TITLE: Gas chromatochraphic detection of Sesquiterpenoids in Dodecane using Perkin Elmer GC 580
AUTHORS: Dennis Dienst, João Rodrigues, Pia Lindberg

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is a quick guide for routine quantitative analysis of sesquiterpenoids in dodecane using an autosampler-assisted gas chromatograph. This protocol has been established in the Lindberg lab at Ångström laboratory (Uppsala University) for direct analysis of dodecane-based </span><span style = "font-style:italic;">ex-situ</span><span>  extracts from cyanobacterial strains producing different types of sesquiterpenoids. The basic protocol was originally described in the master thesis of Sara Nilsson for (</span><span style = "font-style:italic;">E</span><span>)-α-bisabolene quantification [1], and has been extended to (-)-α-bisabolol and (-)-patchoulol.   </span></div><div class = "text-block">This protocol describes the pipeline from the general setup to the generation of quantitative data output for external data processing (e.g. in Excel). Qualitative data analysis is not described. </div><div class = "text-block"><span>[1] Nilsson, S. (2017). (</span><span style = "font-style:italic;">E</span><span>)-α-bisabolene production in </span><span style = "font-style:italic;">Synechocystis</span><span> sp. PCC 6803 (Master thesis). Retrieved from http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-325811</span></div></div>

[STEPS]
?. [Prepare autosampler ]
General Setup fill two wash bottles w/ each 3 mL Dodecane (solvent)place bottles into 'wash' positions 1 & 2  of autosampler platformplace emtpy wash bottles into 'waste' positions 1 & 2 place GC vials (max. 108) into corresponding positions of autosampler platformFor sample preparation check the following protocols:(-)-patchoulol GC sample preparation(E)-α-bisabolene GC sample preparation(-)-α-Bisabolol GC sample preparation
Wear gloves and eyeshields! Dodecane is hazardous to health (GHS08)http://www.sigmaaldrich.com/catalog/product/sial/297879?lang=en®ion=SE
?. [TotalChrom Software: Build a sequence by Template]
open Sequence Editor from TotalChrom Workstationselect 'Clarus 580'call Method (Step 4) of choice for Ch.A and select base file name and file path     → the base file name is used for result (.RST) and raw data (.RAW) files    → the file path should be fix as it will be important for data analysis (Step 8)Spreadsheet:enter sample name into column 'Name'enter your samples numbers into column 'Number' (use alphanumeric numbers)        → align sample numbers with vial numbers in column 'Vial'     → the 'Smart Fill' command is pretty helpful here the data path in columns 'Method' and 'Rpt Fmt File' (Report Format File) should be the same          →  the Report Format File will be crucial for a smooth data output  double-check data path and base file name in column 'Data'   →  the 'Smart Fill' command is pretty helpful here Save vial list as a sequence with .seq extension an .idx file will automatically be generated in parallelTotalChrom uses the base file name from the Setup dialog    → the base file name is used for result (.RST) and raw data (.RAW) files    → the file path should be fix as it will be important for data analysis (Step 8)Spreadsheet:enter sample name into column 'Name'enter your samples numbers into column 'Number' (use alphanumeric numbers)        → align sample numbers with vial numbers in column 'Vial'     → the 'Smart Fill' command is pretty helpful here the data path in columns 'Method' and 'Rpt Fmt File' (Report Format File) should be the same          →  the Report Format File will be crucial for a smooth data output  double-check data path and base file name in column 'Data'   →  the 'Smart Fill' command is pretty helpful here Save vial list as a sequence with .seq extension an .idx file will automatically be generated in parallelTotalChrom uses the base file name from the Setup dialog    → align sample numbers with vial numbers in column 'Vial'     → the 'Smart Fill' command is pretty helpful here the data path in columns 'Method' and 'Rpt Fmt File' (Report Format File) should be the same          →  the Report Format File will be crucial for a smooth data output  double-check data path and base file name in column 'Data'   →  the 'Smart Fill' command is pretty helpful here Save vial list as a sequence with .seq extension an .idx file will automatically be generated in parallelTotalChrom uses the base file name from the Setup dialog          →  the Report Format File will be crucial for a smooth data output  double-check data path and base file name in column 'Data'   →  the 'Smart Fill' command is pretty helpful here Save vial list as a sequence with .seq extension an .idx file will automatically be generated in parallelTotalChrom uses the base file name from the Setup dialog   →  the 'Smart Fill' command is pretty helpful here Save vial list as a sequence with .seq extension an .idx file will automatically be generated in parallelTotalChrom uses the base file name from the Setup dialogExample .seq and .idx files are attached
?. [TotalChrom Software: Setup Instrument]
Start the runMake sure that no 'Editor' window is open      → you cannot simultaneously edit and setup a runOpen the 'Setup' menuSelect 'Sequence' as Setup typeSelect sequence file (.seq) from Step 5Select the same path for 'Raw files' and 'Result files'        → make sure that it matches the path in the Sequence editor's 'Data' column (Step 5)Tick 'Start run when ready'Press 'OK' button exit Setup         → run will start automatically :-)  each sample will take ~30 minthe process can be monitored by clicking the 'Real-Time Plot' button      → you cannot simultaneously edit and setup a runOpen the 'Setup' menuSelect 'Sequence' as Setup typeSelect sequence file (.seq) from Step 5Select the same path for 'Raw files' and 'Result files'        → make sure that it matches the path in the Sequence editor's 'Data' column (Step 5)Tick 'Start run when ready'Press 'OK' button exit Setup         → run will start automatically :-)  each sample will take ~30 minthe process can be monitored by clicking the 'Real-Time Plot' button        → make sure that it matches the path in the Sequence editor's 'Data' column (Step 5)Tick 'Start run when ready'Press 'OK' button exit Setup         → run will start automatically :-)  each sample will take ~30 minthe process can be monitored by clicking the 'Real-Time Plot' button        → run will start automatically :-)each sample will take ~30 minthe process can be monitored by clicking the 'Real-Time Plot' button
?. [TotalChrom Software: Build a method]
Open the Method Editor from TotalChrom Workstation
?. [TotalChrom Software: GC settings]
Define the GC settings in the Method editorIf not yet established - follow the 'Startup' wizard under 'Create new method' using the following settings⇒    use the following settingsInjection T = 250 °CInjection Vol.: 1 µL1 min, 100 °Cramp 5 °C * min-1 to 160 °C,2 min hold,ramp 10 °C * min-1 to 240 °CCarrier gas: N2Flow rate: 50 mL *min-1Go to Step 4
?. [TotalChrom Software: Method Settings]
Define ComponentsGo to 'Components'  →  'New Component...' in the Method Editor Define retention times (RT) of target compounds (e.g. bisabolene, bisabolol and patchoulol) and internal standard (IS) compound (here: β-caryophyllene). The RT and reasonable window settings might be slightly different, due to various factors like carrier gas quality, flow stability, or the wear of the inlet liner (incl. degree of its contamination). as for β-caryophyllene tick 'This component is an internal standard'   as for target compounds select β-caryophyllene as internal standardsave method settings and call method in the sequence setup (Step 5)a method file for sesquiterpenoid analysis is attached
In the example given below, three peaks for β-caryophyllene (BCP are defined, because the commercial standard contains further isomers (>80% BCP purity). For IS calculation only the major peak (7.71 min) is used. The use of an alternative IS compound (e.g. α-humulene) is an option.
?. [TotalChrom Software: Prepare Summary file format]
Summary Report FileTo generate a quantitative output file in .csv format for analysis (e.g. in excel), you first need to generate a 'Summary Report'  template:click the 'Summary' button in the main menu click the 'edit format' buttonthe first two columns should be '1 File Name' and '2 Sample Name'make sure that each component (including IS) is represented by each one column for:       ⋅  retention time (Time [min]) and       ⋅  peak area (Area [µV·s])       ⋅   Adjusted amount a template (.sum file) for parallel analysis of (E)-α-bisabolene, (-)-patchoulol and (-)-α-bisabolol with β-caryophyllene (BCP) as internal standard is attached       ⋅  retention time (Time [min]) and       ⋅  peak area (Area [µV·s])       ⋅   Adjusted amount        → note that for the commercial BCP standard (>80%) three main peaks are annotated in that example, only one of                 which is used as internal standard: RT = 7.71 min
?. [TotalChrom Software: Prepare Summary file format]
Generate Output FileClick 'Summary' button in main menuSelect 'Results From Sequence File'Select Sequence File from Step 5Select 'Results File path' from Step 5Select Results Files path (according to path in Sequence file)Select 'Report format file' (.sum) from Step 7 Go to 'Edit Format' → 'Options' → tick 'Store report in an ASCII formatted file'                         → select '.CSV file' for data analysis in Excel et al.  a 'Summary Report' table will be generated in .pdf format by default (see Fig. below)the creation of a .csv (ASCII) file is noted on the same sheet the .csv file can be imported into Excel via the 'Data' → 'From text' path                        → select '.CSV file' for data analysis in Excel et al.a 'Summary Report' table will be generated in .pdf format by default (see Fig. below)the creation of a .csv (ASCII) file is noted on the same sheet the .csv file can be imported into Excel via the 'Data' → 'From text' path