Nov 06, 2019
(E)-α-bisabolene GC sample preparation
- Dennis DD Dienst1,
- Pia Lindberg1,
- João Rodrigues1
- 1Department of Chemistry - Microbial Chemistry, Ångström Laboratory, Uppsala, Sweden
- CyanoWorld
- Chemistry Method Development Community
Protocol Citation: Dennis DD Dienst, Pia Lindberg, João Rodrigues 2019. (E)-α-bisabolene GC sample preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.mwdc7a6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 30, 2018
Last Modified: November 06, 2019
Protocol Integer ID: 9893
Keywords: GC, bisabolene, dodecane
Abstract
This is a quick guide for the preparation of (E)-α-bisabolene 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 ex-situ extracts from cyanobacterial strains producing (E)-α-bisabolene. 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 (E)-α-bisabolene in the samples of interest.
This protocol is used in combination with the following GC protocol:
Materials
MATERIALS
β-Caryophyllene ≥80%, FCC, FGSigma AldrichCatalog #W225207
(−)-α-Bisabolol; analytical standardAlfa AesarCatalog #95426
HPLC/GC Vials 1.5 mL clear glassVwrCatalog #548-1488
Dodecane Reagent Grade ≥99%Catalog #D221104
Preparation of BCP (β-caryophyllene) internal standard (IS) stocks
Preparation of BCP (β-caryophyllene) internal standard (IS) stocks
| BCP Standard | ||||
| Stock A | 1: 10 Dilution from Original BCP Stock (⇒ 89 mg *mL-1) | |||
| ⇓ | ||||
| Stock B | 281 µL Stock A + 719 µL dodecane (⇒ 25 mg *mL-1) | ⇒ | 1: 100 to samples (2 µL + 198 µL sample) | |
| ⇓ | ||||
| Stock C | 200 µL Stock B ad 19,8 mL Dodecane (⇒ 250 µg * mL-1) | ⇒ | use for preparation of α-bisabolene external standard (ES) series |
Store Stocks in the fridge at ~ 4 °C
Safety information
Dodecane is toxic! Wear protective gloves and goggles. Work under the fume hood, or use a respirator!
Preparation of α-bisabolene standard stocks
Preparation of α-bisabolene standard stocks
| α- bisabolene standard | ||
| Stock I 200 µL | 1:100 dilution from original Stock (⇒ 8.9 µg/µL in Stock C) | |
| ⇓ | ||
| Stock II 2 mL | 179.6 µL Stock I + 1820.4 µL Stock C (⇒ 800 µg * mL-1) |
Safety information
Dodecane is toxic! Wear protective gloves and goggles. Work under the fume hood, or use a respirator!
Preparation of α-bisabolene calibration curve
Preparation of α-bisabolene calibration curve
Example: α-bisabolene external standard (ES) dilution series:
0; 25; 50; 100; 200; 400; 800 µg * µL-1
| High Range Dilution Series: | ||||
| Vol. Stock II | Vol. Stock C | Transfer to vial (3 x) | ||
| 800 µg*mL-1 | 600 | 0 | 200 | |
| 400 µg*mL-1 | 325 | 325 | 200 | |
| 200 µg*mL-1 | 162,5 | 487,5 | 200 | |
| 100 µg*mL-1 | 81,25 | 568,75 | 200 | |
| 50 µg*mL-1 | 40,625 | 609,375 | 200 | |
| 25 µg*mL-1 | 20,3125 | 629,6875 | 200 | |
| 12.5 µg*mL-1 | 10,15625 | 639,84375 | 200 | |
| 0 µg*mL-1 | 0,0 | 600 | 200 | |
| Sum | 1229,6875 | 2610,9375 |
Safety information
Dodecane is toxic! Wear protective gloves and goggles. Work under the fume hood, or use a respirator!
Preparation of biological α-bisabolene samples (in dodecane)
Preparation of biological α-bisabolene samples (in dodecane)
- pipette 198 µL sample to GC vial
- add each 2 μL Stock B
Safety information
Dodecane is toxic! Wear protective gloves and goggles. Work under the fume hood, or use a respirator!