CLAS Goldwater Environmental Lab AACE Software Tutorial

T.Colella 28Jun02

2

Part 1 Introduction

Welcome Welcome to the Bran+Luebbe AACE software package! AACE stands for Automated Analyzer Control and Evaluation Software and is designed to control the Bran+Luebbe TRAACS system. It also provides comprehensive and easy-to-use tools to process and evaluate the data collected from the analyzers. This tutorial provides information and instructions about using the AACE software. The reader is assumed to have a basic knowledge of chemistry and laboratory procedures. A basic computer knowledge and a knowledge of working with Windows 98 is also assumed.

The First Steps with AACE Starting AACE 1. Turn on the computer. 2. When Windows 98 has started, the GEL Database main menu will be displayed. Click “Perform an Analysis”. 3. On the Login form, select “Traacs” from the list of instruments. Select “Tom Colella” as user and “Method Development” as run type. Enter “Software Tutorial” under purpose and click “OK”. 4. On the Traacs Run Log, enter your name as Data File, and select the first choice in both the Method and Sample Type 1 boxes. Press Tab to exit the Sample Type 1 field. The “AACE” command button should now be enabled. 5. Click AACE. The AACE opening screen will appear. After a few seconds, the AACE Login dialog box will appear. Enter “traacs” as both username and password into the appearing AACE Login dialog box and confirm with OK. AACE Main Menu will be displayed.

The AACE Main Menu Screen After starting AACE, the Main Menu screen will be displayed providing the following menu options: File to select the printer, carry out the user management, export data and exit AACE. Configure to enter the configuration of your analyzing system and to choose software options. Set Up to prepare analyses and runs.

3

Run To adjust baseline and sensitivity, to start and stop runs, and to recalculate run results. Retrieve To access reports, charts etc. after a run. Windows To change the arrangement of the windows on the desktop. Help To access context-sensitive on-line help.

Part 2 Setting Up Analyses and Runs Introduction AACE allows you to run different applications (chemistries) with different types of samples on the TRAACS. As the runs for an application (chemistry) will usually be very similar, for each application a so-called Analysis (template) is created; and the runs will be created from this Analysis. Any number of analyses can be defined. Having prepared an Analysis once, you can then prepare runs by copying the Analysis and just adding or modifying run-specific information, for example sample ID’s. Accessing the Set Up Analysis Window To create or edit an Analysis or run, select Set Up - Analysis in the Main Menu or click the Set Up Analysis tool button. A dialog box will open, showing the following information: Analysis: The list on the left shows the analyses that are contained in the AACE/Data directory. Each Analysis is stored in a separate folder. Files: The list on the right shows the Analysis and run files belonging to the Analysis currently selected. There is one file with the extension .anl that contains the general set-up information for the Analysis. For each run, there is a separate file with the extension .run (if you look into these directories from Windows Explorer, for example, you will see that, in fact, each run consists of a number of files with different extensions). File information: Information on the file currently selected, e.g. comment, system, number of channels and number of cups. Creating a New Run To help acquaint you with the software, you will create a new run based on a demo Nitrate/Nitrite analysis template. In this tutorial, you will build the Tray Protocol portion of the run from scratch. Under normal circumstances, you will instead modify an existing template. 1. To select the Analysis for which the run will be created, double-click the Data folder, then double-click the Analysis folder you want, in this case the ‘Tutorial’ folder. Highlight ‘Tutorial.anl’. 2. Click New Run to create a new run based on the ‘Tutorial’ Analysis template, i.e., the settings will be the same as for the master Analysis. The Set Up window for the new run will open, so you can enter or change the parameters for the new run.

4

Note: If you open the Set Up Analysis window of an existing run, the OK button is switched dim, so that any changes you might carry out in the window cannot be stored Entering Main Page Information At the top of the Set Up: Analysis window, click the Main Page tab. Enter the following information: Description: This field is optional. You can type in a comment to provide more information about the Analysis. Run Name: Enter the datafile name to be used as it appears on the Traacs Run Log form. For this tutorial it will be your name. For future analyses, use the format DDMMYY plus the analysis abbreviation and a letter code if more than one of the same type of analysis is run in one day (for example, 01Jan02NO3b for the second nitrate/nitrite run performed on Jan 1, 2002). Entering Channel Information At the top of the Set Up: Analysis window, click the Channel tab. Enter the following information: Calibrants: The calibrant concentrations should be checked to make sure they match the calibration solutions you have prepared. The calibrants should be placed in descending order of concentration, i.e., the calibrant with the highest concentration should come first, followed by the one with the second-highest concentration, etc. Note that this order must be the same on the sampler tray. Use the Decimals field to set the number of decimal places for the calibrant concentrations. The maximum is 4. The Decimals field will also determine the number of decimal places shown in the run chart and the report. For this tutorial, assume that the calibrant values are 0.200, 0.400, 0.600, 0.800, and 1.000 mg/L. Also include a blank of 0.000 mg/L. Sample Limits: Select the check box if you want to limit values for the samples. Then enter the low and the high limit. Samples exceeding these limits will be flagged as high ("+") or low ("-") in the report. It is recommended that you set the limits equal to the high and low calibrants (not blank). Quality Control: Click this button to display the quality control limits. Entering Quality Control Limits On the Channel page, click Quality Control to set limits for a calibrant measured as a sample periodically throughout the run (called a CCV for Continuing Calibration Verification) and determine if the run or channel will be stopped if a limit is exceeded. 1. Click the Action/Condition button for Limit 1 of QC1. Select “<“ for a low limit. Click the Action/Condition button for Limit 2 of QC1. Select “>“ for a high limit 2. Select Continue to continue the run if the limit is exceeded. A warning message will appear so the user can decide whether to proceed or abort the run, but the run will not be stopped automatically.

5

3. Enter the values for the limits in the Limit 1 & Limit 2 fields. These are generally -/+ 10% of the prepared concentration. Assume the CCV is the 0.600 mg/L calibrant for this tutorial. 4. Click OK to store the entries. Entering the Tray Protocol The tray protocol reflects the arrangements of cups on the sample tray. Each Analysis (template) contains a standard ('master') protocol that is as universal as possible. It has been designed for the longest run that can be carried out with a full autosampler. You must modify it to fit each run by inserting or deleting cups, entering sample ID’s, and entering dilution factors if necessary. For later runs using the same or similar tray protocol, you can copy it from a previous run to avoid having to re-type sample ID’s. This is explained under “Saving and Loading a Tray Protocol”. 1. At the top of the Set Up: Analysis window, click the Tray Protocol tab. On the left-hand side, the Tray Protocol shows a table with the following columns: Peak: The peak number. The maximum number is 999. Icon and Type: The icons and abbreviations for the cups inserted in the tray. Descriptions for each cup type are given below under “Entering Cups In the Tray Protocol”. P Primer C Calibrant S Sample B Baseline cup D Drift cup H1, L1,L1 Carryover set QC Quality control standard K Spiked sample N Null cup P Pause E End Cup: The cup numbers correspond to the position of the cup on the sample tray. The maximum number of cups is 120 for the Linear Sampler. A zero in this column means that the sampler stops in the wash solution. Sample ID: To enter the sample names, either select the corresponding field and type a name or use the ID generator to create automatically numbered names or import the names from a .txt file. Additional columns if data fields have been set up for the factor of a manual dilution. You can enter values into the data fields either manually or import them from a file. This can be done when preparing the Tray Protocol, i.e. before a run is started, or later after a run. In this case the run must be recalculated. Options on the right hand side of the Tray Protocol Primer, Calibrants, Samples, Baseline etc.: Use these buttons to enter the cup types and their position on the sample tray. Insert: Insert mode. If Insert is selected, new cups are inserted above the line currently selected, i.e., the cups behind the inserted ones are moved further down in

6

the tray. Move: With Move selected at the same time, the following cups are renumbered. Fix: With Fix selected at the same time, the following cups are not renumbered. Overwrite: Overwrite mode. If Overwrite is selected, new cups overwrite existing cups. If you have overwritten cups accidentally, click Undo to undo the last step. ID Generator to enter automatically numbered sample names. Delete to delete tray information. After clicking on Delete, choose between:

Sample ID to delete the sample ID of the cup currently selected. Line to delete the cup currently selected. As a shortcut, you can also click the right mouse button in the Tray Protocol and select Delete Line from the local menu or you can press Ctrl . Delete on your keyboard. All to delete the complete tray protocol. Delete Block: To delete a certain number of cups, enter the first cup to be deleted in the From field and the last one in the To field. Afterwards click Delete to delete them. Cancel to close the dialog box.

Options at the bottom of the window OK to store the changes and close the Set Up: Analysis window. Load Tray to load a tray protocol from another analysis or run. Save Tray to save the current tray protocol as a file so that it can be loaded into other run. Import to import sample IDs or data for a data field (e.g. the dilution factor) from a .txt file. Configure Import to determine the format of the import file and assign the data fields. Undo to undo the last action. Print to print Main Page, Tray Protocol and Channel information. Entering Cups in the Tray Protocol In the Tray Protocol, use the cup type buttons on the right to enter the cups that will be used on the sampler. For this tutorial, the run will consist of the calibrants listed above (cups 111 – 116) and a set of 40 samples with ID’s #1-40 located in cups 1-40. In addition, QC1 (a CCV) will be run before and after each set of 10 samples, a Drift (the high calibrant) will be run before the first calibrant and as the very last cup, and a Base will be run half-way through the analysis. After reading the following descriptions and instructions, build your tray protocol. Good Luck! Primer Cups The Primer must be the first cup on the tray. It starts the peak window and must reach a certain peak height to be recognized. In most cases the Primer contains the same solution as the highest calibrant. A Primer cup is automatically entered at the beginning of each tray protocol. Calibrants These are the calibration standards of known concentration. Their concentration must be

7

entered on the Channel tab as described above. In a run, their peak height is measured, and sample concentrations are calculated by comparison of their peak height to that of the standards. The calibrants should come immediately after the Primer, in descending order of concentration (i.e., the highest standard first). Insert calibrants as follows: 1. In the Tray Protocol, position the cursor in the line above which the calibrants are to be inserted, then click Calibrants. 2. In the appearing dialog box, select the following options: Calibrant. Click this button to insert a cup with a calibration standard. Double calibrant from 1 cup. The calibrant is aspirated twice from the same cup. Double calibrant aspirated from 2 cups. The results of double calibrants will be averaged. How many: Enter the desired number of calibrants. Include the blank. Start at cup: Enter the number of the cup position where you want to start. The default is the number of the previous cup plus 1. 3. When you have finished your selection, click OK to store your entries and insert the calibrants or Cancel to close the dialog box without inserting calibrants. Samples These are the solutions of unknown concentration. Do not enter any samples before the calibrants, otherwise the results will not be calculated correctly. 1. In the Tray Protocol, click Sample. The Samples dialog box will open. 2. Use the following options: How many: Enter the desired number of samples. Use Cup: Enter the desired cup number of the first sample. The default is the number of the previous cup plus 1. Sequential cups: Select this button if the samples you want to insert will be in successive cups on the tray. The samples will then be automatically numbered in the Tray Protocol. Same cup: Select this button if you want to analyze the same sample several times. Group: This option allows you to define up to six different sample groups and then have statistics calculated for each group (if Statistics is selected on the Main Page). If you want to use this option, select a group number for the samples you are now inserting in the tray protocol. During normal operation (other than this tutorial) you will find that a set of three external source QC measurements are grouped together as group S1.

8

3. When you have finished your selection, click OK to store your entries and insert the samples or on Cancel to close the dialog box without storing. Sample IDs can be entered manually in the Tray Protocol, imported or automatically generated using the ID Generator. Baseline Correction (Base) For baseline correction, the sampler stops in the wash solution for 3 sample periods. On the third period the baseline is read and used for a baseline drift correction. It counts as one peak in the tray protocol. Baseline correction can be programmed as often as required. AACE assumes linear drift between two baseline correction cups and applies a separate correction to each peak. If you want to make a separate baseline correction for the calibrants, place the calibrants in descending order immediately after the Primer, then include a baseline correction after the lowest calibrant. Do not place a baseline correction after the Primer. Depending on the setting in the Post Run page in Configure - Software, at the end of the run the final baseline is automatically taken 3 sample periods after the last peak or as soon as the baseline is stable. In the Tray Protocol, click Baseline. A baseline correction cup will be inserted in the tray. Drift Correction Cups (Drift) A drift standard is a solution of known concentration which is used to measure and correct sensitivity drift. A run must contain at least two drift standards. We recommend placing the first drift standard directly after the Primer and the last one just before the End. When the tray protocol settings are saved, the software automatically checks for the presence of at least two drift correction cups. If only one cup has been set, an error message will occur. You can use as many drift cups as needed and aspirate them from the same cup. AACE assumes linear drift between two drift standards and applies a separate correction to each peak. 1. In the Tray Protocol, click Drift. The Drift Corrections dialog box will open. 2.Enter the desired cup number at Use Cup. The default is the number of the previous cup + 1. 3. When you have finished your selection, click OK to store your entries and insert the drift standard or Cancel to close the dialog box without storing. Carryover Sets For carryover correction, one high peak is followed by two equal lows. It can only be programmed in this sequence. High should normally be a full-scale standard, lows should normally be zero standards or wash solution. It applies to all channels, if selected, and can be entered as often as required: If you use more than one H2L, the average carryover factor is calculated. Carryover cups in the tray protocol override a carryover factor which you may have entered for the Channel. However, this calculated value will not be shown on the Channel page. 1. In the Tray Protocol, click Carryover. The Carryover Corrections dialog box will open. 2. Use the following options:

9

High: Enter the cup number for the high concentration peak. The default is the number of the previous cup plus 1. Low 1: If you use a real cup instead of the wash solution, enter the cup number for the first low concentration peak. The default is 0, which means that the sampler probe stops in the wash solution (no additional cup). Low 2: If you use a real cup instead of the wash solution, enter the cup number for the second low concentration peak. The default is 0, which means that the sampler probe stops in the wash solution (no additional cup). 3. When you have finished your selection, click OK to insert the carryover correction or Cancel to close the dialog box without inserting. Quality Control Cups 10 types of Quality Control cups are available. For a normal run, these are QC1 to QC10. QC1 to QC10 The concentration of these Quality Control standards should stay the same over all runs. Periodically during the run, this standard solution is re-analyzed and called CCV. The results will be compared to the limits entered for the channels. Generally, QC1 is a blank and QC2 is a mid-range calibrant but for this tutorial, you will assign QC1 as the mid-range calibrant. To insert a QC cup: 1. In the Tray Protocol, click QC. 2. In the appearing dialog box, select the type of QC cup. 3. Type in the cup number. 4. Click OK to insert the cup in the Tray or Cancel to abort. Spiked Samples To make sure that the results of an analysis are not influenced by the sample matrix, a known quantity of the substance to determine is added to the sample and the resulting spike is measured. This measurement is required by the EPA (Environmental Protection Agency) for some analyses. You can enter one or several spiked cups with a predefined concentration in your tray protocol by entering Spiked Samples or you can run a spike as a sample and manually calculate the recovery. To insert a Spiked Sample: 1. In the Tray Protocol, click Spiked Sample. 2. In the appearing dialog box the Cup number for spiked sample (sequential number) and the Cup number for reference sample (number of the previous sample) are displayed in the first two lines. In the field Cup position, enter the position of the spiked sample on the tray. 3. Enter the Sample volume [uL] in the corresponding field (the default value is 2000 uL, it can be modified in steps of 100). 4. Enter the Standard volume [uL] in the corresponding field (the default value is 200 uL, it can be modified in steps of 10). The Standard concentration for each channel can be edited on the index cards for the corresponding channels. The value entered on this cards is displayed here. The standard concentration is the same for all spiked samples in a channel.

10

5. Click OK to insert the cup in the Tray or Cancel to abort. Note: A spiked sample can only be entered after a sample in the tray protocol. In all other cases the corresponding button is displayed dim. The software compares the results for the spiked and for the normal sample and calculates the recovery of the added standard. Null Cups Results from these peaks are ignored for all calculations, and are printed as ø in the report. Note: If off-scale samples are likely to occur, you should insert 1 or 2 Null cups before Drift cups, QC cups and Carryovers set to protect them from potential carryover. Null cups can also be used when recalculating results from a run, to cancel a previously programmed cup. In this case, remember to click Overwrite before, to switch to overwrite mode. 1. In the Tray Protocol, click Null. 2. In the appearing dialog box, enter the cup number. The default is 0. If 0 is selected, the sampler goes to wash. 3. Click OK to close the dialog box and insert a Null peak or Cancel to close without inserting. Pauses A pause can be inserted in the tray protocol if you have sample sets larger than the sampler will accommodate and you would like to change sample trays during a run. During a pause, the sampler goes to wash and a message appears on the screen. You can now place a new sample tray on the sampler. Afterwards select OK to start sampling of the next tray. In the Tray Protocol, click Pause. An End cup followed by a new Primer is inserted in the tray. Remember to include a Primer in the new tray. Note: The software does not allow a base pause to be followed by a tray pause. When the tray protocol settings are saved, the software automatically checks for this constellation and if a base pause has been entered before a tray pause, an error message occurs. End Cups This is not a cup, but signifies the end of the run and has to be the last peak on the tray protocol. If you insert it anywhere in the tray protocol, the run will stop at this point. To insert an End peak in the tray protocol, click End. Accessing the Cup Data Browser Window If you double-click a line in the tray protocol, the Cup Data Browser window for the

11

selected cup appears, giving all available information about the cup which is shown in the tray protocol, plus some additional information, e.g. the selected channels. To close the window, click the Close button in the upper-right corner. Entering Sample IDs Using the ID Generator You can automatically enter sample names in the Tray Protocol by using the ID generator: Click ID Generator in the Tray Protocol to open the ID Generator dialog box. Use the following options: No. of Samples: The default number of samples is the same as entered in the tray protocol. If you do not want to number all of them automatically, you can decrease the number. Start Sample determines the first sample to be numbered automatically. The default is 1. End Sample determines the last sample to be numbered automatically. ID: ___________: Type that part of the sample name that will be the same for all samples. Then use # for the digits to be numbered automatically. Example: MSTW### Start Number determines the number at which the automatic numbering will start. Increment determines the step size with which the numbers increase. The default is 1. If you want to have the same sample ID for two adjacent cups (duplicate cups), enter 0.5 here. Append: Use this option if your sample ID contains two parts to be numbered consecutively. First set the parameters for the first part, with Append not being selected. Click OK to return to the sample tray. Then click ID Generator again, click the check box behind Append, type in the second part of the sample ID in the ID field and click OK. The new name is now added to the end of the old one. Example: MSTW###NEW###. OK: Click OK to save the entries and close the dialog box. The sample IDs will appear in the Tray Protocol. Cancel: Click Cancel to close the dialog box without generating sample Ids.

12

Saving and Loading a Tray Protocol If you will use similar tray protocols for different analyses, you can enter a tray protocol once, save it as a file and load this file from another Analysis. To save a tray protocol: 1. Select Save Tray at the bottom of the Tray Protocol. 2. In the appearing dialog box, select the Analysis folder in which the file will be stored. The default is the folder of the current Analysis. 3. Enter the filename in the test field on the right. The default is the Analysis name. When storing the file, the extension .trp will automatically be added to this name. 4. Click OK to close the dialog box and save the tray protocol file or Cancel to close the dialog box without saving. To load a tray protocol: 1. Select Load Tray at the bottom of the Tray Protocol. 2. In the appearing dialog box, select the Analysis folder in which the existing tray protocol is stored. Tray protocol files have the extension .trp. 3. Select the tray protocol file and click OK to close the dialog box and load the tray protocol or Cancel to close the dialog box without loading. Importing Data into the Tray Protocol Sample IDs and entries for data fields can be imported from .txt files into the Tray Protocol. The first step is to configure an import file template (see below ). Afterwards, data can be imported as follows: 1. In the Tray Protocol, insert the samples for the Analysis or run. 2. Click Import at the bottom of the Tray Protocol. 3. In the appearing dialog box, select the directory and the .txt file from which data will be imported. Then click OK. The fields assigned in Configure Import for the Analysis (see below ) will be imported into the Tray Protocol, starting with the first Sample line. Any entries in the fields to be imported will be overwritten. If there are more samples in the Tray Protocol than there are lines in the .txt file, the remaining samples will remain empty. If there are fewer samples than there are lines in the .txt file, the extra lines will be ignored. Printing Analysis, Channel and Tray Protocol Data When setting up an Analysis or Run, you can generate a report with the following data: General Run/Analysis data, as entered on the Main Page, Channel data, e.g. selected corrections and calibrant concentrations, as entered for each channel, Tray Protocol data, e.g. cup types or sample IDs, as entered in the tray protocol. This can be useful before preparing the cups on the sample tray, for example, to ensure

13

that the location of the cups on the sample tray really corresponds to the tray protocol for that run. To print this data, click Print at the bottom of the Main Page, Tray Protocol or Channel. A Report window will open that allows you to preview the report. Print a report of the Run you have just created and bring the printout with you to the TrAAcs training class for review. Saving Changes to a Run Click OK at the lower left of the Set Up:Analysis window to store the changes and close the window. You will return to the AACE main menu screen.

Part 3 The Run Menu

Introduction The Run option in the Main Menu allows you to start and stop runs, to manually control the analyzer modules, adjust the baseline, and to recalculate run results. By selecting Run in the Main Menu you can access the following options: Chart to open Charting windows for the channels and check or adjust baseline, reagent absorbance and sensitivity before a run. When Charting has been started or a run is in progress, you can manually control some analyzer modules and transmit commands. You can, for example, switch valves or change the pump speed. You will learn these operations during the TrAAcs training class. Start to start a run. Stop to stop a run or Charting. Quick Start to start a run without having to prepare it in advance. Recalculate to recalculate the results of a run after changing the run parameters or correcting calibrant concentrations.

Starting a Run using Run - Start You can either start a run by using Run - Start (see below) or, if it is a routine run with the same cups as in the Analysis, by using Run - Quick Start. Since this instrument has multiple users with sample sets that usually differ from run to run, we generally use Run – Start and rarely, if ever, use Quick Start. Because you have not yet been trained to operate the TrAAcs hardware, you can not start a real run today. But, you should now simulate starting the run that you have created to acquaint you with the process.

14

To start your run using Run – Start proceed as follows: 1. Normally, you would first confirm that the system is pumping reagents and the sampler tray is loaded according to the tray protocol, observe the bubble pattern and make any other final checks that may be necessary before starting the run. 2. Assume all conditions for starting a run appear satisfactory. Either: Click Run in the Main Menu, then point to Start and click the system you want, OR In the System window on the desktop, click Run. 3. The appearing dialog box shows the Analysis folder currently selected on the left and the runs belonging to this Analysis on the right. To select another Analysis, double-click the Data folder, then double-click the Analysis folder you want. The runs belonging to this Analysis will appear in the list on the right. If you scroll down through the run list, you will see that run information is displayed in the lower-right corner of the dialog box, telling you if the run file has already been used. 4. To start a run, double-click the run you want (in this case, the one you just created using your name as the run name) in the list on the right, or click the run and then click OK. Note: You can select a file that has already been used, for example to overwrite a run that was aborted. In this case you will be asked if you really want to use the file. Select Yes. Afterwards confirm again with Yes that you want to overwrite the file. Since AACE has not established communication with the analyzer (TrAAcs should be turned off), it will now try to connect. This attempt will fail and result in an error message prompting you to turn TrAAcs off and on and retry. Normally, this would not be necessary since communication would already be established before starting your run. Click OK and continue reading the description below for now. Under normal operation, the Start Run dialog box would now appear. The dialog box offers the following options: Run Name to enter or edit the run name. Operator to type in the operator name. Comment to enter a comment for easy identification of the run. Autobase to automatically adjust the baseline before a run. OK to start the run. Cancel to close the dialog box without starting the run. Help to open help about this dialog box.

15

After clicking OK a chart for each channel appears. Modifying the Tray Protocol While the Run Is in Progress Even while a run is in progress, you can access the tray protocol and enter sample IDs or delete or add samples: Access Set Up - Analysis and select the Analysis and the run. Then click the Tray Protocol tab and edit the tray protocol as desired. The changes become effective when you select OK at the bottom of the window. Caution: Before selecting OK make sure the sampler is aspirating from a cup that is at least 2 cups away from the position where you want to insert the new ones. Otherwise the inserted cups will be ignored. Note that sampler status and cup position are shown in the lower-right corner of the System window. You cannot insert cups if the programmed run has already finished and AACE has added samples to be reanalyzed after an off-scale sample to the tray protocol. Stopping a Run A run will stop automatically when all the samples have been analyzed and the final baseline has been taken. The window with the curve will disappear, and a message saying that the run was successfully completed will appear instead. You can also stop a run at any time before it has been finished: Click Stop in the System window on the AACE desktop, OR Click Run in the Main Menu, then point to Stop and click the system you want. Recalculating a Run The Run - Recalculate option allows you to recalculate run results, e.g. if you want to leave out certain samples, standards or corrections or if you want to move the peak marker in a run. To demonstrate, remove (null) the 0.40 ppm calibrant from the run “Ammonia High Range Phenate\20Feb02HiNH4“ to delete the corresponding point from the calibration curve and recalculate the data by following the directions below. Compare the correlation coefficient of the recalculated run to that of the original to determine how the recalculation affects the data. 1. Select Run - Recalculate in the Main Menu or click the Run: Recalculate tool button. 2. The appearing dialog box shows the Analysis folder currently selected on the left and the runs belonging to this Analysis on the right. To select another Analysis, double-click the Data folder, then double-click the Analysis folder you want. The runs belonging to this Analysis will appear in the list on the right. To select the run to be recalculated, double-click the run you want in the list on the right, or click the run and then OK. 3. The Recalculate dialog box will open, showing the details for the run. A default name for the run will be generated, which can be changed manually. The default name consists of the original run name followed by "R1", "R2", etc. for different recalculations of the run. Note that the run description is changed to "Recalculated from run..."

16

You can now change run details, for example: …On the Channel page, you can correct wrong calibrator concentrations, for example, or deselect corrections (e.g. carryover) if the peaks did not seem OK. The concentration values for the corresponding cups will then be ignored for the calculations (it is not necessary to null the cups in the tray protocol). …On the Tray Protocol page, you can null a calibrator or a correction cup (e.g. for sensitivity drift) if the peak does not seem OK. Note that overwrite mode is automatically active. Select the desired cup and click Null. Nulled calibrators are marked by an "X" in the Type column, all other nulled cups by an "N". ...In the View Chart window, you can move the peak marker. 4. Click OK. A dialog box will open, asking if you wish to start the recalculation now. 5. Click Yes to start the recalculation process or No to abandon. A report will automatically be generated (using the original raw data and the modified run data) and displayed. The same options are available as for a run report in the Retrieve - Report option as described below. For now, close the report by clicking Exit.

Part 4 The Retrieve Menu

Introduction After the completion of a run you have different possibilities of retrieving run data, using the Retrieve option in the Main Menu. It provides the following menu options: View Chart to display the charts of a run Print Chart to print the charts Calibration Curve to display and print calibration curves Report to display and print reports listing all relevant data for a run Raw Data to display and print the raw data of a run Charting Values to display and print the Charting values before a run was started QC Chart to display and print QC charts

Retrieving a Chart Any time after the completion of a run or even during a run you can have the run chart displayed on the screen. Display the chart for the recalculated run (‘20Feb02HiNH4R1’) as follows: 1. Select Retrieve - View Chart in the Main Menu or click the Retrieve: View Chart tool button.

17

2. The appearing dialog box shows the following information: Analysis: The Analysis folder currently selected is the one that was selected when you last accessed the Retrieve option. You will need to look in the ‘Ammonia High Range Phenate’ folder to find your recalculated run. Files: The runs belonging to the Analysis currently selected. A message will be shown on the right if the result file is not available, because the run was set up but not started. File information: Information on the run file currently selected, e.g. comment, system, number of channels and number of cups. To open the chart, double-click the run you want (‘20Feb02HiNH4R1’) in the list on the right, or click the run and then OK. The chart will appear on the screen. At the top of the window, the run name is displayed. The window shows a part of the concentration chart of the run. The top of the chart contains information about each peak: the peak number, the peak type, e.g. P for Primer, C for Calibrant, S for Sample or Q for all kinds of QC cups, the corrected or uncorrected concentration of the peak, as shown in the status line. Notice that the 0.40 ppm calibrant peak (peak 10) has an ‘X’ indicating it has been nulled. The tool buttons at the top of the View Chart window provide the following functions: Show/hide peak start: To display vertical lines indicating the beginning of a new peak period. This is useful if a run contains a series of near-zero samples and to show if time slippage occurred during a very long run. Show/hide peak window: To show the peak window in red, i.e., that part of each peak during which the peak-picking program was looking for the peak. Show/hide peak marker: To show a green marker where the peak-picking program took a peak. Curve/points: To display the chart either as a curve or as the individual points, which the computer logged. Corrected/uncorrected results: To show the concentration results before or after corrections (e.g. carryover or sensitivity drift). The status line at the bottom of the window will show if the results displayed are the corrected or uncorrected ones. Show mouse position: To indicate the current mouse position. Select the tool button and then move the mouse pointer in the chart. A red marker will move along the chart, and the status line will show the time in seconds, the A/D counts and the concentration for the current mouse position. Zoom in to enlarge a part of the chart: Click this tool button, then click in the chart. With each click, the chart will be zoomed in further. Zoom out after zooming in the chart: Click the tool button, then click in the chart. If you zoomed in with several clicks, you will need several clicks as well until the original size is restored.

18

Original size: To display the chart in its original size after zooming in the chart. Move chart: Click the tool button, point in the chart, and then press and hold down the left mouse button. As you move the pointer from left to right, the chart will move correspondingly (you can also use the scroll bar at the bottom of the window for the same purpose). Expand time axis: Click this tool button to increase the time scale: the number of peaks visible will decrease. Compress time axis: Click this tool button to decrease the time scale: the peaks will be displayed closer together, so that more peaks will fit in the window. Move marker: This option can be used to move the green peak marker if the software did not set it at the correct position. This function is described below in more detail (see ‘Moving Peak Markers’). Recalculate and save: Click this tool button if you have moved peak markers and want to store the new position. See ‘Moving Peak Markers’ below for more details. Show calibration curve: Click this tool button to open a window showing the calibration curve (see ‘Retrieving a Calibration Curve’ below) Print chart: Click this tool button to print the chart (see ‘Printing a Chart’ below). Print report: Click this tool button to display and print the run report (see ‘Retrieving a Report’ below). Help: Click this tool button to open the help text for this window. To close the window, click the Close button in the upper-right corner. Moving the Peak Marker Peak markers can be moved only in recalculated runs. Original run data cannot be changed. Continue to work with ‘20Feb02HiNH4R1’ for this section of the tutorial. In the View Chart window you can move the green peak marker to a new position if you think the peak should have been picked in a different place. A moved marker is changed from green to red in the chart, and the value in the report is marked with an M to indicate that it was taken manually. For this example, first take note of the values for peaks 14, 15, and 16. These are QC standards with a theoretical concentration of 0.50 ppm. The current values should be 0.48, 0.48, and 0.49, respectively. Follow the directions below to move the peak marker for peak 8, the 1.2 ppm calibrant, from 636 seconds to 630 seconds (hint: use the ‘Show Mouse Position’ function as described above). 1. Click the Move marker tool button. 2. Position the mouse pointer on the peak marker you want to move. Press and hold down the left mouse button and move the pointer to the desired position. Then release the button. A red peak marker will appear in the plot. 3. To save the new position, click the Recalculate and save tool button. The original green marker will disappear and the red marker will turn green. If an unknown was moved, the new

19

concentration will be shown above the peak. If a calibrator or correction cup was moved, the concentrations of all peaks of the run are recalculated. In this example, notice that because you moved a calibrant marker, all peaks were recalculated. The values for peaks 14 – 16 should now be 0.49 ppm. Now, try moving the peak 7 (1.6 ppm calibrant) marker from its current position, 599 sec. to 585 sec. After you recalculate, view the Calibration Curve. The correlation coefficient should change from 0.9996 to 0.9995 since the marker is now on the shoulder of the peak. Caution: Once you have selected the Recalculate and save tool button, the original peak positions cannot be restored. In Retrieve - Report and Retrieve - Raw Data, moved peaks are marked with an 'M'. Close the chart by clicking the Close button. Printing a Chart To print charts after a run, use one of the following ways:

Select Retrieve - Print Chart from the Main Menu or click the Retrieve: Print Chart tool button. Afterwards select the Analysis folder and then the run file to be printed. OR

In an open View Chart window, click the Print tool button. Retrieving a Calibration Curve You can view the calibration curve any time after the completion of a run or even during a run, as soon as the calibrants have been analyzed. Calibration curves of recalculated runs can be accessed as well. 1. Select Retrieve - Calibration Curve in the Main Menu or click the Retrieve:Calibration Curve tool button. Note that the same tool button can also be selected in Run and in View Chart windows. 2. The appearing dialog box shows the Analysis folder currently selected on the left and the runs belonging to this Analysis on the right. To select another Analysis, double-click the Data folder, then double-click the Analysis folder you want. The runs belonging to this Analysis will appear in the list on the right. To open the calibration curve, double-click the run you want in the list on the right, or click the run and then OK. The calibration curve will appear on the screen. On the left, the correlation coefficient, the calibration fit, the calibration coefficients (for the linear, quadratic or cubic regression, depending on the calibration fit), and selected corrections are displayed. Print the calibration curve for your recalculated run. Retrieving a Report The run results can be displayed and printed after the end of a run. Follow the directions below to print a report of the recalculated run. 1. Select Retrieve - Report in the Main Menu or click the Retrieve:Report tool button. Note that the same tool button can also be selected in View Chart windows.

20

2. The appearing dialog box shows the Analysis folder currently selected on the left and the runs belonging to this Analysis on the right. To select another Analysis, double-click the Data folder, then double-click the Analysis folder you want. The runs belonging to this Analysis will appear in the list on the right. To open the report, double-click the run you want in the list on the right, or click the run and then OK. The top of the window contains the following tool buttons: Fit in window: To display the complete page. 100 %: To display the report in the original size. Page width: To display the report so that the complete page width is visible. First page: To display the first page of the report. Previous page: To display the previous page of the report. Next page: To display the next page of the report. Last page: To display the last page of the report. Print report : To print the report. Exit: To close the report window. Examine the report to find the following information, if available: General information about the run, e.g. run name, Analysis, system, operator and start and stop time. Channel information: For real channels, data field. Cup information: Peak number, cup number, sample ID, dilution factor, weight and concentration for the real channels and the results for the data fields. The concentration values can be followed by one of the following characters: * concentration value is offscale, + concentration value is higher than sample limit (if entered for the Channel), - concentration value is lower than sample limit (if entered for the Channel), P QC/CLP cup passed, F QC/CLP cup failed, N concentration value not calculated (for example for the Primer) or not used, R resample after offscale, M peak marker was moved manually, D diluted sample. QC Limits: A list of the limit values (appears only if QC cups were included in the run). Corrections: The corrections for each channel, if any. Statistics: The statistics calculated for groups of samples, if selected.

21

Spiked Sample Report: Reports of spiked samples (K cups), if selected. Spiked samples are reported each on three lines, with the first line giving the sample name with the extension .Added., the second line giving the Quantity of standard found, and the last line giving the actual recovery. To close the window, click the Close button in the upper-right corner.

Part 5 Shutdown Before closing the software, please delete the recalculated run file that you have created during this tutorial as follows:

1. Go to Setup Analysis and highlight ‘20Feb02HiNH4R1’ 2. Click the Delete button

Exiting AACE and Switching Off the Computer 1. On the TrAAcs Run Log in the GEL Main Database, enter “AACE Tutorial” in the Comments text box and enter “0” in the # of Samples field. 2. Click Run Aborted-Not Billable to close the log form. 3. Close any open dialogs and windows. If Charting or a run were still running, you would stop them by clicking the Stop button in each channel window or by clicking Run - Stop and then selecting the relevant system. 4. To exit AACE, select File - Exit in the Main Menu or click the Exit tool button in the Toolbar. 5. To Shut down the computer, click the Start button in the lower left-hand corner of the screen, and then click Shut down. 6. In the appearing dialog box, select Yes to Shut down the computer. Note: Upon shutdown, a file synchronization program will run to back up Traacs data files to the GEL_Main PC. You must shut down the TrAAcs PC after each use to guarantee that your files are backed up. 7. A screen message lets you know when you can safely turn off your computer. If the computer locks-up upon shutdown, turn off the power. This happens occasionally after the file sync program runs.

Part 6 File Formats List of Files for Analyses and Runs All important Analysis and run data is stored in a number of individual files. The following files can be found in the AACE\Data subfolders: Analysis *.anl stores Main Page, Channel and Tray Protocol information. *.ini stores some general settings. e.g. the system configuration. When you create an Analysis, a new folder with the same name is automatically created, and the files for all runs belonging to the Analysis will be stored in this folder.

22

Run *.run stores Main Page, Channel and Tray Protocol information for the run. *.res stores the uncorrected concentration results. *.cor stores the corrected concentration results. *.raw stores the A/D values. *.sta stores the statistics, if sample groups were entered. *.txt is generated if the run is exported in text format. *.slk is generated if the run is exported in SYLK format. *.tmp is generated if the raw values are exported. Congratulations! You have completed the tutorial and are on your way to mastering the TrAAcs 800 Autoanalyzer…