Dark Chocolate… The Real Dark Secret

Mark Pieterse LECO South Africa 2015

Presentation Outline : • General facts • Method of Extraction • GC-TOFMS Methodology • Results • Conclusion

Where is Cocoa Grown ?

Global Production :

Bean to Bar : • Pods are collected and split open • A farmer can expect 20-50 beans per pod,

depending upon variety • Takes approximately 400 beans to produce 500g of

Chocolate • Harvested beans are fermented for 3-7 days • Fermented beans are then dried, sold, packed and

transported • Roasting and Grinding- Cocoa liquor produced • Cocoa liquor pressed ready for Chocolate

manufacture

Dark Chocolate…

• More cocoa solids than sugar • Greater health benefits • More intense flavours

LECO GC-TOFMS : Fastest Type of Mass Analyzer • Requires fast data acquisition system • Leco Pegasus HT: Capable of 500 Full Mass Range

Spectra/s • Ability to properly define narrow chromatographic peaks

(Hi Speed GC) • Delivers the data density needed for True Signal

Deconvolution® • Full range mass spectrum is collected at all times without

sacrificing speed or sensitivity….no need for SIM for best sensitivity

• Spectral Continuity, no spectral skewing

Method of Extraction : • 2 grams of each sample weighed out into headspace vial • Each sample incubated at 37ºC for 15 minutes • Fibre used for SPME was PDMS-DVB-Carboxen • Sampled with SPME for 20 minutes • Thereafter sample was desorbed in GC inlet and

analysed

GC-TOFMS Method : GC: Agilent 7890 Gas Chromatograph Column: 30m x 0.25mm x 0.25µm Restek Stabilwax Inlet: Splitless @ 220°C Carrier Gas: 1.50 mL/min, Constant Flow GC Oven: 40°C (5 min hold), 7°C/min to 220°C, (5 min hold) MS Transfer Line: 200°C MS: LECO Pegasus HT Ion Source: EI at -70 eV Ion Source Temperature: 220°C Spectral Acquisition Rate: 20 spectra/s Acquired Mass Range: 30-550 m/z Instrument Control and Data Review: ChromaTOF Instrument Control and Data Review: ChromaTOF

Typical Dark Chocolate Chromatogram:

200 400 600 800 1000 1200 1400 1600 18000

5e+006

1e+007

1.5e+007

2e+007

2.5e+007

3e+007

3.5e+007

4e+007

Time (s)TIC

Does Chromatographic Peak Size Count ?

400 600 800 1000 1200 1400 1600 18000

1e+006

2e+006

3e+006

4e+006

5e+006

6e+006

Time (s)TIC

Marks & Spencer Dark Chocolate 72% : Mix of Flavours

• Aldehydes impart chocolate flavour : 2-Methylbutanal and 3-Methylbutanal • Furanoid : Flowery, fruity taste, : trans-Linalool

oxide • Pyrazines generally a roasted odour or flavour : Methylpyrazine

Aldehydes impart chocolate flavour : 2-Methylbutanal and 3-Methylbutanal

150 152.5 155 157.5 160 162.5 165 167.5 170

1e+006

2e+006

3e+006

4e+006

5e+006

Time (s)

Buta

nal,

2-m

ethy

l-

Buta

nal,

3-m

ethy

l-

TIC

Furanoid Flowery, fruity taste: trans-Linalool oxide

937.5 940 942.5 945 947.5 950 952.5 955 957.5 960

-0

20000

40000

60000

80000

100000

120000

140000

160000

Time (s)

3-Fu

rald

ehyd

e

2-Fu

ranm

etha

nol,

5-et

heny

ltetra

hydr

o-à,

à,5-

trim

ethy

l-, c

is-

Pyra

zine,

tetra

met

hyl-

59

Pyrazines Roasted : Methylpyrazine

681 682 683 684 685 6860

10000

20000

30000

40000

50000

60000

70000

80000

Time (s)

Pyra

zine

, m

ethy

l-

2-H

epta

nol,

acet

ate

94

Willie’s Cacao Madagascan Gold, 71% - Notes of Summer fruits :

• Alcohols – Flowery, Floral and Fruity: 1-Pentanol

• Esters – Honey, Sweet, Fruity and Floral : Acetic acid, 2-phenylethyl ester

Alcohols – Flowery, Floral and Fruity: 1-Pentanol

663 664 665 666 667 668 669 670-0

5000

10000

15000

20000

25000

30000

35000

40000

Time (s)

1-Pe

ntan

ol

55

Esters – Honey, Sweet, Fruity and Floral : 2-phenylethyl ester Acetic acid

1299 1300 1301 1302 1303 1304 1305 1306

-0

200000

400000

600000

800000

1e+006

1.2e+006

Time (s)

Acet

ic ac

id, 2

-phe

nyle

thyl

est

er

104

Visual Difference :

200 400 600 800 1000 1200 1400 1600 1800 20000

500000

1e+006

1.5e+006

2e+006

2.5e+006

3e+006

Time (s)TIC Madagascar 71%:1 TIC Marks Spencer 72%:1

Aldehydes : 2-Methyl Butanal

155 156 157 158 159 160

-0

20000

40000

60000

80000

100000

120000

140000

160000

180000

Time (s)

Buta

nal,

2-m

ethy

l-

Buta

nal,

2-m

ethy

l-

57 Marks Spencer 72%:1 57 Madagascar 71%:1

Alcohols: 1- Pentanol

663 664 665 666 667 668 669 670 671 672

-0

5000

10000

15000

20000

25000

30000

35000

40000

45000

Time (s)

1-Pe

ntan

ol

55 Marks Spencer 72%:1 55 Madagascar 71%:1

Pyrazines: Methyl-Pyrazine

681 682 683 684 685

10000

20000

30000

40000

50000

60000

Time (s)

Pyra

zine,

met

hyl-

2-He

ptan

ol, a

ceta

te

Pyra

zine,

met

hyl-

94 Marks Spencer 72%:1 94 Madagascar 71%:1

The Difference : Linalool Oxide, Marks Spencer

945 946 947 948 949

5e+006

1e+007

1.5e+007

2e+007

2.5e+007

3e+007

3.5e+007

Time (s)

Pyra

zine,

tetra

met

hyl-

Linalo

ol Ox

ide

Pyra

zine,

tetra

met

hyl-

TIC Madagascar 71%:1 TIC Marks Spencer 72%:1

Conclusion : • Chocolate is a very complex sample, not as simple

as it tastes • SPME provides a snapshot • GC-TOFMS can find a multitude of components • We have only seen the tip of the iceberg • Due to mass spectra produced fingerprinting of

compounds is even more beneficial • Provides more accurate results

Acknowledgements : • SENSORY FX • KERRY Flavours • SAAFoST

Thank you!