screen should look like Figure 31. Page 23 of 37

24 Figure 31 Step 5 of the Suitability Wizard The Suitability section allows you to calculate parameters and measure the quality of the individual chromatogram. This could be the asymmetry of peak or the area of the largest peak. The results of these calculations can then be compared against a preset standard level. If the calculation is not in tolerance, a message will be displayed on the report. Once you have made your entries in a method you can pull down the File, Save Method entry and save the method under the name that you gave it. When you save a method, the Galaxie software will check to see if there are any internal errors in it. For example, if you have specified an internal standard method and you have not specified an internal standard peak, the method will not be saved until you fix the problem. It will not catch errors such as acquisition time longer than run time of the instruments. Making a Single Injection Once you have built a method, you can make a run. Make sure that the one for which you built a method is ready to be run on the appropriate instrument. The instrument needs to be on and equilibrated before you start a run. The simplest way to make a run is to use Quick Start. Click on Acquisition Quick Start as shown in Figure 32. Alternatively, you can click on the Quick Start Icon. Page 24 of 37

25 Figure 32 Quick Start and Quick Start Icon You will now display the Quick Start Menu shown in Figure 33. Figure 33 Quick Start Menu If you are associated with all of the projects in a group, a slightly different screen will appear. It will ask you for the Project name. On this screen you will enter the system that you will work on and the method that you want to use. Note for our instruments we are not using projects. Projects are utilized with big companies where lots of data is being generated. Make these entries and click OK. You will see a screen that is similar to the screen in the Acquisition section of the method. This is shown in Figure 34. Page 25 of 37

26 Figure 34 Acquisition parameters screen. You can now change any of the parameters and click the Start button. DO NOT DO THIS. THE INSTRUMENT IS NOT TURNED ON. When you start the run, the screen in Figure 35 will be displayed. Figure 35 Galaxie Run Screen Page 26 of 37

27 The browser window displays all of the instruments that are available for the individual logged into the system. By selecting the Detector, the status screen on the right is displayed. To display only the chromatogram or status, the appropriate box below the instrument can be checked. Here both boxes are checked. The top half of the status screen displays the chromatogram. The icons on top of the display allow the user to change the way the chromatogram is displayed. The bottom half of the display shows the status of the individual modules that make up the instrument. Selecting the buttons on the left side of the screen will display the status of each module. The system is ready to begin collecting data. If an autosampler is present, it will begin making the injection. If manual injection is being used you will have to fill the loop injector, make the injection and start the system either by starting one of the modules on the system or by having the injector provide a start signal. Questions to Answer See Handout Page 27 of 37

28 Using a Sequence - Overview The following is covered in this section: Building a New Sequence Sequence Editing Tools Sequence Entries Bracketing Running a Sequence Building a New Sequence For this section, the computer needs to be connected to an instrument (the LC or GC). Open a new Sequence by selecting File, New, New Sequence. You will see a screen as shown in Figure 36. Figure 36 New Sequence Wizard Selecting the System First select the system that will use the sequence. This must be one of the systems that you are allowed to use. Once you have selected a system, press Next. The next screen (not shown here) will ask how many lines you want in the sequence. These lines correspond to unknowns, blank baselines (if any) and standards. If you have five unknowns that you want to inject and you want to inject each of them 2 times, you will want to enter 5 (the number of unknowns), not 10 (the number of injections). It is not necessary to have the correct number of lines entered here. You can always add or delete lines from a sequence later. Now click on the Next button to display the screen in Figure 37. Page 28 of 37

29 Figure 37 Entering a Name and Description for the Sequence. Enter a name for the new sequence and a description. The description will help you remember why the sequence was built. Now click on OK. The main screen for editing a sequence will be displayed as shown in Figure 4. Figure 38 Main Sequence Editing Screen Figure 38 shows the top of the main screen. The status of the sequence is listed on the top of the table. The sequence shown here is in IDLE. The other states that a sequence may be in are listed in the table below. Page 29 of 37

30 Status IDLE BEFORE RUN RUNNING COMPLETED ERROR WAITING FOR FINISHING RUN STOPPING RUN AFTER RUN STOPPED PAUSED SEQUENCE RESET CONNECTION LOST CONNECTION RECOVERED Meaning The sequence has not been started or is still being edited. The control parameters have been sent to the system and it is getting ready to start. The Chromatogram is being acquired. The sequence has been finished without any errors. A problem has occurred during acquisition. The sequence has been aborted at the user s request and the run will be stopped after completion of the current line in the sequence. The run is being stopped. The message appears when a system has been stopped manually or paused. The sequence has been stopped manually. The message appears when a sequence has been paused manually. The sequence has been reset and can be started. The connection with the acquisition server has been lost. The connection with the acquisition server was lost and is now recovered. Sequence Editing Tools To make entries into the sequence, you may either enter the information manually, or select the information from a drop-down menu. This is shown in Figure 39. Figure 39 Manual entry of data or selecting data from the drop down menus. Page 30 of 37

31 You may enter information in one line of a table and fill it down into other consecutive lines of the table. This is shown in the next three figures. Enter the data you want to copy into multiple lines and move the cursor so it points to the upper right as in Figure 40. Figure 40 Entering the First Line of Data While holding the left mouse button down, point to the first data entry and pull the cursor down to select all of the lines on which you want to make entries. This is shown in Figure 41. Figure 41 Selecting the Boxes Where Information Will be Copied Click on the right mouse button and select Fill block. This is shown in Figure 42. Figure 42 Filling the Block of Entries Page 31 of 37

32 The icons at the top of the sequence also aid in editing the sequence. They are shown in Figure 9. The Cut, Copy, and Paste Icons operate normally. Even though this screen looks similar to Excel, it does not behave like Excel. The two left-most icons allow you to clear part or all of a sequence. The up and down arrow allows you to reorder the lines in a sequence. Samples are run in the order of the lines in the sequence. The right-most icon allows you to hide columns in a sequence. You might want to do this for columns which you will not use in this sequence. The entries you hide will not be hidden on any other sequence you create. Figure 43 Special Editing Icons for a Sequence The icon second from the right has two functions. When you have selected a series of cells related to a textual entry, like a Run name prefix, the icon will fill in all of the selected cells with the entry in the first cell. However, when you have selected a numerical entry, such as the Run Suffix, the window in Figure 44 will be displayed. Figure 44 Auto Fill Block Window Here you can choose to fill the cells with an incremented entry. For the Run Suffix, you can increment the value by 1 and you will get sequentially-numbered data files after you run the sequence. Page 32 of 37

33 Sequence Entries Many of the entries that are made into the sequence are duplicates of entries that are made into a method. In all cases, the entry into the sequence supersedes any entries made into the method itself. Read through the table below and follow along making appropriate entries into the sequence. Entry No Enabled Method Method Properties Run name (Prefix) Run ID (Suffix) Description Run time Injection Number Vial Rack Inj Vol Sample Type Calibration Level ISTD Value User Input Divisor Multiplier Sample mass Dead Time Function The number of the sequence line. This is the order in which the samples are injected. If this entry is unchecked in a line, that line will not be run. The method that will be used for the sample. This is a list of certain parts of data handling that will be ignored when this line is run. See Figure 45 for an example. Name of the file in which data will be stored. Number attached to the data file to distinguish it from other files. Free-form entry of information that will be saved with that particular data file. Total acquisition time for the run in minutes. Number of times that a sample will be injected. The run index suffix will be incremented automatically. If the autosampler identifies samples by vial number, then this will designate the vial to be injected. If not, this is a free-form information entry Rack number for the autosampler. Volume to be injected if this can be controlled by the autosampler. Standard, unknown (the amount is unknown), or blank (for injection a blank sample) If the injected sample is a standard, choose whether to add the points to the current existing calibration curve, to delete all of the old points or to delete all of the points for the particular calibration level. If the injected sample is a standard, choose which calibration levels it is. Used to enter the internal standard amount for each peak. Note, if your method is not designated as an internal standard method, you will not be able to view this input. Specific user input variables are entered here. Used to divide all the quantities calculated for an unknown sample. Used to multiply all the quantities calculated for an unknown sample. This is often used to change units (from Micrograms to Milligrams). Total mass of the sample. If specified, all of the peaks results will be divided by the sample mass. Time required for an unretained material to be eluted from the column. It is used to calculate certain suitability parameters. Page 33 of 37

34 In the method properties table you will be able to change some of the properties specified in the method on an individual sample basis. Figure 45 shows the Method Options screen you get when you click on Method Options. Figure 45 Method Options Screen If a box is checked, then the method action will occur. Those boxes that are grayed out are method actions that were not programmed into the method. Bracketing Normally in a sequence, standards are run before samples. When all of the standards have been run, the calibration curves are calculated for each peak and stored in the method. Then as the unknowns are run, results are calculated. If bracketing is used, unknowns and standards are intermixed with some standards before the unknowns, and some standards after the unknowns. In this mode, the system must wait until all of the standards have been run before calibration curves can be calculated and results determined. To use this feature, it is necessary to specifically program the sequence to do bracketing. To select bracketing as an option, click on the Bracketing check box on the top of the sequence. A new column will be created at the end of the Sequence table entitled Bracket as shown in Figure 46. Page 34 of 37

35 Figure 46 Bracket Building In this table you will designate how bracketing is to be done. The simplest form of bracketing is to use all of the standards in a given run to calculate all of the unknowns in the run. To do this, you only have to click on the Overall bracketing check box in the upper left hand corner of the Bracket builder. If you do that, all of the entries in the bracketing column will be set to B1, the only bracket. Another form of bracketing is to run several different brackets throughout the run. If you want to use several brackets, you have to designate different brackets in the bracketing column. To have multiple brackets but not have them overlap, select the bracket builder as shown in Figure 46, select bracket B1 and click on the OK button. Once you have done that, you will get B1 in the first line. Now you can fill B1 into the first 6 lines. This is shown in Figure 47. This will use the first two standards, line 1 and 2, and the standards in line 5 and 6 to calculate the calibration curve for the unknowns in lines 4 and 5. Figure 47 Creating Two Non-overlapping Brackets. Click on the small square next to the bracket column and display the bracket builder. Select the Next bracket button and the value B2 will be displayed. Then click OK and B2 will be displayed in line seven. B2 can then be copied for the remainder of the lines. Standards in line 7, 8, 11, and 12 will be used to calculate the calibration curve for the unknowns in line 9 and 10. The last form of bracketing is overlapping bracketing. In overlapping bracketing, standards are shared between two brackets. For example, we can have a set of samples that look like those in Figure 48. Page 35 of 37

36 Figure 48 Setting Bracket 1 In this example we want to use standards on lines 1, 2, 5 and 6 to determine the calibration curve for the unknowns on line 3 and 4. We do that the same way we did previously, selecting the small box next to the bracketing line, selecting the first bracket as B1, clicking on the OK button and then filling B1 into the first 6 lines. Now we want to use the standards on line 5, 6, 9 and 10 to determine the calibration curve for the unknowns on line 7 and 8. To do this, click on the small box next to the bracketing line on line 5, the line with the first standard we want to use for the second bracket. We now click on the Next bracket button in the Overlapped brackets box and click OK. This will enter B1 and B2 in line 5 as shown in Figure 49. Now you can copy line 5 to line 6 and get two entries for B1 and B2. This means that standards on lines 5 and 6 will be used for both Bracket 1 and 2. Page 36 of 37

37 Figure 49 Selecting B2 as the bracket for the Last 2 Unknowns and Standards To complete the process, select line 7, enter the bracket builder, and select bracket B2. Then click on OK, and fill lines 8, 9, and 10 with the entry in line 7, B2. This will create the overlapping brackets. Running a Sequence DO NOT DO THESE STEPS, the instrument is not turned on. To start a sequence, click on the green triangle above the sequence table. This will start the sequence. When a sequence starts, the line that is being run is in displayed in yellow. All of the lines which have been already run, are colored in green. While the sequence is running, you can modify the sequence by adding or deleting lines so long as they are after the yellow line (the line currently running). You can use the Pause icon to stop the sequence but allow the current running acquisition to finish. If you restart the sequence after it has been paused, it will restart on the current line. You can use the Stop icon to stop the sequence and to stop the currently running acquisition. Questions to Answer See Handout. Page 37 of 37