Adventures in Hop oil Extraction using Pressurized Liquid Extraction Jared Harkins, 1 Katy Orr 1 Sierra Nevada Brewing Company 1
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Adventures in Hop oil Extraction using Pressurized Liquid Extraction
Jared Harkins,1 Katy Orr
1 Sierra Nevada Brewing Company
1
Study Location
Arthur’s PassNational Park
State Highway 73
2
Taramakau Valley (east-extending valley)
3
Wind roses for east-extending valley
Tota
l PA
Hs
Co
nc
(ng
g-1lic
hen
)
Distance from road (km)
0
10
20
30
40
50
60
70
80
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
20
30
40
50
60
70
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.
6
Hawdon Valley (north-extending valley)
7
Wind roses for north-extending valley
Tota
l PA
Hs
Co
nc
(ng
g-1lic
hen
)
Distance from road (km)
0
20
40
60
80
100
120
140
0 2 4 6 8 10 12
• Wind direction is predominantly down-valley.
• Significant drop in concentration when transect enters narrow valley.
8
Perc
ent
of
ho
url
y w
ind
s
0
20
40
60
80
100
East-extending valley
0
20
40
60
80
100
West-extending valley
0
20
40
60
80
100
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
10
20
30
40
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60
70
0 2 4 6 8 10 12
0
20
40
60
80
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120
140
0 2 4 6 8 10 12
0
10
20
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0 2 4 6 8 10 12
Tota
l PA
Hs
Co
nc
(ng
g-1lic
hen
)
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
17
Method - Simultaneous Distillation Extraction
18
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
20
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
21
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
22
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
23
Literature• A Rapid Solvent Extraction Method for Hop Essential Oils
• Kai C. Lam, Gail B. Nickerson, and Max L. Deinzer
• J. Agric. Food Chem. 1988, 34, 63-66 83
• Accelerated Solvent Extraction: A Technique for Sample Preparation
• Bruce E. Richter,* Brian A. Jones, John L. Ezzell, and Nathan L. Porter
• Anal. Chem. 1996, 68, 1033-1039
• Artefact Production in the Likens-Nickerson Apparatus when Used to Extract the Volatile Flavorous Components of Cod
• Alister S. McGill and Roy Hardy
• J. Sci. Fd Agric. 1977, 28, 89-92
• Comparative Gas Chromatographic–Mass Spectrometric Evaluation of Hop (Humulus lupulus L.) Essential Oils and Extracts Obtained Using Different Sample Preparation Methods
• Magdalena Ligor & Mantas Stankevičius & Anna Wenda-Piesik & Kęstutis Obelevičius & Ona Ragažinskienė & Žydrūnas Stanius &Audrius Maruška & Bogusław Buszewski
• Food Anal. Methods DOI 10.1007/s12161-013-9767-5
• Compounds Contributing to the Characteristic Aroma of Malted Barley
• Andrew D. Bealt and Donald S. Mottram*
• J. Agric. Food Chem. 1994, 42, 2880-2884
• Detection of Certain Hop Oil Constituents in Brewing Products‘
• S. T. LIKENS and G. B. NICKERSON", Oregon Agricultural Experiment Station, Corvallis, Oregon
• Proceedings of the American Society of Brewing Chemists 5-13
• Optimized Likens-Nickerson Methodology for Quantifying Honey Flavors
• Amina Bouseta and Sonia Collin
• J. Agric. Food Chem. 1995, 43, 18904897
• Recovery of Less-volatile Chemicals from Pure Fat Using a Simultaneous Steam Distillation-extraction Apparatus
• Fernando E. Figuerola* and Takayuki Shibamoto
• Agric. Biol Chem., 47 (12), 2933-2934, 1983
• Fermentation Science: Oregon State University (Presentation)
• Thomas H. Shellhammer, Daniel C. Sharp
• Simultaneous distillation–extraction: from birth to maturity—review
• Alain Chaintreau*
• Flavour Fragr. J. 2001; 16: 136–148
• Superheated water extraction, steam distillation and Soxhlet extraction of essential oils of Origanum onites
• Mustafa Z. Ozel & Hilal Kaymaz
• Anal Bioanal Chem (2004) 379: 1127–1133
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