8L, Gas Chromatography (GC)

L3-1

CHEM 8L, Lecture 3 Experiment 2, Day 2 – Gas Chromatography (GC) Analysis of Citrus Oil

Last time… Exp 2, Day 2 - Steam distillation (AKA Co-Distillation) of Terpenes (Citrus Oil) *Green (Environmentally-Friendly)*

- Water-based extraction of terpenes - Minimized waste (you eat the oranges, we compost the peels) - Alternative to solvent-based extraction (toxic, wasteful; more on that in Exp 3)

Citrus Oil Analysis Week 1 - Percent Recovery: (mass of oil) / (mass of peels) x 100% = very low!! Typical recovery of 0.5 – 3 g, 1-10% recovery or less Week 2 - Terpene Identification and Percent Composition by GC CHROMATOGRAPHY - family of techniques for separating components from a mixture based on distribution between a mobile & stationary phase

Stationary Phase (S) Mobile Phase (M)

Metric Sample Mixture Separated by…

Selectively binds to components in

sample Selectively pushes sample along (S)

Gas, GC (Exp 2) Liquid

Gas

Retention Time (tR)

Boiling Point ↓bp, faster (smaller) tR

Thin-Layer, TLC

(Exp 3) Solid

Liquid

Retention Factor (Rf)

Polarity ↓polar, higher Rf Column

(8M, Exp 1)

How does GC help you understand citrus oil? 1. Analyze known standard terpenes - How long does each terpene spend on the column? Retention Time (tR)

Ex. limonene has specific tR on specific instrument, no matter whether it’s pure limonene or mixture containing limonene, based on boiling point

2. Terpene components of citrus oil separate in heated GC column 3. What’s in your oil?

- Compare tR of peaks in oil to tR of standards 4. Quantitative Analysis of Your Citrus Oil - Calculate the area under each peak - Relative Percent Composition: xx% α-pinene, xx% β-pinene, xx% limonene

8L, Gas Chromatography (GC)

L3-2

Inner Workings of Gas Chromatograph

B has ↓ bp, moves faster, ↓ tR A has ↑ bp, moves slower, ↑ tR

8L, Gas Chromatography (GC)

L3-3

Chromatograph Analysis 1. Corrected Retention Time (tR’) for Peak Identification

2. Peak Integration for Sample Composition

Integration: Peak Area = h x wh/2

% Composition of A = Area of component A Total area of all components

air

solvent

sample

injection baseline

distance from air to sample

(cm)

8L, Gas Chromatography (GC)

L3-3

Chromatograph Analysis 1. Corrected Retention Time (tR’) for Peak Identification

2. Peak Integration for Sample Composition

Integration: Peak Area = h x wh/2 % Composition of A = Area of component A Total area of all components

Next week in lab: Exp 2, Day 2 ** Coordinate with partner – bring freshly prepared citrus peels ** Due at the door:

- Exp 2, Day 1 pre-lab questions & notebook pages - Exp 1 report – cover page (last pg of Exp 1 PDF), typed responses to post-lab

questions, carbon copies of notebook pages

air

solvent

sample

injection baseline

distance from air to sample

(cm)

tR' (sec) =

distance from air to sample

(cm) x

chart speed1 min

2.5 cmx

60 sec

1 min

•

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8L, Gas Chromatography (GC)

L3-3

Chromatograph Analysis 1. Corrected Retention Time (tR’) for Peak Identification

2. Peak Integration for Sample Composition

Integration: Peak Area = h x wh/2 % Composition of A = Area of component A Total area of all components

Next week in lab: Exp 2, Day 2 ** Coordinate with partner – bring freshly prepared citrus peels ** Due at the door:

- Exp 2, Day 1 pre-lab questions & notebook pages - Exp 1 report – cover page (last pg of Exp 1 PDF), typed responses to post-lab

questions, carbon copies of notebook pages

air

solvent

sample

injection baseline

distance from air to sample

(cm)

tR' (sec) =

distance from air to sample

(cm) x

chart speed1 min

2.5 cmx

60 sec

1 min

•

thequeylKhliii.is

( too little ) In

c. :µWL

Ii

:¥¥knIe

•

tR' (sec) =

distance from air to sample

(cm) x

chart speed1 min

2.5 cmx

60 sec

1 min

8L, Gas Chromatography (GC)

L3-3

Chromatograph Analysis 1. Corrected Retention Time (tR’) for Peak Identification

2. Peak Integration for Sample Composition

Integration: Peak Area = h x wh/2 % Composition of A = Area of component A Total area of all components

Next week in lab: Exp 2, Day 2 ** Coordinate with partner – bring freshly prepared citrus peels ** Due at the door:

- Exp 2, Day 1 pre-lab questions & notebook pages - Exp 1 report – cover page (last pg of Exp 1 PDF), typed responses to post-lab

questions, carbon copies of notebook pages

air

solvent

sample

injection baseline

distance from air to sample

(cm)

tR' (sec) =

distance from air to sample

(cm) x

chart speed1 min

2.5 cmx

60 sec

1 min

•

thequeylKhliii.is

( too little ) In

c. :µWL

Ii

:¥¥knIe

•

8L, Gas Chromatography (GC)

L3-4

This week in lab: Exp 2, Day 1 ** Coordinate with partner – bring freshly prepared citrus peels ** Due at the door:

- Exp 2, Day 1 pre-lab questions & notebook pages - Exp 1 report – cover page (last pg of Exp 1 PDF), typed responses to post-lab

questions, carbon copies of notebook pages

Biosynthesis of Terpenes and Cannabinoids

High Performance Liquid Chromatography (HPLC): Quality Control (QC) in Cannabis Industry

OP2O73-

Isopentyl diphosphate(IPP)

2 x

α-Pinene155 oC

β-Pinene165 oC

Limonene175 oC

γ-Terpinene183 oC

PP i

CO2HOH

O

H

H

CO2OH

O

H

H

Tetrahydrocannabinolic acid (THCA, decarb 120 oC)

Tetrahydrocannabinol (THC Δ-9), bp 155 oC

Decarboxylation (decarb)

PP i

OH

HO

CO2H

Olivetolic Acid

OH

HO

CO2H

Cannabigerolic Acid(CBGA)

Plants, esp. citrus

Cannabis

MONOTERPENESCollect via steam distillation

OP2O73-

Geranyl diphosphate (GPP)

HO

HOH H

CBDA, decarb 130 oC

OH

O

CBNA, decarb 140 oC

CO2H

CO2H

CANNABINOIDSCollect via CO2, EtOH, or butane extraction