ACD/Spectrus Processor: Chromatography Data Irina Oshchepkova and Laura Zepeda Advanced Chemistry Development, Inc. Toronto, ON, Canada www.acdlabs.com Introduction The following document outlines how to utilize ACD/Spectrus Processor for processing and reporting chromatograms. Importing Raw Data Launch Spectrus Processor from the Start menu or shortcut icon. Method 1: Use Open Data Panel There are several ways to import the data; we would recommend using the Open Data Panel (Document Tree) located on the left hand side. To use it, click on All Files and go to the folder containing the data you wish to process. There you can click and drag the folder to the workspace. Also if you have all your chromatographic or MS data within a directory, you can star that folder and make it a favorite, so you do not have to scroll the Document Tree to find it in the future. Click the empty star next to the folder to mark it as a favorite.
It will now appear on the Document Tree as a separate tab: Method 2: Import chromatogram data files On the main toolbar, click ( ) to open the Import dialog box. In the Look In list, locate the folder containing the chromatogram data files. Select the data file you wish to import, and click ( ). Note: If an expected data file is not shown in the folder content list, select (Auto Detect) in the File of Type list at the bottom of the Open/Import Document window. This will show all files irrespective of extension. Method 3: Drag and drop the file of supported format If a chromatographic data file is of a supported format, it can be dragged and dropped to the Spectrus Processor window. Use Windows Explorer to navigate to the data file. ADVANCED CHEMISTRY DEVELOPMENT, INC. (ACD/LABS) 2
Method 4: Use Connect to Also, raw data from three Chromatographic data systems (CDSs) can be opened in the Spectrus Processor window via the Connect to option from the File menu. This option works when CDS (Waters Empower, Dionex Chromeleon or Total Chrom) and ACD/Labs software are installed on the same computer. Method 5: Use ACD/Empower Add-on and ACD/Chromeleon Add-on In addition, ACD/Labs provides (by request) add-ons for Empower and Chromeleon to allow easy conversion of data stored in the mentioned CDSs into ACD/Labs format (*.esp or *.spectrus) for import into Spectrus Processor. ACD/Chromeleon Addon is based on Export functionality within Chromeleon and uses the Chromeleon SDK. ACD/Chromeleon Addon does not write/save anything inside Chromeleon, it just pulls data from Chromeleon and saves it in *.spectrus format. ADVANCED CHEMISTRY DEVELOPMENT, INC. (ACD/LABS) 3
Processing Chromatographic Data Peak Detection For this example, open the wine1.esp file provided in the \\ACD2017\EXAMPLES\spectrus\CHROM\TUTORIAL\WINE installation folder. Select the Peak by Peak icon found on the Workflow toolbar, Peak Detection section. Quick Start Guide Point to the peak and when you see a red arrow, click. Tops of small peaks (below a predefined threshold or on the tail of a larger peak) may not be recognized. To detect those peaks, zoom in the area (with Horizontal or Rectangular zoom tool SHIFT key then click. ), hold the ADVANCED CHEMISTRY DEVELOPMENT, INC. (ACD/LABS) 4
All results will be automatically updated to the Table of Peaks. Within this table, right click to edit peak names, delete peaks, and observe the result of deconvolution. ADVANCED CHEMISTRY DEVELOPMENT, INC. (ACD/LABS) 5
Note: To view the table of peaks in your workspace, go to Show Tables and click Peaks. Selecting Peaks Now we will add a peak that was not detected on the shoulder of another peak (at 9.7 min). Select rectangular zoom from the toolbar to zoom into a smaller section of the chromatogram. To select the small peak on the shoulder, hold the SHIFT key and click on the peak to detect it. ADVANCED CHEMISTRY DEVELOPMENT, INC. (ACD/LABS) 6
In similar way, add a peak at 3.95 min. The peak is almost invisible and it is hard to see the edges or integrated area. Undo the last action by clicking ( ) or delete this peak from the Table of Peaks. Still in Peak by Peak mode, click in the beginning of the peak and holding the mouse, drag across to the right edge of the future peak, then release the mouse: ADVANCED CHEMISTRY DEVELOPMENT, INC. (ACD/LABS) 7
Adjusting Peak Integration To adjust integration ranges, hover over a selected peak to see a red boarder that denotes the peak range. You can adjust the start/stop integration ranges by making the red boarder larger or smaller. Changing Peak Labels To change the peak labels that are displayed above a peak, go to the Options menu, choose Chromatogram Preferences, and in the Labels tab, choose your settings. For example, here we have changed the peak labels to show names rather than retention time: Name all the peaks of interest and adjust the peak areas if needed. ADVANCED CHEMISTRY DEVELOPMENT, INC. (ACD/LABS) 8
Deconvolute Peaks Zoom in to the area from 6 to 11 min. We will deconvolute peaks 6, 7 and 8. From the Analysis menu, select Deconvolution and click Deconvolute. ADVANCED CHEMISTRY DEVELOPMENT, INC. (ACD/LABS) 9
On the chromatogram window, drag a box around the co-eluting peaks to be deconvoluted. Note that when the mouse is released, the new values for peak areas are updated in the table. ADVANCED CHEMISTRY DEVELOPMENT, INC. (ACD/LABS) 10
To view new deconvoluted curves under the peaks, we need to hide the colored peak areas. To do so, from View menu, select Chromatogram Labels and clear Show Peak Areas. To see the colored areas again, select Show Peak Areas. ADVANCED CHEMISTRY DEVELOPMENT, INC. (ACD/LABS) 11
Annotation Quick Start Guide You can annotate single peaks and whole regions of an open spectrum, chromatogram, or curve. 1. In the Interpret group of the Workflow bar, click Annotation ( ). Note that the mouse pointer turns to ( ). 2. Drag over the chromatogram region to select the area to be annotated or click the required peak. 3. In the Edit Annotation dialog box that appears, type an annotation text, and then in the Layer No. list, select a layer number for the current annotation. 4. Click OK. The annotation is attached to the specified region or peak. ADVANCED CHEMISTRY DEVELOPMENT, INC. (ACD/LABS) 12
Attaching Structures Quick Start Guide The user can import structures by copying and pasting from ChemSketch or from a variety of third party chemical drawing packages, such as ChemDraw or ISIS, into Spectrus Processor. Once imported, if the structure needs to be modified, you do not have to go to ChemSketch, just edit it within the Chemical Structure window. To work with structures, we will need the Chemical Structure Window (Show Window > Structure) and the Table of Structures (Show Table > Structure) ready: ADVANCED CHEMISTRY DEVELOPMENT, INC. (ACD/LABS) 13
Start drawing structures (one by one) in the Chemical Structure window and they will appear in the Table of Structures. You can copy/paste the structure or just drag the ChemSketch file into this window. To bring a group of structures at once, right click within the Table of Structures and Import Structures from an *.sdf file with several structures in it (use WINE.SDF, provided in the installation folder). ADVANCED CHEMISTRY DEVELOPMENT, INC. (ACD/LABS) 14
Now click the first line in the table, hold the mouse and drag it over to the corresponding peak. When you release the mouse, the structure will be assigned to this particular peak and will get the tr value in the Table of Structures. Click Yes to see the name updated in the Table of Peaks. The structure line goes down in the Table of Structures. Continue the assignment procedure for the rest of the peaks. If you have not manually assigned names to each peak prior to this, you will not be warned about the name change (for example, going from unknown to ethanol). ADVANCED CHEMISTRY DEVELOPMENT, INC. (ACD/LABS) 15
Editing Method Parameters The final preparation step is to edit and save HPLC or method parameters. Quick Start Guide 1. On the top toolbar, select the Edit/Show Chromatogram Parameters shortcut button ( ). Click OK. Note that the Type of the chromatogram may be already recognized by Spectrus Processor and you will see only the next window: The window could be blank or show you some information about Sample, Injection volume and Detector that comes from the instrument. To import parameters from a file, select ( ) in the HPLC Parameters window. ADVANCED CHEMISTRY DEVELOPMENT, INC. (ACD/LABS) 16
Select the appropriate HPLC Parameters file, then click ( ). ADVANCED CHEMISTRY DEVELOPMENT, INC. (ACD/LABS) 17
2. To input parameters manually, type the information in the fields. Note: For further optimization in LC Simulator, dwell volume, dead time, and flow rate must be specified. 3. Entry of solvent data is strongly recommended. Click the Mobile Phases ( ) button. You will see a new window. 4. To manually enter solvents, right click the Solvents section and Add a new line first. In the % column type the number; in the Name column, you can either type the name or select it from the drop-down list box. The example above loaded mobile phases automatically when the HLPC parameters were loaded. 5. For Buffers, right click and Add a new line first. In the Value column, type the data; in the Unit column, you should select the units of measurement; and, in the Name column, you can either type the data or select it from the drop-down list. 6. The Calculate button helps you to calculate ph based on the information provided for buffers. Note: This works only when the buffers are chosen from the drop-down list of standard buffers. 7. By default you see only one phase window which is good for isocratic conditions. To add more phases, navigate your mouse pointer to the Phase A tab and right click. Then choose Add. ADVANCED CHEMISTRY DEVELOPMENT, INC. (ACD/LABS) 18
8. Second Phase B is added and the window for gradient program appears on the right: 9. The gradient program table displays the data of your mobile phases. Note that the last phase in each table line is updated automatically if you select the Automatically Correct Last Phase check box. The incorrect data is highlighted in red. 10. An example of the complete Mobile Phases dialog box is shown below. ADVANCED CHEMISTRY DEVELOPMENT, INC. (ACD/LABS) 19
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11. You can save all of the information by clicking Save at the very bottom of the HPLC Parameters window (in *.pa format). You can send this file to your colleague. To load the parameters from a file, click Load and navigate to the.pa file. Click OK to close Mobile Phases. Click OK for HPLC Parameters window. 12. Save the processed file ( ). ADVANCED CHEMISTRY DEVELOPMENT, INC. (ACD/LABS) 21
Generating a Standard Report In this section we will generate a simple (standard) report. There is a Report button ( ) on the bottom toolbar. The same button is used to generate either a standard report or a more sophisticated report based on the user template. First, we need to set the options for Standard Report. Click the small black arrow next to the Report button to see the shortcut menu: Make sure the Standard option is checked and then click Report Setup In the Report Page Setup window, go through all tabs and specify objects and their styles, then click OK. The settings are saved to be used for the same data type in the future. Click the Report button ( ). It instantaneously generates the report and displays it in the ChemSketch window. You can then change the sizes and location of the report's objects in the ChemSketch window as well as edit their style in the corresponding Objects Panel dialog box; displayed as you double-click an object. ADVANCED CHEMISTRY DEVELOPMENT, INC. (ACD/LABS) 22
From ChemSketch you can print the report ( ), save it in ChemSketch format ( ), or produce a PDF version ( ). Note: Larger reports automatically create a multipage report. On the bottom bar, use the arrow buttons ( ) to navigate between the pages of the report. The report can be transferred to Microsoft Word by selecting Edit > Select All and then Edit > Copy and pasting the object in Microsoft Word. To generate a report directly in PDF format, from the File menu point to Export Report to PDF > Standard. ADVANCED CHEMISTRY DEVELOPMENT, INC. (ACD/LABS) 23
Conclusion Quick Start Guide The process described above is a very basic overview of the main workflow in ACD/Spectrus Processor for chromatographic data. Many more actions and options exist that may be valuable in your process. For a more in-depth view of any of the individual processing features, please consult the quick start guide screen when you open Spectrus Processor or go to the Help menu. Please also visit www.acdlabs.com for more details. If your software is on a computer connected to the internet, our technical support team can be easily contacted by selecting Send Bug Report/Feature Request under the ACD/Labs menu, filling out the appropriate information and sending via Web or Mail. ADVANCED CHEMISTRY DEVELOPMENT, INC. (ACD/LABS) 24