Adventures in Hop oil Extraction using Pressurized Liquid Extraction

Jared Harkins,1 Katy Orr

1 Sierra Nevada Brewing Company

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Study Location

Arthur’s PassNational Park

State Highway 73

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Taramakau Valley (east-extending valley)

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Wind roses for east-extending valley

Tota

l PA

Hs

Co

nc

(ng

g-1lic

hen

)

Distance from road (km)

0

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0 2 4 6 8 10 12

• Predominance of northeast winds means up-valley component to wind direction.

• Concentrations fairly constant throughout valley.

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Waimakariri Valley (west-extending valley)

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Tota

l PA

Hs

Co

nc

(ng

g-1lic

hen

)

0

10

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40

50

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0 2 4 6 8 10 12

Distance from road (km)

Wind roses for west-extending valley

• Wind direction is predominantly down-valley.

• Linear decrease in concentration away from the road.

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Hawdon Valley (north-extending valley)

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Wind roses for north-extending valley

Tota

l PA

Hs

Co

nc

(ng

g-1lic

hen

)

Distance from road (km)

0

20

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0 2 4 6 8 10 12

• Wind direction is predominantly down-valley.

• Significant drop in concentration when transect enters narrow valley.

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Perc

ent

of

ho

url

y w

ind

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East-extending valley

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West-extending valley

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North-extending valley

Up-valley

Down-valley

Up-valley

Down-valley

Up-valley

Down-valley

Percentage of up-valley versus down-valley winds also helps explain PAH concentration trends

0

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0 2 4 6 8 10 12

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Tota

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Hs

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g-1lic

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Distance from road (km) 9

Purpose

To quantitatively measure the amount of hop oils present before and after torpedoing to determine total extraction efficiency. Obtaining absolute quantification also allows insight into the total extract potential.

Extraction ≠ Content

● No technique achieves total extraction.o To achieve absolute quantitation, you cannot assume that your extract is

equal to total content.● Every extraction technique has bias.

o Hops have hundreds of unique compounds which extract at different efficiencies depending on the technique.

Internal Calibration Curve:

CT/CS

A T/A

S

CT = conc of targetCS = conc of surrogateAT = area of targetAS = area of surrogate

conc target actual conc surr actual x conc surrogate dmeas'

conc target dmeas'

• This is how cal curves are currently set up in our Agilent software.• This is why you provide surrogate conc.• Reported concentration has automatically been adjusted for losses

that occur during sample prep (if you spiked surrogates beforeextraction)!

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Surrogate Standards

A surrogate is a molecule chosen to mimic the extraction behavior of a target molecule.

A known amount of surrogate is injected onto the sample before extraction. The extraction efficiency of that surrogate is indicative of the efficiency of the target compound. Surrogates also correct for loss during workup, cleanup, etc.

The perfect surrogate standard is one which has the same extraction characteristics as the target. Isotopically labeled compounds have the same chemical properties as their non-labeled counterparts, yet can be distinguished by mass spectrometry. The perfect surrogate!

Imperfect Surrogates

Isotopically labeled surrogates are best, however labeling can be difficult depending on the molecule. This leads to standards that can be expensive or impossible to find.

The next best option is to choose surrogates based on similar chemical properties

Monoterpenes

Surrogate Choices

Oxidized SesquiterpinoidsSesquiterpenes

These 3 surrogates were chosen as representatives for the hop oil compounds of interest.

Oxidized Monoterpenoids

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Method - Correcting with Surrogates

Injection of known amount of surrogate

Extraction of surrogate and target

[Amount of Target Recovered]

[Amount of Surrogate Recovered]÷[Amount of Surrogate Injected]

“Correction Factor”

Method - Accelerated Solvent Extraction

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Method - Simultaneous Distillation Extraction

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Experimental Design

Concept:1. Do extractions on unused hops and determine total oil content by

GCMS2. Do extractions on spent torpedoed hops and determine oil content,

again by GCMS3. Find the difference and celebrate!

Final Experimental Conditions

• ASE• 2 extraction phases: Hexane

followed by Dichloromethane

• Pressure: 1600psi

• Temperature: 50°C

• Static Time: 5 minutes

• Volume: ca. 100mL

• Mass Fresh/Spent: 1.0090g/1.0658g

• SDE• Extraction Solvent:

Dichloromethane

• Cold Finger: ca. 0°C

• Nitrogen Flow: ca. 2mL/min

• Purge Time: 45 minutes

• Run Time: 45 minutes

• Mass Fresh/Spent: 1.0417g/1.3233g

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Results

ASE ConcentratedMyrcene Caryophellene Linalool

Spent Unspent Spent Unspent Spent UnspentMass % 0.104 0.296 0.008 0.010 0.013 0.014

% Extracted Myrcene 64.8 20.3 8.8

SDE ConcentratedMyrcene Caryophellene Linalool

Spent Unspent Spent Unspent Spent UnspentMass % 0.066 0.216 0.014 0.031 0.010 0.014

% Extracted Myrcene 69.4 54.8 24.2

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ResultsASE Concentrated

Myrcene Caryophellene Linalool

Spent Unspent Spent Unspent Spent Unspent

Response 1723785 2600466 129369 251032 16908 42165

Mass of Hops (g) 1.066 1.009 1.066 1.009 1.066 1.009

Mass Corrected Percent 62.8 48.8 38.0

SDE Concentrated

Myrcene Caryophellene Linalool

Spent Unspent Spent Unspent Spent Unspent

Response 5394404 8126851 649089 903812 21346 43013

Mass of Hops (g) 1.323 1.042 1.323 1.042 1.323 1.042

Mass Corrected Percent 52.3 56.5 39.1

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Acknowledgements

• Sierra Nevada Brewing Company• R&D Lab

• Jared Harkins

• Tom Nielson

• Gil Sanchez• Ashlynn Fulton

• QA Lab

• Filtration• James Conery

• Chris Lindsey

• Andrew Duensing• Zak Driscoll

• Oregon State University• Rachel Hotchko

• Chemistry Department at CSU, Chico• Randy Miller

• Lisa Ott

• David Ball• Carol Buckman

• Blaine Wells

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