11 © 2009 PerkinElmer© 2009 PerkinElmer© 2009 PerkinElmer© 2012 PerkinElmer

Determining Flavors and “Defects” in Beer by Headspace Trap/Gas

Chromatography/Mass Spectrometry

Andrew Tipler, Chromatography R&D ManagerLee Marotta, Field Application Scientist

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This presentation will describe a system that provides an almost comprehensive analysis of flavor compounds and defects in beer Theory Design and operation Beer Application

The system comprises the following components: A headspace trap sampling system A gas chromatograph A mass spectrometer

Content

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Ethanol - FID

Defects Vicinal Diketones (‘butterscotch’) - ECD Dimethyl Sulfide (sulfury character) – FPD or SCD Aldehydes (oxidation ‘cardboard’ products) - FID Thiols (skunkiness) - SCD

Flavor Compounds – MS (characterization) Alcohols Ketones Esters Acids Turpenes

Fermentation Markers Diacetyl

Replacing Multiple Systems with One …

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The authors would like to thank Bill Yawney of the LongTrail Brewery, Vermont, for his advice, inspiration and some of the analytical data used in this presentation

Acknowledgement

55 © 2009 PerkinElmer

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Headspace Instrumentation – Theory, Design and Operating Principles

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Principles Behind Headspace Sampling

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Besides actually making beer, much of the fun is associated with drinking it!

It’s nutritious, it makes the world easier to live in and it tastes good.

Taste is obviously subjective but we beer connoisseurs generally consider the following when drinking a fine beer: Don’t drink out of the bottle Don’t cool the beer to Arctic temperatures Use an appropriately shaped glass Don’t fill the glass completely

Tasting Beer

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Besides actually making beer (of course), much of the fun is associated with drinking it!

It’s nutritious, it makes the world easier to live in and it tastes good.

Taste is obviously subjective, but beer connoisseurs will generally consider the following when drinking a fine beer: Don’t drink out of the bottle Don’t cool the beer to Arctic temperatures Use an appropriately shaped glass Don’t fill the glass completely These are all done to ensure that the beer

aroma is involved in the tasting process (beer aroma, or ‘nose’ as it’s called, is an important part of the formal beer-judging process)

Tasting Beer

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Headspace sampling is a bit like smelling the aroma

Step 1 – put beer sample into a vial and seal it

Step 2 – heat the vial for a period of time at a constant temperature

Step 3 – extract some of the vapor and analyze it by gas chromatography

Headspace Sampling

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During the equilibration step, molecules distribute themselves according to their partition coefficients

Molecules with low partition coefficients favor the vapor (headspace) phase whereas molecules with high partition coefficients favor the liquid (sample) phase

Partition coefficients are reduced as the temperature is increased

At equilibrium, the concentration in the headspace phase is proportional to the original concentration in the sample

Determining the composition of the headspace phase enables the composition of the sample to be established.

Theory

Liquid Sample

Compound

K=Cl/Cv

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Key Components in the PerkinElmer Headspace System

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Since polar compounds in water (or beer) have very high partition coefficients – often less than 0.5% of the compound in the sample may pass into the headspace.

With headspace without the trap, only a small fraction of the total headspace vapor will enter the column

The headspace trap technique can enhance detection limits by 100 times by withdrawing the entire HS volume and enabling several injections from same vial to be focused on trap

Enhanced Sensitivity with the Headspace Trap

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Sample Vial Thermal Equilibration

column

Headspace Sampler Gas Chromatograph

detector

seal

vial

oven

valve

trap

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Vial Pressurization

column

Headspace Sampler Gas Chromatograph

detector

seal

valve

vial

oven

trap

column isolation

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Trap Load

column

Headspace Sampler Gas Chromatograph

detector

seal

valve

vial

oven

trap

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Vial Re-Pressurization

column

Headspace Sampler Gas Chromatograph

detector

seal

valve

vial

oven

trap

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Trap Re-Load

column

Headspace Sampler Gas Chromatograph

detector

seal

valve

vial

oven

trap

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Dry Purge

column

Headspace Sampler Gas Chromatograph

detector

seal

valve

vial

oven

trap

1919

Trap Desorption

column

Headspace Sampler Gas Chromatograph

detector

seal

valve

vial

oven

trap

Trap desorbed in Opposite direction

2020 © 2009 PerkinElmer

Analyzing Beer

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6.50 8.50 10.50 12.50 14.50 16.50 18.50 20.50 22.50 24.50 26.50 28.50 30.50 32.50 34.50Time0

100

%

Beer Flavors HS_GC_MS Scan EI+ TIC

2.95e1016.59;70

6.0446 12.02

4311.43

61

10.1543

22.3170

16.7655

18.1743 19.26

43

30.3388

26.018822.42

43

31.54104 33.77

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Pro

pyl a

ceta

teE

thyl

pro

pano

ate

Dim

ethy

l Sul

fide

Dia

cety

l

Eth

yl A

ceta

te

Isob

utan

ol

Isop

enta

nal

Isoamyl alcohol

2-M

ethy

l-1-b

utan

ol

Isob

utyl

ace

tate

Eth

yl b

utyr

ate

Isoa

myl

ace

tate

2-M

ethy

lbut

yl a

ceta

te

Eth

yl h

exan

oate

Lina

lyl a

nthr

anila

te

Eth

yl o

ctan

oate

á-P

hene

thyl

ace

tate

Met

hyl g

eran

ate

Eth

yl c

aprin

ate

Nice peak shapesGood peak separationRequired detection limitsRepeatable responseLinear response

Beer - Component Identification by Mass Spectrometry

Sample Size: 5 mLSample Temp: 70oCSample Load: 1 cycle Trap Load Temp: 25oCDry Purge: 6 minTrap high Temp: 300oCNeedle Temp: 160oCT Line Temp: 180oCColumn Flow: Pressure Pulse: 2mL/min for 0.4minAnalytical Flow Rate 1mL/min

Mass Range: 30 to 300 amu

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Sensitivity is AMAZING by HS Trap/GC/MS

DMS at 10 ppbSignal to Noise is 56940 to 1

Diacetyl at 10 ppbSignal to Noise is 1067 to 1

2323 © 2009 PerkinElmer

Hops EvaluationBeer Comparisons

Supplier InformationBrewing Investigation

Data of Beer Analysis from Long Trail Brewery

Thank you Long Trail and Bill Yawney

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Hop Volatile Comparison

1 2 3 4 5 6 7 8 9 10 11 120

2

4

6

8

10

12

Hop Volatile Compound ComparisonAverage Amounts - Sample 1 vs Sample 2

Sample 1Sample 2

Volatile Compounds

pp

m

Co

mp

ou

nd

#

Compound Name (Longer names have been

truncated)1 1R-à-Pinene2 β-Myrcene3 β-Pinene4 à-Phellandrene5 2,6-Dimethyl-1,3,5,7-octatetra6 Limonene7 1,4-Cyclohexadiene, 1-methyl-48 Furan, 3-(4-methyl-3-pentenyl)9 Copaene

10 1,6,10-Dodecatriene, 7,11-dime11 Bicyclo[3.1.1]hept-2-ene, 2,6-12 Caryophyllene

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Beer Volatile Comparison

Co

mp

ou

nd

N

ame

Ret

enti

on

T

ime

1-Propanol 8.022-Butanone, 4-hydroxy 9.721-Propanol, 2-methyl 10.451-Butanol, 3-methyl 12.541-Butanol, 2-methyl 12.80Propanoic acid ethyl ester 13.63n-Propyl acetate 13.74Mixture of methyl butanols 14.77Mixture of methyl butanols 14.93Acetic acid, 2-methylpropyl ester 16.28Butanoic acid, ethyl ester 17.371-Butanol, 3-methyl-, acetate 20.421-Butanol, 2-methyl-, acetate 20.55Hexanoic acid, ethyl ester 24.49Acetic acid hexyl ester 24.84Heptanoic acid, ethyl ester 26.93Acetic acid, heptyl ester 27.22Phenyl ethyl alcohol 27.45Octanoic acid 28.15Octanoic acid, ethyl ester 28.95Acetic acid, 2-phenylethyl ester 30.12Ethyl9-decanoate 32.11Decanoic acid ethyl ester 32.27Caryophyllene 33.93Alpha caryophyllene 34.55Decanoic acid ethyl ester 35.86

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Research and Development Project

Name RTDimethyl sulfide 8.3231- Propanol 9.3581- Propanol 9.591Acetic acid, anhydride with formic acid 10.1311-Propanol, 2-methyl 12.104Butanol, 3-methyl 13.004Formic acid 13.987Vinyl butyrate 14.4371-Butanol, 3-methyl 14.7001-Butanol, 2-methyl-, (S) 14.962Acetaldehyde, O-methyloxime 15.487Vinyl butyrate 17.528Butanoic acid, ethyl ester 19.178Butanoic acid, 3-methyl-,ethyl ester 21.406Cyclobutanone, 2,2,3-trimethyl- 21.744Cyclopentane 21.872Propanoic acid, 2-methyl-,2-methylpropyl ester 23.642Pentyl glycolate 25.938β-Myrcene 26.133Propanoic acid, 2-methyl-,2-methylbutyl ester 26.530Limonene 27.446Methyl 2-methyl hexanoate 28.286Butanoic acid, 2-methyl-,2-methylbutyl ester 28.631Decanoic acid, ethyl ester 28.924Octanoic acid, ethtyl ester 30.251Benzenebutanal 31.4522,6-Octadienoic acid, 3,7-dimethyl-, metyl ester, (E) 32.525Decanoic acid, ethyl ester 33.717

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Supplier Comparison

2828 © 2009 PerkinElmer

PerkinElmer Fermentation Experiment

Testing the process using HS/GC/MS

American Pale Ale

12mL sample taken approximately every eight hours starting from time zero (prior to adding the yeast) during the brewing process

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Grains Maris Otter Pale Malt Munich Malt Crystal Malt

Hops Chinook Centennial Amarillo Nelson Sauvin

Yeast SafAle American Ale 05 dry yeast, no starter

O.G. 1.058

IBU 45

Process Single infusion mash at 67°C Fermentation at 19-20°C

The ‘Profile’ Beer: American Pale Ale

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Results of Components Changing with Time

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Activity of Two Components over 111 Hours of Sampling

Dimethyl Sulfide (DMS) 2,3-Butanedione (Diacetyl)

Plot: Detector Response –vs- Time

Time Interval: Every Eight Hours

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Interesting Component … 3-Methyl-1-Butanol

0 20 40 60 80 100 1200

100000000

200000000

300000000

400000000

500000000

600000000

Plot: Detector Response –vs- Time

Time Interval: Every Eight Hours

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Concentration of Four Components through Fermentation

Time DMS Diacetyl 2,3-Pentanedione trans 2-Nonenal(hours) (PPB) (PPB) (PPB) (PPB)

Zero 16.5 81 15 4.77 18.8 1577 16 3.9

15 4.6 2779 37 3.823 3.0 2183 52 3.931 3.2 1658 59 3.839 3.0 862 43 3.947 4.0 715 85 4.053 3.6 422 93 3.9

66.5 3.3 249 41 3.970.75 3.7 341 129 4.0

79 3.4 109 72 3.987 3.4 95 91 3.9

95.5 3.6 74 65 3.9103 3.3 44 49 3.9111 3.2 48 64 4.0

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Specific Gravity Measurements over 95 hours

Profile Beer Specific Gravity

Time, hrs S.G Attn %0.0 1.058 0.07.0 1.058 0.0

15.0 1.054 6.923.0 1.052 10.331.0 1.050 13.839.0 1.042 27.647.0 1.035 39.755.0 1.030 48.364.5 1.025 56.970.8 1.023 60.379.0 1.020 65.587.5 1.016 72.495.0 1.014 75.9

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Quantifies defects

Characterizes beer by Providing component identity – WHAT IS IT? Qualitative information Providing relative component ratio information Providing concentration (quantitative) information – How much?

Enables the investigation of other beers – What makes my neighbors beer so very delicious?

Optimizes process control

Conclusion

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lee.marotta@perkinelmer.comandrew.tipler@perkinelmer.com