Fragrances -Analytical Strategy Off-note in a fragrance … · Confidential and proprietary...

Off-note in a fragrance raw materialFragrances - Analytical Strategy

17 November 2015

1. Analytical strategies to identify the off-note in low concentration and low odour threshold

2. Headspace techniques - strategies to “obtain” the off-note and to identify its chemical structure

3. Odour threshold values and how they can be measured

4. Analyse flavour compounds in peppermint chewing gum

17 November 2015Confidential and proprietary business information of Givaudan 2

Introduction

� GC-MS on appropriate columnlooking for known and unknown constituents based on mass spectrum and library search spectrum comparison

� Olfactive analysis: GC-Sniff/FIDDetector = human nose and FID

� Headspace enrichmentIsolation by preparative HPLC or GC

� Structure determination by NMR of isolated sampleor synthesis of hypothetical structure, verified by NMR, and comparison with GC-MS

� Determination of odour threshold values


Workflow

� Menthone naturally found in essential oils of mint plants but in small quantities� synthesis from Menthol

� ~14.- Fr/kg Natural Menthol~15.- Fr/kg Menthone

� 50% of world production of l-Menthol is obtained as natural Menthol>>1000 tons

� Fragrances: small concentration used of both Menthol and Menthone in perfumesFlavours: high amount of Menthol in e.g. chewing gum, only small amounts of Menthone (bitter taste)


Menthone Background Information

S. A. Haut, A convenient preparation of pure menthol and menthone isomers. J. Agric. Food Chem. 1985, 33, 278-280.

� Analytical techniques such as GC-MS and NMR usually detect diastereomers

� More complex techniques such as e.g. chiral GC column or Mosher reagents for NMR is needed to possibly differentiate enantiomers

� Cheap chemistry � usually we have

racemic mixtures


Stereochemistry Menthone

MenthoneOdour: minty, refreshing, diffusive

Iso-MenthoneOdour: powerful, refreshing, clean, minty


Stereochemistry Applied to GC Separation Menthone

RT: 0.00 - 20.00

0 2 4 6 8 10 12 14 16 18 20Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

8.35

8.45

NL:3.06E6m/z= 35.0-350.0 - m/z= 39.5-40.5+43.5-44.5 MS k92582e

Isomenthone

Menthone

� Diastereomers usually separated by GC

2 stereocentres � 2(2-1) = number of diastereomers


2. Headspace techniques




Tasks – Analytical Strategies


� Sample is usually a liquid � dilution in methyl tert-butyl ether (MTBE)

Identification of the Off-note


� ~2 µg/µl concentration of sample

� GC with split � Sniff and FID

GC-Sniff / FID Technique


� GC-Sniff/FID using appropriate GC column type: e.g. apolar (DB5) as used here or polar (Wax)

Identification of Off-note by GC-Sniff / FID

Zommed GC-FID/Sniff Chromatogram:

RT: 2.00 - 7.33

2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

5.21

3.72 5.37 5.68 5.863.803.29

NL:2.23E6m/z= 35.0-350.0 - m/z= 39.5-40.5+43.5-44.5 MS K92583


� GC-MS on apolar GC column (DB5 type)

Identification of Off-note by GC-MS

GC-MS Chromatogram:

Menthone

Iso-menthone

Menthol

RT: 2.00 - 7.33

2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0Time (min)

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

0.32

0.34

0.36

0.38

Rel

ativ

e A

bund

ance

NL:2.23E6m/z= 35.0-350.0 - m/z= 39.5-40.5+43.5-44.5 MS K92583


� GC-MS on apolar GC column (DB5 type)


Zoomed GC-MS Chromatogram:

MenthoneIso-menthone

Menthol

Odour found in this region


� Mass spectrum of the “odour region”


K92583 #453-455 RT: 4.36-4.37 AV: 3 SB: 2 4.41-4.41 NL: 1.22E2T: + c EI Full ms [25.00-460.00]

40 60 80 100 120 140 160 180 200 220 240 260 280m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

120

13432

10741 1219353 92 1177360 161 231156 1739751

Molecular ion M•+ ?

Library hit?


� Headspace trapping on a Porapak-filter � 1-10 Liter headspace sampled� volatile fraction enriched

� Filter eluted with solvent (MTBE)

� Sample analyzed by GC-Sniff/FID

� Off-note enriched by preparative GC

Isolation of Off-note

� Isolated sample for NMR analysis� minimum 1 µg needed� 14.0926 Tesla � 600 MHz instrument(1.6 mm probe head, 1H, 2D experiments)


� 1H NMR and 2D NMR; e.g. HSQC, HMBC, COSY, NOESY

NMR Spectrum of Isolated Sample


Example for 2D NMR Experiments

HMBC

COSY

NOESY

HSQC


� Mass spectrum of isolated sample � serves as reference spectrum for internal library database

Mass Spectrum for Library Database


� We know that the off-note is more volatile because it is enriched in first distillation fraction

� It has a green/mossy smell

� It should originate from natural source as it is not present when synthesizing from synthetic menthol

The Story from the Factory


The Off-note – Literature Search

M. Ishihara, T. Tsuneya, M. Shiga, S. Kawashima, K. Yamagishi, F. Yoshida, H. Sato, K. Uneyama. New pyridine derivatives and basic components in spearmint oil (Mentha gentilis f. cardiaca) and peppermint oil (Mentha piperita). J. Agric. Food Chem.1992, 40, 1647-1655.


� Add mass spectrum to library

Database for Future Search


Odour Threshold – Analyzed by GC-Sniff / FID

P.M. Müller, D. Lamparsky. Perfumes: Art, Science & Technology. Elsevier Applied Science. 1991. Chapter 6: N. Neuner-Jehle, F. Etzweiler. The Measuring of Odors.

� Off-note pyridine Boiling point 185°C

� Menthone Boiling point 210°C

GC odour threshold done based on dilution series and sniffing by panelists� important value for evaluation of new molecules

GC odour threshold: 0.15ng

� More sample amount needed than for GC odour threshold measurement� usually pure reference material

from synthetic chemists

� Concentration calculated from odorant’s vapour pressure and from dilution ratios assuming vapour saturation in sample generator� dilute sample for good range� measure vapour pressure� check vapour saturation by

headspace analysis


Odour Threshold – Analyzed by Olfactometer

P.M. Müller, D. Lamparsky. Perfumes: Art, Science & Technology. Elsevier Applied Science. 1991. Chapter 6: N. Neuner-Jehle, F. Etzweiler. The Measuring of Odors.

� Usually 15-20 panelists needed for evaluation

� Individual values given as mldiluted sample/min� approx gaussion distribution� 1 person is anosmic


Odour Threshold – Typical Result Obtained

� Odour threshold calculated (geom. mean) as ng/L air (25°C)

� From this value and vapour pressure: 2-Isopropyl-4-methylpyridine: 0.11 ng/L (25°C)

Chewing gum typically consists of

� Gum base

� Artificial sweeteners (e.g. aspartame, sorbitol, mannitol, xylitol)

� Softeners (e.g. glycerine, vegetable oil)

� Flavours (0.4-2%; e.g. ethyl hexanoate, limonene, menthol, menthone, cinnamic acid, vanillin, … )

� Colours


Composition of Chewing Gum

Side Trip to Flavour World

Peppermint vs Spearmint

� Peppermint oil produced by steam distillation of herb Mentha piperita L.

� US origin

� 34-46% menthol15-27% menthone2.5-7% menthyl acetate

� Used for toothpaste and chewing gum


Flavour Compounds in Chewing Gum

� Spearmint oil produced by steam distillation of herb Mentha spicata L.and Mentha gracilis Sole (“Scotch”)

� China, India, and US origin

� 9-15% limonene60-70% carvone0-0.2% menthone0.1-0.5% Viridiflorol

� Used for toothpaste and chewing gum

H. Surburg, J. Panten. Common Fragrance and Flavor Materials. 2006. 5th edition. Wiley-VCH.

How to characterise flavour compounds in chewing gum?


Analysis of Flavours in Chewing Gum

Headspace solid phase microextraction(HS-SPME)

Extraction with solvents

Advantages + solvent free+ selective+ simple

+ use of internal standard

Disadvantages - Quantification - Dirty matrix directlyinjected into GC-MS

� Analysis done by GC-MS

S. W. Wong, B. Yu, P. Curran, W. Zhou, Characterising the release of flavour compounds from chewing gum through HS-SPME analysis and mathematical modelling. Food Chem. 2009, 114, 852-858.

Extraction method

� Making a powder of chewing gum

� Extraction with methanol/water mixture containing an internal standard� not sticking together� dissolves flavours and e.g. sorbitol but not gum base

� Addition of saturated NaCl solution and dichloromethan� organic phase, evaporate to concentrate and inject into GC-MS


Chewing Gum Extraction Method


Off-note in a Fragrance Raw Material

http://www.chemistryviews.org/S. Lin; Wonderlab_Comic__Mass_Spectrometry. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Confidential business and proprietary information of Givaudan, may not be copied or distributed to anyone without the express written permission of GivaudanConfidential business and proprietary information of Givaudan, may not be copied or distributed to anyone without the express written permission of Givaudan

Thank you

ContactSusanne [email protected] Schweiz AGFragrances S&T / Ingredients Research Dübendorf

Fragrances -Analytical Strategy Off-note in a fragrance … · Confidential and proprietary...

Documents

Transcript of Fragrances -Analytical Strategy Off-note in a fragrance … · Confidential and proprietary...