TITLE: (-)-patchoulol GC sample preparation
AUTHORS: Dennis Dienst, Oliver Mantovani, Pia Lindberg

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is a quick guide for the preparation of (-)--patchoulol samples and external standards in dodecane for GC analysis. 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 (-)-patchoulol. The protocol includes the preparation of stock solutions for the internal standard β-caryophyllene (BCP) and the external standard. The preparation of a fresh external standard series is suggested for each sample analysis. The calibration curve is required for quantification of (-)-patchoulol in the samples of interest. </span></div><div class = "text-block">This protocol is used in combination with the following GC protocol:</div><div class = "text-block"><a href="https://dx.doi.org/10.17504/protocols.io.kj2cuqe" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">dx.doi.org/10.17504/protocols.io.kj2cuqe</span></a></div></div>

[STEPS]
?. [Preparation of BCP (β-caryophyllene) internal standard (IS) stocks]
ABCD1&nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;BCP Standard&nbsp;&nbsp;2Stock A1: 10 Dilution
from Original BCP Stock
(⇒ 89 mg *mL-1)
&nbsp;&nbsp;3&nbsp;&nbsp; &nbsp;&nbsp;⇓ &nbsp;&nbsp;&nbsp;&nbsp;4Stock B&nbsp;281 µL&nbsp;Stock A + 719 µL dodecane
(⇒ 25 mg *mL-1)
&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; ⇒&nbsp; &nbsp;1: 100 to samples (2 µL + 198 µL sample)56&nbsp;⇓&nbsp;&nbsp;7Stock C200 µL Stock B &nbsp;ad 19,8 mL Dodecane
(⇒ 250&nbsp; µg *&nbsp; mL-1)
&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;⇒use for preparation of&nbsp;&nbsp; &nbsp;α-bisabolene&nbsp;external standard (ES) seriesStore Stocks in the fridge at ~ 4 °C
ABCD1&nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;BCP Standard&nbsp;&nbsp;2Stock A1: 10 Dilution
from Original BCP Stock
(⇒ 89 mg *mL-1)
&nbsp;&nbsp;3&nbsp;&nbsp; &nbsp;&nbsp;⇓ &nbsp;&nbsp;&nbsp;&nbsp;4Stock B&nbsp;281 µL&nbsp;Stock A + 719 µL dodecane
(⇒ 25 mg *mL-1)
&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; ⇒&nbsp; &nbsp;1: 100 to samples (2 µL + 198 µL sample)56&nbsp;⇓&nbsp;&nbsp;7Stock C200 µL Stock B &nbsp;ad 19,8 mL Dodecane
(⇒ 250&nbsp; µg *&nbsp; mL-1)
&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;⇒use for preparation of&nbsp;&nbsp; &nbsp;α-bisabolene&nbsp;external standard (ES) series
Dodecane is toxic! Wear protective gloves and goggles. Work under the fume hood, or use a respirator!https://pubchem.ncbi.nlm.nih.gov/compound/dodecane#section=Handling-and-Storage
?. [Preparation of patchoulol calibration curve]
Example: Patchoulol external standard (ES) dilution series: 0; 25; 50; 100; 200; 400; 800  µg *  µL-1  ABCD1High Range Dilution Series:&nbsp;&nbsp;&nbsp;2&nbsp;Vol.&nbsp;&nbsp;Stock P-IIVol. Stock CTransfer to vial (3 x)3800 µg*mL-160002004400 µg*mL-13253252005200 µg*mL-1162,5487,52006100 µg*mL-181,25568,75200750 µg*mL-140,625600200825 µg*mL-120,3125629,6875200912.5 µg*mL-110,15625639,84375200100 µg*mL-10,060020011Sum1218,752581,2&nbsp;
ABCD1High Range Dilution Series:&nbsp;&nbsp;&nbsp;2&nbsp;Vol.&nbsp;&nbsp;Stock P-IIVol. Stock CTransfer to vial (3 x)3800 µg*mL-160002004400 µg*mL-13253252005200 µg*mL-1162,5487,52006100 µg*mL-181,25568,75200750 µg*mL-140,625600200825 µg*mL-120,3125629,6875200912.5 µg*mL-110,15625639,84375200100 µg*mL-10,060020011Sum1218,752581,2&nbsp;
Dodecane is toxic! Wear protective gloves and goggles. Work under the fume hood, or use a respirator!https://pubchem.ncbi.nlm.nih.gov/compound/dodecane#section=Handling-and-Storage
?. [Preparation of (-)-patchoulol standard stocks]
AB1Patchoulol standard2Resuspend 10 mg stock powder in 100 µL Dodecane10 mg/ 100 µL
= 100 µg/µL3456&nbsp;7Stock P-I
200 µL20.57 µL  Stock solution (100 µg/µL) 
+ 179,43 µL Stock C  &nbsp;⇒ 10.284&nbsp; µg/µL8&nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;⇓9&nbsp;10Stock P-II
2 mL&nbsp; 155,6 µL Stock P-I&nbsp;  + 1844.4 µL Stock C
(⇒ 800 µg&nbsp; * mL-1)
AB1Patchoulol standard2Resuspend 10 mg stock powder in 100 µL Dodecane10 mg/ 100 µL
= 100 µg/µL3456&nbsp;7Stock P-I
200 µL20.57 µL  Stock solution (100 µg/µL) 
+ 179,43 µL Stock C  &nbsp;⇒ 10.284&nbsp; µg/µL8&nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;⇓9&nbsp;10Stock P-II
2 mL&nbsp; 155,6 µL Stock P-I&nbsp;  + 1844.4 µL Stock C
(⇒ 800 µg&nbsp; * mL-1)
Dodecane is toxic! Wear protective gloves and goggles. Work under the fume hood, or use a respirator!https://pubchem.ncbi.nlm.nih.gov/compound/dodecane#section=Handling-and-Storage
?. [Preparation of biological patchoulol samples (in dodecane)]
pipette 198 µL sample to GC vial add each 2 μL Stock B
Dodecane is toxic! Wear protective gloves and goggles. Work under the fume hood, or use a respirator!https://pubchem.ncbi.nlm.nih.gov/compound/dodecane#section=Handling-and-Storage