Contents

The following text styling is applied in this document. Commands, paths or filenames are denoted by: command, or path\filename.ext. Buttons in the graphical user interface are shown as [Button]. Keys on the keyboard are denoted by [Key]. A parameter to be set is denoted by parameter, and a value of a parameter or an option in a menu is denoted by option.

## Installing and starting Chrombox Q

### Windows computers

• Download the installation and unzip the archive qq.zip
• Move folder qq to the preferred destination, e.g. C:\CHROMBOX\. This will be the Q-root folder
• If Installed on a local disk or on a memory stick Chrombox Q can usually be started by using the "Chrombox Q.exe" file in the Q-root folder.

If installed on a network disk you may have to use one of the methods described below:

• Find the file qstart.m in the folder …\qq\various and move it to somewhere in your Mathlab path. This is the only file that needs to be in the Matlab path. Possible destinations may be found by starting Matlab and typing path.
• Open the qstart.m and edit the last line after the run command so that it points to the file qq_startscript (see example below).
• You should now be able to start Chrombox Q by typing qstart in the Matlab command window.

An example of qstart.m is shown below:

xxxxxxxxxx% Startupscript for Chrombox Q% Starts startscript by the run command.% Startscript must be located in the Q root. % ostart must be in the matlab searchpath.% run C:\CHROMBOX\QQ\qq_startscript​run C:\CHROMBOX\QQ\qq_startscript

You can also create a desktop shortcut by copying the shortcut to Matlab and adding the following to the destination /automation /r qstart An example of how it can look is shown below:

C:\MATLAB6p5\bin\win32\matlab.exe /automation /r qstart

### Mac computers (OS X)

• Download the installation and unzip the archive qq.zip

• Move the folder CC to the preferred destination, for example /Users/yourname/Documents/CHROMBOX/QQ, This will be the Q-root folder

• The shell script macstart_q.command stored in the Q-root folder can be used to start the program if the file is executable and Matlab can be started with the terminal command ./matlab. Note that the extension .command may be hidden in Finder.

• To check if Matlab can executed by ./matlab open the terminal and type ./matlab. If Matlab does not start you can do the following:

• Put a symbolic link to Matlab in your path by opening the terminal and typing sudo ln -s /Applications/MATLAB_RXXXXx.app/bin/matlab /usr/local/bin where RXXXXx should be replaced by the Matlab version number, for example "R2017a". Alternatively, open Applications in Finder. Locate Matlab, right-click and select Show Package Contents. Open the folder bin and locate the application file matlab. In terminal type sudo ln -s without pressing enter. Thereafter drag the matlab application file to the terminal. Ensure there is a space between "-s" and "/Applications" and press enter.
• To make macstart_q.command executable, do the following:

• Open the terminal. Use cd to change directory to the Q root where the macstart_q.command is located or open the terminal at the Q root folder if that is an option. Type chmod +x macstart_q.command. Alternatively, type chmod +x without pressing enter and drag the macstart_q.command file from Finder to the terminal. Ensure there is a space between "+x" and "macstart_q.command" and press enter.

• Thereafter double-click on macstart_q.command in Finder to start the program. Depending on your security settings you may get the following message: "macstart_q.command can’t be opened because it is from an unidentified developer". To solve this, open System Preferences – Security and Privacy – General and press [Open anyway] next to the message regarding the file. An alternative way of allowing the file to be executed is to open the file in TextEdit and saving it again. Then it will no longer have status as downloaded from the Internet.

As an alternative to the above procedure, Chrombox Q can be started by the following method:

• Find the file qstart.m in the folder …/qq/various and move it to somewhere in your Matlab path. Possible destinations may be found by starting Matlab and typing path.
• Open the qstart.m and edit the last line after the run command so that it points to the file qq_startscript (see example below).
• You should now be able to start Chrombox Q by typing qstart in the Matlab command window.

An example of qstart.m is shown below:

xxxxxxxxxx% Startupscript for Chrombox Q% Starts startscript by the run command.% Startscript must be located in the Q root. % ostart must be in the matlab searchpath.% run /Users/yourname/Documents/CHROMBOX/QQ/qq_startscript.m​run /Users/yourname/Documents/CHROMBOX/QQ/qq_startscript.m

### Linux computers

• Download the installation and unzip the archive qq.zip

• Move folder QQ to the preferred destination, for example /home/yourname/CHROMBOX/QQ, This will be the Q-root folder

• The shell scripts linstart_q.sh stored in the Q-root folder can be used to start the program, if the file is executable and Matlab can be started with the terminal command matlab.

• On Ubuntu you can use the following procedure to make linstart_q executable:

• Right-click on the file and select Properties. Select Permissions and Allow executing file as program.
• It should now be possible to start Chrombox Q by double-click on linstart_q.sh and selecting the option run in terminal. If you don’t get the run in terminal option while double-clicking the file you will have to edit the preferences in the file manager. Choose Edit in the menu for Files, thereafter Preferences and select the Behaviour tab. Select Ask each time as the option for executable text files.

• There is also a file linstart_q_term.sh in the Q-root folder. The difference between linstart_q and linstart_q_term is that linstart_q runs the application disconnected from the terminal while linstart_q_term runs in the terminal. Chrombox Q will continue to run if you close the terminal if it was initiated by linstart_q, while it will close together with the terminal if it was initiated by linstart_q_term.

As an alternative to the above procedure you can also start Chrombox Q by qstart.m as described for Mac computers above.

### Starting Chrombox Q from the Matlab desktop (on all systems)

On all operating systems you can use the following procedure to start Chrombox Q.

• Start Matlab in the regular way, so that the Matlab desktop is opened.

• Change the current working directory of Matlab to the Q-root folder, either by the line showing the working directory or by browsing in the panel in the left side of the Matlab desktop.

• You can now start Chrombox Q by one of the following methods:

• Select qq_startscript.m in the panel showing the contents of the working directory, right-click and select run.
• type run qq_startscript in the Matlab command window.

In a minimized Matlab session (running in terminal without Matlab desktop) you can use the cd command to set the working directory and run qq_startscript to start the program.

### Changing settings

• The program should normally start without the need to change any settings. But you may want to adjust parameters such as window size. These are specified in the qq_localsettings file in the Q-root folder.
• Open qq_localsettings (.sdv or .csv) in an editor such as Notepad and edit the paths for raw data, etc, if necessary.
• An example of “qq_localsettings” is shown below. Parts to check or edit are shown in blue.
xxxxxxxxxxdefaultfolders; 1; Overrides path settings with default values (qq_root\export,library,method,reports,results,qqraw)defaultmethod; Default; Method to load on startuppath_agilent; ; Folder for MS chemstation rawdatapath_cdf; ; Folder for NetCDF (AIA format) rawdatapath_export; ; Folder for exported datapath_jeoljmc; ; Folder for JEOL .jmc export folderspath_library; ; Folder for spectral libraries and data defining compound classespath_matlab; ; Folder for Matlab datapath_method; ; Folder for methodspath_nist; ; Folder for NIST librarypath_qqraw; ; Folder for Chrombox Q raw data (Matlab format)path_reports; ; Folder for Chrombox Q reportspath_results; ; Folder for Chrombox Q results datatracker; 0; For development purposes, 0 or 1user; Anonymous; version; Q-16-05; Code version to usewindowpos; [0.025 0.07 0.95 0.9]; Window position and size [leftposition lowerposition width height] in fractions of screen size
• windowpos is position of the window in fractions of the screen size. The two first numbers in the vector is the position of the lower left corner. As specified above the lower left corner is 10% from the bottom of the screen and 10% from the left. The height and width is 75% of the screen size. Ensure that the sums of numbers 1 and 3 and numbers 2 and 4 are less than 1.
• If defaultfolders is set to 1 the program will use the standard setup for subfolders and it is not necessary to edit the paths even if they are not correct. If the parameter is set to 1 you will have to specify the location of each path for data and methods. Data can be read from other folders than the ones are specified. Folders can also be changed by using the [Settings] option within the program.
• version refers to the current version of the code. The parameter can also be updated from within the program.
• If you have created a method that you want to import on startup you specify this as defaultmethod.

### Updating

• Unzip the archive with the new code.
• The folder containing the code, e.g. Q-16-05 should be placed in the folder code in the Q root folder.
• Open the file qq_localsettings.sdv (may also have .csv extension) that is found in the q root folder and update the version to the folder name of the new code. The part to be edited is shown in blue in the example below.
• Note that it is not necessary to delete the folders with old code. Keeping these allows you to run previous versions if necessary.

The part to edit in qq_localsettings.sdv is between the two semicolons in the line shown below.

xxxxxxxxxxversion; Q-16-05; Code version to use

Alternatively, you may select the new code by the following procedure:

• Open Chrombox Q
• Press the [Settings] button down in the right corner
• Select [Directories]
• Select the code version and press [Save local settings]
• Restart Chrombox Q.

### Using Chrombox Q with NIST MS-Search 2.0.

On Mac (OS X) and LInux, first install Wine to be able to run NIST MS-Search and verify that you can run windows exe-files. Thereafter proceed as described for Windows below. On Mac, the installer for WineBottler is a convenient way to install Wine. On Mac you currently need the beta version (Q-16-05b) of Chrombox to use the NIST MS-Search.

• Install the NIST software or copy an existing install (the NISTxx folder with all its content) to a separate location, where xx refers to the library version, e.g. NIST05.
• Update the path to the NIST library in Chrombox Q. This can be done in the qq_localsettings.sdv file or you can click [Settings] down in the right corner and choose [Paths / Version]. After editing the path to the NIST library you press [Save local settings]. The edit field for the path will be yellow if the path is not valid.
• If the file autoimp.msd exists in the NISTxx\MSSEARCH folder, open it with a text editor (e.g. Notepad) and ensure that it points to an existing directory where you have write access.
• If the file don't exist, create a mass spectrum, for instance by double-click in a chromatogram in the main window of Chrombox Q. Thereafter right-click in the mass spectrum and select Search NIST from the menu. This should give you the option to create a new autoimp.msd. Select [Yes] to create the file. You will typically get a warning message saying “NIST MS database directory invalid.”. Ignore this and press [OK]. Thereafter you will get the option to select the NIST MS database directory. Select the folder MSSEARCH under NISTxx and press [OK]. The program should open and display the search result.

## Tutorial 1. Basic functions

The main purpose of this tutorial is to get used to the most basic functions in the program. Fatty acid methyl esters (FAME) in a reference mixture will be identified using an existing libraries and an already calibrated method.

### 1.1. Startup and select method

• Start Chrombox Q as wxplained in "Installing ans starting Chrombox Q". The main window should look as in Figure 1.1.
• Start by selecting the method Tutorial-1.
• Select [Load Data] which will take you to the main window for importing and converting raw data.

• The [Load] button takes you to the main window for loading raw data files
• Ensure that the current folder is the TUTORIAL_1-2 folder. If a different folder is selected you can navigate by pressing the [Dir Up] button or by right-click in a directory in the directory and files list. You can also scan for subfolders of the search path that contain the right data type by checking the Scan box next to the search path.
• Select the file Tutorial-1 in the table in the middle
• Press [Load Sel.] and the file opens.
• Press thereafter [Accept] that will take you back to the main window.

### 1.3. Baseline subtraction

• Before proceeding the baseline must be subtracted by pressing the [Auto Basel.] in the main window.

After baseline subtraction the chromatogram should look like in Figure 1.3.

### 1.4. Peak region detection

• The next thing you have to do is to detect the peaks in the chromatogram. Press [Detect] in the main window and the Peaks window shown in Figure 1.4 will open.
• Use the threshold slider to adjust the threshold line so that it is well above the baseline, but covers all peaks (see figure 1.4). You can also adjust the threshold by clicking on the red line.
• Thereafter press [Detect].
• Inspect all peaks. You can zoom in on a peak by clicking on it and thereafter pressing [()] followed by [+] on the zoom line under the chromatogram. You can navigate through the chromatogram by the slider on the zoom line or by the [<] and [>] buttons down in the right corner.
• If you are not satisfied with a peak region you can edit it by right-click on the rectangle marking the peak area or by clicking on the red vertical lines that mark the selected peak region.
• When all peaks are ok you zoom out by [-], press [Lock all] and thereafter [Accept].

### 1.5 Quantification

• After detection the peaks must be quantified. In this case the sample is a reference mixture where all peaks are resolved. The peaks can therefore be quantified by pressing the [All Single] button.
• Press [Close] when the last peak has been quantified.
• Press thereafter [Results] in the main window.

### 1.6. Identification and reporting

The results window is shown in Figure 1.6. .The first thing you have to do is to calculate retention indices for the peaks. The retention indices for fatty acid methyl esters are equivalent chain lengths (ECL).

• The calibrations are stored in the method. Select the calibration Tut-1_cal among the stored calibrations. Press thereafter the [Use Sel.] to activate the selected calibration.
• Press [Calc. RI] to calculate retention indices for the compounds in the mixture. By selecting “Ret. Index” as label you can view the retention indices for each peak. The ECL scale uses the saturated FAMEs as calibration compounds. The saturated FAMEs 12:0, 14:0, 15:0, 16:0, 17:0, 18:0, 20:0, 22:0, 23:0 and 24:0 should have ECL values close to integer values corresponding to the number of double bonds in the fatty acid chain. You can also choose to use retention index as x-axis instead of retention time by selecting the scale on the zoom line.
• When retention indices are calculated it is time to identify the compounds. First press [Settings] on the library line. Verify that Z_BP-20 is selected as search library. You can also add HI-2009 if it is available. Check that Gaussian function is selected under Match retention and that the interval is set to 0.9. Press thereafter [Close] and the libraries will be loaded to the memory.
• Select the first peak, either by clicking on it or by using the arrow-buttons [<] and [>]. This will show the mass spectrum of the compound.
• Press [Search Lib] to search the library. This will take you to the Identify window shown in Figure 1.7. The figure shows the results for the third peak (14:1 n-5).

The compound list shows the 40 best matches from the library. The correlation plot shows similarity between the spectrum from the chromatogram and the library spectra. Ideally, all masses should be near the diagonal blue line.

The match plot shows calculated scores for the compounds in the compound list in decreasing order. Compounds with the same identity as the spectrum selected in the list (the one with highest score by default) are shown in red. Other compounds are shown in blue. For a reliable identification the situation should be similar to the one shown in the figure, where all the best matches have the same identity, there is a clear difference to the next compound and the best matches are above the threshold value marked by the green field.

The info box show additional info, such as the difference in retention indices and the source of the matching compound. In the list of search parameters you can set how the library search should be performed, for instance if and how retention indices should be used. By selecting Exclude instead of Gaussian funct. you can see the effect of omitting the retention indices and match the compounds using only spectra.

• Press [Accept] to accept the suggested or selected ID, [Close] to return without identifying the peak and [Reset] to delete the ID of an already identified peak.
• Go through all peaks in the chromatogram and identify them. Identified peaks will be drawn in green or red (if they are calibration compounds). Unidentified peaks are drawn in blue.

If you are confident about the content of your sample you can also use the [Search all] option that will identify all peaks with match above the threshold, without opening the Identify window.

• When finished with the identification you can report the result by pressing the [Report] button. Various report formats can be selected in the field next to the button. A report in will open if the Open option is checked or if Auto open is set as default in the method.
• Press [Close] to leave the Results window.

You can save your results by [Save results] in the main window and recall the results by typing * or any relevant search string, such as Tut* in the search string field, and thereafter import the selected file by the [Load] button.

## Chrombox Q – Tutorial 2

The purpose of this tutorial is to get used to peak detection and quantification in real samples, which usually have a more challenging peak pattern than reference mixtures. The sample is fatty acid methyl esters (FAME) from a fish oil. You will use the same method as in Tutorial 1.

### 2.1. Startup and select method

• Start the program, select the method Tutorial-1, load the file Tutorial-2 and perform baseline subtraction in the same way as explained in Tutorial-1.

After baseline subtraction the chromatogram should look like in Figure 2.1

### 2.2. Peak region detection:

• Press [Detect] in the main window, which will take you to the window for peak region detection.

The challenge with this sample compared to the reference mixture is that there is large variation in the peak size. Some peaks are also partially overlapping. In the peak detection window the best way to detect the peaks in usually to start with a high threshold value and then gradually reduce the threshold.

• Start by adjusting the threshold slider so that the value is around 100 000 and press [Detect]. The result should look approximately like in Figure 2.2.
• Select the first peak, press the [()] button on the zoom line and zoom in on the peak. Verify that the marked area look reasonable and check all peaks by stepping through the chromatogram with the [>] button.
• When you have reached the last peak, press [Lock All]. This ensures that the already detected peak regions will not be affected by later detection of smaller peaks.
• Reduce the threshold to around 25 000.
• Zoom in on the chromatogram so that approximately 2-3 min is shown on the x-scale. Move to the start of the chromatogram using the slider on the zoom line. Adjust the Y-scale so that the threshold is clearly shown. -0.1 to 1 e5 are appropriate values. When you edit the Y-scale the scale will be locked at this level. You can toggle between locked mode and auto-scaling by the [Lock] and [Auto] buttons on the zoom line.
• Press [Detect Peaks]. You can adjust the selected regions by right-click in field that mark the regions or by first left-clicking on the field and thereafter clicking on the red vertical lines that mark the beginning and end of a peak region. The three first peaks should look approximately as shown in Figure 2.3.
• When you are satisfied with the peaks, press [Lock Visible] to protect the peaks. Be sure all the peaks you want to protect are visible in the window when you apply [Lock Visible]. An alternative to [Lock visible] is to lock the peaks one by one by [Lock Selct.].
• Scroll through the chromatogram and detect peaks that are above the threshold, adjust the marked area if necessary, and lock the peaks when the areas are ok. Some of the peaks will need adjustments of the areas.

There are several alternative methods for adding and adjusting peak regions. If you left-click in the chromatogram above or below a peak while the shift button is pressed on the keyboard a peak region is added around the selected peak. Left-click in front or behind a single or a group of peaks while the [ctrl] button is pressed on the keyboard adds a region with user defined width. Right-click on the area that marks a peak region provides a menu with several more options.

• Right-click on the area that marks a peak region also provides options for viewing the average spectrum or the ion traces of the selected peak region.

When the end of the chromatogram is reached there should be approximately 50 peaks. There are many minor peaks that are not detected at this level, but the threshold level is sufficient for this exercise.

• Zoom out to full view and verify that all peaks are locked (green areas). Press thereafter [Accept] to return to the main window.

### 2.3. Quantification

• Press the [Quantify] button to go to the Quantification window.

Contrary to the reference mixture in Tutorial 1, where all chromatographic peaks were resolved, you may now have overlapping or partially resolved peaks. You must therefore evaluate in each case whether the peak is pure or not. In cases with overlapping peaks you have four possibilities

• If there are parts of the cluster where pure spectra can be found you can resolve the cluster by using the estimates of the pure spectra (S)
• If there are ions that are unique for the compounds you can resolve the peak cluster by using the selective ions (C)
• If none of the two criteria above are fulfilled, but there is a clear valley between overlapping peaks you can go back to the peak cluster by pressing the [Peakwindow] button and split the peak
• If none of the three criteria above are fulfilled the cluster cannot be resolved and you must quantify it as a single peak or use a manual split in the peak window.
• The first peaks are typically pure peaks. Quantify these by pressing the [Single Peak] button. If the Accept single option next to the button is active the program moves to the next peak. If this is off you have the possibility to view the spectrum by pressing the [Resolved S] button after quantification, but you must press the [Accept] button to accept the solution.

At approximately 21 minutes there is a peak with a deviating peak shape with a shoulder to the left, but the two overlapping peaks have nearly identical spectra and there is no valley between them (Figure 2.4). The only option is therefore to quantify them as one peak or to jump to the peak window and split the peak manually.

The next peak cluster is found around 24.7 min. In this case there is a clear shoulder to the right, but there are also impurities in the main peak.

• If you press the button [Init Est.] you will get a plot similar to Figure 2.5 where the program tells where the purest spectra are likely to be found (the vertical lines) and which ions that may be selective (thick lines). The minor impurity under the main peak is not even partially resolved. The spectra can therefore not be used for resolution, but you may use the ions by selecting Use C followed by the [Resolve] button and the suggested chromatographic solution is shown. If you inspect the spectra by pressing [Resolved S] you will see that there are negative ions, which means that the solution is not good.
• Press the [Refine] button to refine the solution. You should then get spectra similar to the ones in Figure 2.7. By right-clicking in the spectra you can do a preliminary search in the libraries to check if the spectra match any known compounds.
• Press [Resolved C] to check that the resolved chromatographic profiles make sense. The blue profile is flat and wide because the peak actually consists of two isomers (cis and trans 7-methyl-6-Hexadecenoic acid) that cannot be distinguished by mass spectrometry.
• Press [Accept] to move to the next peaks.

At around 27.7 there is a new peak cluster that clearly consists of two peaks that are partially resolved (Figure 2.7). When peaks are only partially overlapping like this the best strategy is usually to use the estimates of the spectra for resolution.

• Press [Init Est.] followed by Use S and [Resolve] to quantify the two peaks.
• Press [Accept] to move on to the next peaks.

At around 36.3 there is a new cluster of two severely overlapping peaks (Figure 2.8). In this case there are selective ions but the peaks are too overlapping to give good estimates of the spectra.

• Press [Init Est.], Use C, followed by [Resolve]. Press [Resolved S] to inspect the spectra. If you now press [Refine] you will see that the spectra are confounded again (the masses 185 and 187 appear in the spectrum to the left). In some cases you may get better results by omitting the Refine procedure. It is therefore often necessary to evaluate the results before and after [Refine].
• In this case you will get the most accurate spectra by doing a refinement without the Closure option. Repeat the procedure [Init Est.] and [Resolve]. Uncheck the Closure option and press [Refine]. Press [Resolved S] to inspect the spectra again. Deselecting the Closure option may give you more accurate spectra, but in some cases you may get worse estimates of the chromatographic areas.
• Press [Accept] and move on to the next peaks.

At approximately 48.4 min there is a new cluster with one large and one small peak. There is a quite good resolution between the peaks and this cluster is best resolved using the spectra.

• Press [Init Est.], Use S, [Resolve] and [Refine] (with Closure activated). The minor peak is from a furan fatty acid. You can verify this by doing a preliminary search in the library (right-click on the spectrum).

The remaining peaks are rather pure and can be quantified as single peaks.

• When you reach the last peak, press [Close] to get back to the main window.

### 2.4. Identification and reporting

• Press [Results] to get to the Results window.
• Select the calibration Tut-1_cal and press [Use Sel.]. Press thereafter [Calc. RI] to calculate retention indices. Select Ret. Index as labels and check that the index for the highest peak is around 16.0. This is the 16:0 FAME, which has ECL value of 16 by definition.
• Press [Settings] and check that you have activated the same libraries as in Tutorial 1, that retention scale is retention index, and that Match Retention is set to Retention index. Press [Close] and the libraries will be loaded to memory.
• You can now try to identify the peaks by using the [Search All] button. It may still be necessary to check the identities of the peaks. If any labels are marked orange it means that a compound was identified twice. Large peaks may be skewed and get inaccurate retention indices.
• In the beginning of the chromatogram there are two minor peaks that were not identified. These are the saturated FAMEs 8:0 and 10:0. These are outside the range of the retention index calibration that starts at 12:0. You can identify these by searching only the spectra. Select one of the peaks and press [Search Lib]. In the Identify window, select Exclude under Retention match and accept the result if it is correct. Repeat for the other peak.
• You can now press [Report] to report the results, which should look approximately as in the table below (with additional columns for peak width and asymmetry).

## Tutorial 3. Baseline subtraction and calibration

The purpose of this tutorial is to learn advanced baseline subtraction using CODA, building a retention index calibration from scratch, and use the calibration to identify compounds in a mixture of PAH.

### 3.1. Importing data

• Start the program, select the method Tutorial 3, load the file ALKANES and return to the main window by pressing [Accept].

The chromatograms show a dilute sample of every n-alkane from C9 to C40. The last compounds are completely hidden in the column bleed.

### 3.2. Baseline removal

• Open the baseline window by pressing [Baseline] in the main window.

The window should look like in Figure 3.1. Important functions are CODA and the baseline finder. CODA [Windig et al. Anal. Chem. 68 (1996) 3602] is a filter for removal of ions that are basically found in the background. The baseline finder is a background subtraction function.

• Press [CODA] using a Thresh: (threshold) level of 0.8. This will remove approximately 320 ions of the original 526 ions in the raw data, but it has limited influence on the column bleed.
• Gradually increase the threshold level in steps of 0.02 and press [CODA] after each step. You should see a gradual improvement in the background level as more ions are removed.
• At a threshold level of 0.92 you will see a dramatic reduction of the background when the ions from the column bleed are filtered away, and small peaks at the end of the chromatograms will be visible.
• The chromatogram should now look as in Figure 3.2
• After filtering by CODA, press [BL Finder] to subtract the background of remaining ions.
• Press thereafter [Accept].

### 3.3. Peak region detection:

• Press [Detect] in the main window, which will take you to the “Peaks” window.
• Peaks are detected as in Tutorial 1 and 2. A threshold level of approximately 5000 is suitable.

It may be necessary to adjust the borders of the last peaks that is poorly separated from the noise.

• Click on the last peak. Select [()] on the zoom line and zoom in on the peak using [+]. Right click on the green field and select move borders to adjust the start and end of the peak. Repeat for the second last peak and scroll thereafter through the chromatogram by the [<] button and adjust peaks if required.
• Zoom thereafter out to full view and check that you have 32 peak regions that appear with fairly regular intervals. The detected peaks should look like in Figure 3.3.
• Thereafter press [Accept].

### 3.4. Quantification

• Since the sample is a reference mixture that contains no peak overlap you can quantify the peaks by pressing [Quantify] in the main window and [All single] in the quantify window. Press [Close] when the last peak has been reached.

### 3.5. Building the calibration.

• Press [Results] to open the results window.

• Press thereafter [Settings] and make sure the following are set:

• Libraries to search in should be only Alkanes.
• The Store spectra in RAM option should be selected. This usually speeds up library searches because the libraries do not have to be read from disk each time a search is performed.
• Retention scale should be Retention index.
• Since no retention indices are yet calculated, Match retention must be set to Exclude.
• Interval should be set to 90. This is 100 times higher than the interval used in the previous tutorials, because you will now use Kovats’ indices where the distances between homologous n-alkanes are 100, while the distance is 1 for the ECL scale used with FAME.
• Root should be set to 2. This will take the square-root of all the spectra before they are compared, which will increase the influence of minor ions in the spectra. Negative signals in the spectra are also deleted when root is set higher than 1.
• Press [Close] when settings are appropriate.
• Select the first peak. This should be the C9 alkane with a visible molecular ion of 128.

• Press thereafter [Search Lib]. The peak should be clearly identified as n-nonane. Press [Accept] to accept the result.

• Since the sample contains all n-alkanes from C9 to C40, the next peaks should be C10, C11, C12, etc. Identify these the same way as n-nonane.

• As the chain length increases, the spectra will be more similar and the molecular ion may be weak or absent. For the last compounds you may have to look further down in the list to find the right compound. A way to avoid that is to follow the procedure below:

• After for instance C20, when the spectra are becoming more similar, Press [Calibr] and [Calc. RI] in the Results window. This will give you a temporary calibration where the next peaks can be identified by extrapolation.
• Select the next unidentified peak. Press [Search Lib] and select Gaussian function under Retention match. This should give you a clear result because retention indices are now applied in addition to the spectra.
• When the identities start to become less clear (for instance with score values less than 95) you repeat the procedure with [Calibr] and [Calc. RI] and continue.
• When the last compound is identified, press [Calibr] and [Calc. RI] again. You should then have a calibration that shows a fairly linear relationship between retention index and retention time (Fig. 3.4).

• Press [Store] to store the calibration in the method

• Close the window and press [Save Results].

• Since you may need the calibration later you must also save the updated method with the calibration. Do this by selecting [Settings] in the lower right corner of the main window. Press thereafter [Save Meth.] followed by [Close].

• Press the [Load] button and import the file PAHMIX

### 3.7. Background removal

• Press [Baseline] to go to the baseline window. Press [CODA] using the same parameters as last time and press thereafter [BL Finder].
• Press [Accept] to accept the solution and return to the main window.

### 3.8. Peak region detection

• Press [Detect] to go to the Peak detection window.
• Set the threshold level to approximately 40000 and detect the peak regions by pressing the [Detect] button.
• Select the first peak and zoom in on this using the [()] and the [+] buttons.
• Any peaks before 10 minutes are not PAH and should be deleted using the [Delete selct.] button.
• Scroll through the chromatogram using the [>] button and check that starts and ends of the peaks are properly set. Adjust if necessary.
• Press [Accept] when finished.

### 3.9. Quantification

• Press [Quantify] that will take you to the quantification window and quantify the peaks.

• There are several overlapping peaks. Proposed solutions are given below:

• Two-peak cluster from approx. 21.55-21.80 min: This is two peaks with very different spectra. It can easily be resolved by pressing the [Resolve] button followed by [Accept].
• Two-peak cluster from approx. 32.40-32.70 min: Same as above
• Two-peak cluster from approx. 37.40-37.75 min: This is of two peaks with almost identical spectra and deconvolution will not work since it require differences in the spectra. The best way to solve it is to press the [Peakwindow] button that will take you back to the Peak detection window. Press [Split Select] to split the peak. Press thereafter [Accept], which will take you back to the quantification window. You can now quantify the cluster as two single peaks.
• Two-peak cluster from approx. 38.10-38.45 min: Same solution as above.
• Two-peak cluster from approx. 42.15-42.55 min: This is of two peaks with different spectra and the peak overlap is small. Both [Resolve] and splitting the peak in the peak window will work. Using [Resolve] is usually the simplest solution.
• Two-peak cluster from approx. 46.80-47.10 min: This is a problem with two severely overlapping peaks and no differences in the spectra. There is no ideal solution for these cases, but the best option is split it in the Peak window and quantify the two parts as single peaks. Areas may not be accurate but spectra and retention times will usually be ok.
• Two-peak cluster from approx. 53.05-53.40 min: This is of two peaks with similar spectra and the peak overlap is small. Splitting the cluster in the Peak window and quantifying the two peaks individually is the best option.
• Tree-peak cluster from approx. 60.50-61.05 min: This is a problem with two peaks with similar spectra and one peak with different spectra. There are two solutions for this. You can go to the peak window and split the last peak from the two other. This will give you a peak cluster consisting of two peaks with different spectra that can be resolved by pressing the [Resolve] button and one isolated peak that can be quantified as single. The other option is to press [Resolve] with all three peaks in the cluster, thereafter right-click on the profile with two peaks. Choose Select split position, place the axis-cross in the valley and press the left mouse button. This will give you three peaks (as shown in figure 3.5). Note that if you use [Refine] before the peak splitting you must turn off the Unimod (unimodality) option, since this option forces profiles to have only one maximum.
• Press [Close] when the last peak/cluster is quantified.

### 3.10. Identification

• Press the [Results] button in the main window
• Select your stored calibration ALKANES and press [Use Sel]. Press thereafter [Calc RI] to calculate retention indices for each peak. Change the labels to retention indices.
• Press the [Settings] button and to the following: Select POP-Rxi-1, which is the right library for the applied column, as the only library in the Search in list. The library contains spectra and retention indices of various organic environmental pollutants.
• Check that Match retention is set to Gaussian function, that Retention scale is set to Retention Index, that Spectr. Searchparam is set to MF1: Correlation and that Interval is set to 90. Close the settings window.
• You can thereafter identify the peaks one by one, or by the [Search All] button.
• Press the [Report] button with the options TXT and Open. Inspect your report to verify that all peaks are different PAHs. The list of expected compounds is shown in Table 3.1.