Terpenes Analysis of Essential Oils using GC-VUV An Automated, Speed-based Approach Alex Hodgson, Senior Applications Chemist VUV Analytics, Inc.
VUV Absorption Spectroscopy • Simple absorption spectroscopy in a new spectral region • Vacuum ultraviolet (VUV) spectroscopy is a universal technique that provides unique spectral fingerprints • High energy, low wavelength exposure produces electronic transitions between σ→σ* , n →σ* , and π→π* molecular orbitals • Compound identification is unambiguous, even for structural isomers • Chromatographic compression leads to higher sample throughput • Software deconvolution of coelutions reduces priority on chromatographic separation • Chromatography runs can be deliberately compressed
Introduction to Terpenes • Class of organic compounds produced by plants (and some insects) • Primary constituents of essential oils, which contribute heavily to fragrances and flavors • Widely utilized in the food and fragrance, cosmetics, and pharmaceuticals industries • Composed of multiple units of isoprene (C 5 H 8 ) • Monoterpenes – 2 isoprene units (C 10 H 16 ) • Sesquiterpenes – 3 isoprene units (C 15 H 24 ) • Very high prevalence of structural isomers
Monoterpenes Sesquiterpenes Sesquiterpenoids Monoterpenoids α -Pinene β -Myrcene Limonene β -Caryophyllene α -Humulene Eucalyptol p -Cymene Linalool trans-Nerolidol Guaiol
Headspace/Solid-phase Microextraction (SPME) Setup Gerstel MPS2 Autosampler VUV Analytics Agilent 6890 Gas VGA-100 Chromatograph
Headspace Run Conditions • Gerstel MPS2 • Incubator: 80°C for 10 minutes, 250 rpm agitation (10 sec on, 1 sec off) • Syringe: 90°C, 2.5 mL injection volume • Agilent 6890 GC • Inlet: 250°C, split 2.5:1 • Column: Restek Rxi-624Sil MS (30 m x 0.25 mm, 1.40- μm) • Flow: 4 mL/min helium , constant flow • Oven: 60°C, hold 0.1 min; 23.8°C/min to 300°C (run time - 10.2 min ) • VUV Analytics VGA-100 • Makeup gas: 0.25 psi N 2 • Flow cell and transfer line: 275°C • Acquisition rate: 4 spectra/sec • Acquisition range: 125-240 nm
Terpenes Standard (Restek) Class Compound RT (min) Formula MW Sources Monoterpene α -Pinene 3.87 C 10 H 16 136.23 Coniferous trees, rosemary, eucalyptus Camphene 4.03 C 10 H 16 136.23 Camphor, neroli, valerian β -Myrcene 4.21 C 10 H 16 136.23 Thyme, cardamom, hops, cannabis β -Pinene 4.25 C 10 H 16 136.23 Nutmeg, rosemary, sage 3-Carene 4.43 C 10 H 16 136.23 Turpentine, rosemary, cedar α -Terpinene 4.50 C 10 H 16 136.23 Allspice, juniper, marjoram cis-Ocimene 4.53 C 10 H 16 136.23 Basil, lavender, clary sage Limonene 4.58 C 10 H 16 136.23 Citrus fruits, mint trans-Ocimene 4.64 C 10 H 16 136.23 Basil, lavender, clary sage γ -Terpinene 4.77 C 10 H 16 136.23 Citrus fruits, cumin, Syrian oregano Terpinolene 4.98 C 10 H 16 136.23 Allspice, citrus fruits, juniper
Terpenes Standard (Restek) Class Compound RT (min) Formula MW Sources Monoterpenoid p -Cymene 4.60 C 10 H 14 134.22 Cumin, thyme Linalool 5.21 C 10 H 18 O 154.25 Mint, cinnamon, rosewood Isopulegol 5.67 C 10 H 18 O 154.25 European pennyroyal, corn mint Geraniol 6.27 C 10 H 18 O 154.25 Almonds, palmarosa, rose Sesquiterpene β -Caryophyllene 7.32 C 15 H 24 204.35 Cloves, rosemary, hops, cannabis α -Humulene 7.53 C 15 H 24 204.35 Sage, ginseng, tobacco Sesquiterpenoid cis-Nerolidol 7.85 C 15 H 26 O 222.37 Neroli, jasmine, lavender trans-Nerolidol 8.01 C 15 H 26 O 222.37 Neroli, jasmine, lavender Guaiol 8.37 C 15 H 26 O 222.37 Cypress, pine, cannabis α -Bisabolol 8.73 C 15 H 26 O 222.37 German chamomile, balsam poplar
0.5 1. α -Pinene 12. Terpinolene 125-160 nm 7 2. Camphene 13. Linalool 11 8 170-240 nm 3. β -Myrcene 14. Isopulegol 4. β -Pinene 15. Geraniol 3 5. 3-Carene 16. β -Caryophyllene 5 6 9 0.4 6. α -Terpinene 17. α -Humulene 7. cis-Ocimene 18. cis-Nerolidol 4 17 8. Limonene 19. trans-Nerolidol 16 12 9. p -Cymene 20. Guaiol 1 10. trans-Ocimene 21. α -Bisabolol 2 0.3 11. γ -Terpinene Detector Response 10 Last analyte elutes before 0.2 9 min! 13 0.1 19 20 14 18 21 15 0 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 Time (min)
Mass Spectra of Coeluting Isomers 41 β -Myrcene 93 69 121 136 93 β -Pinene 41 69 121 136
Absorbance Spectra of Monoterpene Isomers 1 α -Pinene β -Pinene β -Myrcene Limonene cis-Ocimene trans-Ocimene 0.9 0.8 0.7 Normalized Absorbance 0.6 0.5 0.4 0.3 0.2 0.1 0 130 140 150 160 170 180 190 200 210 220 230 240 Wavelength (nm)
Spectral Deconvolution of Coeluting Peaks 0.4 125-240 nm Limonene α -Terpinene p -Cymene 0.35 cis-Ocimene trans-Ocimene 0.3 0.25 Detector Response 0.2 0.15 0.1 0.05 Summary: XXX • 0 XXX • 4.44 4.46 4.48 4.5 4.52 4.54 4.56 4.58 4.6 4.62 4.64 4.66 XXX • Time (min)
Region 1 – Single Analyte Spectral Matching 0.22 0.2 0.18 0.16 0.14 Absorbance 0.12 0.1 0.08 0.06 0.04 Summed Retention Region Spectrum Summed Target Spectrum Best Fit; R^2 = 0.99890 0.02 alpha-Terpinene 0 130 140 150 160 170 180 190 200 210 220 230 240 Wavelength (nm)
Region 2 – Summation of Spectra from Coelution #1 Summed Retention Region Spectrum 0.33 Summed Target Spectra Best Fit; R^2 = 0.99923 0.3 alpha-Terpinene cis-Ocimene 0.27 0.24 0.21 Absorbance 0.18 0.15 0.12 0.09 0.06 0.03 0 130 140 150 160 170 180 190 200 210 220 230 240 Wavelength (nm)
Region 3 – Summation of Spectra from Coelution #2 1.2 Summed Retention Region Spectrum 1.1 Summed Target Spectra Best Fit; R^2 = 0.99962 Limonene 1 p-Cymene 0.9 0.8 0.7 Absorbance 0.6 0.5 0.4 0.3 0.2 0.1 0 130 140 150 160 170 180 190 200 210 220 230 240 Wavelength (nm)
Region 4 – Single Analyte Spectral Matching 0.5 0.45 0.4 0.35 0.3 Absorbance 0.25 0.2 0.15 0.1 Summed Retention Region Spectrum Summed Target Spectrum Best Fit; R^2 = 0.99771 0.05 trans-Ocimene 0 130 140 150 160 170 180 190 200 210 220 230 240 Wavelength (nm)
VUV vs MS – Tale of the Tape Who has the edge? Higher flow limit Lower relative LOD Ease of use/ Ease of maintenance 3-dimensional information Unique spectra for all measurable compounds Isomer differentiation
VUV Methods – Run Conditions Agilent 6890 GC VUV VUV VUV Method 1 Method 2 Method 3 1 µL injection • Inlet: 250 ° C, split 10-300:1 Col Flow (mL/min • 1.4 2 4 He) Column: Restek Rxi-1ms • (30 m x 0.25 mm, 0.25-µm) Oven Start (°C) 40 40 40 VUV Analytics VGA-100 Initial Hold (min) 0.2 0.2 0.1 Makeup gas: 0.25 psi N 2 • Oven Ramp Flow cell and transfer line: 275°C • 9.6 20.4 30.9 (°C/min) Acquisition rate: 5 spectra/sec • Oven Final (°C) 250 250 250 Acquisition range: 125-240 nm • 22.1 10.5 6.85 Run Time (min)
VUV Run Results Peak # Analyte 1 α -Pinene 2 Camphene 3 β -Pinene 4 β -Myrcene 5 3-Carene 6 α -Terpinene 7 p -Cymene 8 Eucalyptol 9 Limonene 10 cis-Ocimene 11 trans-Ocimene 12 γ -Terpinene 13 Terpinolene 14 Linalool 15 Isopulegol 16 Geraniol 17 β -Caryophyllene 18 α -Humulene 19 cis-Nerolidol 20 trans-Nerolidol 21 Caryophyllene oxide 22 Guaiol 23 α -Bisabolol
VUV Methods Comparison – Tea Tree Oil VUV Method 1 VUV Method 2 VUV Method 3 VUV Average Compound (mg/mL) – (mg/mL) – (mg/mL) – CV (%) (mg/mL) 22.1min 10.5min 6.85min α -Pinene 20.6 21.5 23.0 21.7 4.48 α -Terpinene 77.9 80.4 84.8 81.0 3.57 β -Caryophyllene 2.09 2.26 2.03 2.13 4.46 β -Myrcene 7.20 7.72 7.95 7.62 5.14 β -Pinene 5.83 5.85 5.21 5.63 5.30 Eucalyptol 21.0 25.0 22.8 22.9 7.24 γ -Terpinene 161 172 193 175 7.53 Limonene 6.38 5.62 7.03 6.34 9.08 p -Cymene 25.2 27.9 33.3 28.8 11.6 Terpinolene 27 29.7 23.1 26.6 10.3
MS Methods – Run Conditions Agilent 6890 GC MS Method MS Method 1 2 1 µL injection • Inlet: 250 ° C, split 20-975:1 • Col Flow (mL/min He) 1 1.4 Column: Restek Rxi-1ms • Oven Start (°C) 40 50 (30 m x 0.25 mm, 0.25-µm) Transfer line: 280 ° C • Initial Hold (min) 1.5 1 Agilent 5973N MS Ramp 1 (°C/min) 1 15 EI Source: 300 ° C • Ramp 1 End (°C) 77 -- Quad 1: 200 ° C • Ramp 2 (°C/min) 7 -- Scan range: 50-250 • Oven Final (°C) 250 250 63.2 14.3 Run Time (min)
MS Run Results Peak # Analyte 1 α -Pinene 2 Camphene 3 β -Pinene 4 β -Myrcene 5 3-Carene 6 α -Terpinene 7 p -Cymene 8 Eucalyptol 9 Limonene 10 cis-Ocimene 11 trans-Ocimene 12 γ -Terpinene 13 Terpinolene 14 Linalool 15 Isopulegol 16 Geraniol 17 β -Caryophyllene 18 α -Humulene 19 cis-Nerolidol 20 trans-Nerolidol 21 Caryophyllene oxide 22 Guaiol 23 α -Bisabolol
VUV vs MS Comparison – Tea Tree Oil MS Method 1 Average w/ all VUV runs Compound CV (%) (mg/mL) – 63.2min (mg/mL) α -Pinene 26.3 22.8 9.56 α -Terpinene 91.2 83.6 6.04 β -Caryophyllene 1.58 1.99 12.6 β -Myrcene 7.44 7.58 3.76 β -Pinene 6.36 5.81 7.00 Eucalyptol 30.2 24.8 14.0 γ -Terpinene 205 183 9.48 Limonene 7.84 6.72 12.2 p -Cymene 35 30.4 13.1 Terpinolene 31.1 27.7 11.0
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