gas chromatography + olfactometry basic principles and use in...

Gas Chromatography + olfactometry

basic principles and use in the food QC + RD

Summer School:

“Food Safety, Quality and Nutrition Course”

21st July 2014 ČZU Praha

Jan Marek – Shimadzu Handels GmbH

GC,GCMS Sales & Support Bohemia

Analytical & Measuring Instruments Division



CONTENTS

Gas Chromatography (GC) basic principles

Columns, Injectors, Detectors

Some special options – sampling, detection

Mass spectrometry (MS) for GC

Multidimensional techniques (MDGC, GCxGC)

GC + GCMS of flavour and fragrance

GC – Olfactometry

Conclusion

2

Gas Chromatography - basic principles

Analytical instrumental

techniques for organic compounds

UV-VIS, FTIR, NMR, MS, …. (titrations, etc.)

MIXTURES → SEPARATION BEFORE DETECTION

And so separation techniques are used,

wide range of combinations +

“multidimensional techniques“

GC (FID,...), GC/MS, LC (HPLC, UHPLC), LC/MS, ELECTROMIGRATION (Electrophoresis, ITP)


Introduction - Principle and structure of GC

CHROMATOGRAPHY IS ACTUALLY ONE OF THE MOST

IMPORTANT SEPARATION TECHNIQUE



Stationary Phase and Mobile Phase ( carrier gas )

Mobile phase and stationary phase contact through phase boundary

Different compounds have different affinities to stationary phase and mobile phase

Difference of moving velocity results in

separation!

(interactions with MF are not so importent in GC but very much in LC! )

Mobile phase

Stationary phase

Strong Weak

Flow of MF (carrier gas)



Chromatography : Analytical method

Chromatograph : Instrument

Chromatogram : Obtained

“picture”

Chromatographer : Analyst

Terminology

Gas Chromatography - why and when ?

Qualitative analysis ・ What components？

Quantitative analysis ・ How much of the component ?

Purpose of GC, GC/MS Analysis

Volatile and/or volatilised compounds only *

* Even other materials are possible - pyrolyse of polymers etc.

Gas Chromatography - basic principles Introduction -

Principle and structure of GC - Analytical method

Gas Chromatography - basic principles Introduction -

Principle and structure of GC - Analytical method


Introduction - Principle and structure of GC - Instrument

Heated Injection Port (with innert liner)

Sampling device-

syringe/autosampler

Column oven (thermostat) with a column –

capillary or packed


Introduction - Principle and structure of GC - columns

Structure of GC (for capillary column)

MS spectrometer

in case of GC/MS

Split unit manual and/or electronic

Flow controllers manual and/or electronic

Flow controllers manual and/or electronic

(Solenoid valve unit)



Detectors

TCD

BID*

Inorganic gases, � Low sensitivity for organic compounds

Inorganic gases, � High sensitivity for organic compounds

FID Organic compounds ( higher sensitivity than TCD and linearity) mostly used detector

ECD Good sensitivity for high electron affinity compounds (halogenated compounds)

FPD Good sensitivity for phosphorous compounds and sulfureted compounds

FTD Good sensitivity for nitrogen compounds

* Barrier discharge Ionisation Detector

FPD data with packed column

Analytical Conditions

Column : ββ’-ODPN 25% on Chrormosorb W

60/80mesh 3.2mmφ×3.1m , Glass

COL.Temp. : 70℃

INJ.,DET.Temp. : 150℃

Carrier gas : N2 , 30mL/min

Detector : FPD S-mode

MeSH : Methyl Mercaptan

DMS : Dimethyl Sulfide

DMDS : Dimethyl Disulfide

5 10 15 m i n

H2S

MeSH DMS

DMDS

0

Standard gas 0.5mL injection

(1mg/L, N2 base)

The example of FPD S mode data acquired with typical packed column

for analyzing bad smell.



Detectors

Barrier discharge Ionisation Detector



Detectors

Barrier discharge Ionisation Detector Acetaldehyde

Meth

anol

Ethanol

Ace

tic A

cid

Form

ic A

cid

Water

BID

100 ppm concentration each comp. in water, 1:24 split

analysis, 0.5 μL sample volume

FID



Injection techniques

Split injection Split vent is open. Only a part of sample is introduced to column. Used for high concentration samples Mostly used

Splitless injection Split vent is closed Almost all amount of sample is introduced to column. Used for low concentration samples

OCI injection Samples are directly injected to column. Used for samples including both low boiling components and high boiling

components. Used for thermally decomposed samples.

PTV injection Low temperature at injection port Solvent is removed at injection port. Used for large volume injections( a few L a few tens – a few hundreds L)




OPTIC-4 Most powerful multimode inlet

LINEX Automated Liner Exchanger


Introduction - Principle and structure of GC - Chromatogram



SVOC (Phthalate + Siloxane)



Direct TD – Coffee


Introduction - Principle and structure of GC/MS

Ionization Part Ionizes sample molecules

in vacuum

Mass Separation part

Separates ions

according to their

masses

Ion Detection Part Detects ions



EI ELECTRON IMPACT IONIZATION

EI Mass Spectrum

UF sensitivity Patented ASSP function

ensures high sensitivity

during high speed

scanning

UF scanning High speed calculation

process enables

20,000 u/sec scan

speeds

UF sweeper

(colision cell) High speed CID cell with

minimum 1 ms dwell

time achieves 600

trans/sec

High-Performance Supported by "UF technologies"

Triple Quadrupole Gas Chromatograph Mass Spectrometer

GCMS-TQ8030/40

Quadrupole mass filters Ion source

Detector

GC-MS/MS operation modes

Q1 Q3 Collision Cell

MRM

CID SIM SIM


Precursor Ion Scan

CID SIM Scan


Q3 Scan

Transfer Transfer Scan

Product Ion Scan

CID Scan SIM


Chromatograms (Flavor and Fragrance)

Volatiles (Potpourri Fragrance Compounds) on SLB-IL60

SLB-IL60 has similar polarity, but employs different interaction mechanisms.

In addition to unique selectivity, the SLB-IL60 allows a faster analysis with the same run conditions.

30 sigma-aldrich.com/il-gc

PEG

SLB-IL60


Essential Oils (Lemon) on SLB-IL59, 3 ºC/min Ramp Rate

SLB-IL59 employs different interaction mechanisms than a PEG.

This results in unique selectivity compared to PEG.



Essential Oils (Lemon) on SLB-IL59, 5 ºC/min Ramp Rate





Essential Oils (Cold Pressed Lemon) on SLB-IL60




PEG

SLB-IL60


Essential Oils (Distilled Lime) on SLB-IL60




PEG

SLB-IL60


Essential Oils (Patchouli) on SLB-IL60




PEG

SLB-IL60


Essential Oils (Peppermint) on SLB-IL59





Essential Oils (Kennewick Peppermint) on SLB-IL60




PEG

SLB-IL60


Essential Oils (Willamette Peppermint) on SLB-IL60




PEG

SLB-IL60


Essential Oils (Petitgrain) on SLB-IL60, 50 ºC Initial Oven Temp.




PEG

SLB-IL60


Essential Oils (Petitgrain) on SLB-IL60, 75 ºC Initial Oven Temp.




PEG

SLB-IL60


Essential Oils (Spearmint) on SLB-IL59, 50 ºC Initial Oven Temp.





Essential Oils (Spearmint) on SLB-IL59, 80 ºC Initial Oven Temp.





Essential Oils (Native Spearmint) on SLB-IL60




PEG

SLB-IL60


Essential Oils (Scotch Spearmint) on SLB-IL60




PEG

SLB-IL60


Introduction - Principle and structure of GCxGC,MDGC

How to solve the problem

with

complete separation ?

GCxGC, MDGC

multidimensional separations…

Un

iver

sity

of

Mes

sin

a, It

aly

Comprehensive Two-dimensional GC Workshop

Peter Quinto Tranchida and Luigi Mondello

University of Messina, Italy

e-mail: [email protected]

23-24 February, 2010

Charles University, Prague

Conferences and Seminars:\I:\2010\023) GCxGC workshop Praha\Course

Un

iver

sity

of

Mes

sin

a, It

aly

At present, one-dimensional chromatography is the most commonly applied

method for the separation of real-world samples. It is generally accepted that 1D

chromatography methods provide rewarding analytical results on what can be

defined generically as “low-to-mediumly-complex samples”.

1950s: bergamot

oil and a packed GC column 1990s: bergamot

oil and capillary GC


The World and Chromatography

Un

iver

sity

of

Mes

sin

a, It

aly

GC x GC is an “on-line” multidimensional technique that enables a bidimensional

analysis of the entire initial sample, through continuous heart-cutting. The leap from

heart-cutting MDGC to comprehensive GC was acheived in 1991 by Liu and Phillips who

developed a new transfer system: the thermal modulator.

IT WAS AN EYE-OPENER FOR MANY ANALYTICAL CHEMISTS

Comprehensive 2D GC

Lui ZY and Phillips JB. J. Chromatogr. Sci. 1991, 29, 227-231


Un

iver

sity

of

Mes

sin

a, It

aly

DB:0

DB:1 C18:0

sec

min

C15:0

C16:0

C17:0

C19:0

C20:0

C21:0

C22:0

C23:0

C24:0

C25:0

C26:0

C15:1 C16:1w9

GC x GC olive oil result: 24 FAMEs,

some of which totally unexpected

C16:1w7

C17:1w7

C18:1w9tr

C18:1w9c

C18:2w6c,tr

C18:2w6c,c C18:3w3

C19:1 C20:1


Un

iver

sity

of

Mes

sin

a, It

aly

GC x GC hazelnut oil result: 33

FAMEs

DB:0

DB:1

DB:2

DB:3

s

min

C18:0

C15:0

C16:0 C17:0

C19:0

C20:0

C21:0

C22:0

C23:0

C24:0

C25:0

C26:0

C15:1

C16:1w9 C16:1w7 C16:1w5

C17:1w7

C16:2

C18:1w9tr C18:1w9c

C18:1w7

C17:2

C17:3

C19:1

C18:2w6c,tr

C18:2w6c,c

C20:1

C18:3w3

C20:2

C22:1

C22:2

C24:1

GC x GC hazelnut oil result: 31

FAMEs


Un

iver

sity

of

Mes

sin

a, It

aly

C16:1w7

C17:0

Extra-virgin

olive oil

Hazelnut

oil

C16:1w7

C17:0

C16:1w5



Introduction - Principle and structure of GCxGC

GCMS-TQ8030 Triple Quadrupole Gas Chromatograph Mass Spectrometer

2. Fastest Speed

Using Fast Q3 Scan and GCGC

Qualitative analysis using mass

spectrum from GCGC/Q3 scan

(20,000 u/sec)

Separate and analyze high matrix samples with GCGC

Switch to GCGC/Q3 MRM mode for trace

quantitative analysis of target components

Data currently being acquired at University of Messina.

To be presented at ISCC (capillary symposium) in May.

l


Introduction - Principle and structure of MDGC/MS

Easy setting software

MDGC divertible to normal

GC and GC/GCMS

High Performance

Separation with 2 columns

Hardware achieves perfect switching

MDGC/GCMS-2010

Heart-cut SEPARATION ON 2ND COLUMN

Multi Dean‘s Switching

Multi Dean‘s Switching-Cut Mode

P－ΔP2 ＜ P－ΔP1

Pressure＝P－ΔP1

APC Pressure＝P

Pressure (ΔP2)

Valve

1st DET 2nd Column 1st Column

Pressure (ΔP1)

Pressure＝P-ΔP2

Sample


Introduction - Principle and structure of MDGC/MS

Heart cutting

Barbara d’Acampora Zellner and Luigi Mondello

Université de Messina

Wil van Egmond

ATAS GL, Veldhoven, The Netherlands

http:/pharma.unime.it/foodchem

[email protected]

Grasse, 7th April 2005

Olfactometric Perception by GC-O of Chiral Compounds

What are Chiral Compounds?

Any carbon atom bonded to four different groups is termed a chiral or an assymetric carbon.

Molecules containing one or more of these carbon centers are considered chiral molecules.

Chiral centers can exist in two forms called enantiomers. These two forms are non superimposable mirror images of each other, but both have similar properties.

Life and Chirality

In extremely simple terms, chirality is "handedness“, that means, the existence of left and right opposition, as the

hands, that are mirror images and therefore "chiral".

“[...] I call any geometrical figure, or group of points, chiral, and say it has chirality, if its image in a plane mirror, ideally realized, cannot be

brought to coincide with itself [...]“

From: Lord Kelvin, Baltimore Lectures on Molecular Dymnamics and the Wave

Theory of Light (1904).

Life and Chirality

Enantiomers may show different behaviour when interacting with living beings.

As the enantiomers of chiral drugs, often characterised by different pharmacological activity, one enantiomer being usually bioactive, whereas the other either inactive or inhibitory.

Chirality and Odour Perception

In the beginning of the 20th century chemists in the Flavour and Fragrance Industry recognized that certain enantiomeric chemicals, such as menthol and carvone presented different and also differentiating organoleptic properties.

In 1961 Rienäcker and Ohloff published the first data regarding the enantioselective perception of (+)-b-Citronellol as having a citronella-like odour, while (-)-b-Citronellol had a geranium-like odour.

(Rienäcker, R. and Ohloff G. Angew. Chemie, 73, 240, 1961)

In the 1960's, a number of processes had been developed for the synthesis of the desired enantiomer, e.g. (-)-menthol from optically active terpenoids.

Chirality and Odour Perception

However, based on erroneous theories of olfaction, the premise that optical enantiomers could have different odours was not generally accepted by various academics until the 1980's.

Prior to GC and other measurements of purification techniques, the purity of the enantiomers used in odour evaluations was not precise. In addition, a high enantiomeric excess is nearly always required in organoleptic evaluations.

Flavour and Fragrance Compounds

Numerous substances presenting different functional groups, and frequently chiral centres.

Enantiomers can differ in:

odour quality odour activity odour intensity

Sensorial Properties of Chiral Compounds

eliciting identical or different odour

sensations

described by value of odour threshold

one of the isomers is odourless

Sensorial Properties of Enantiomers

O

H

H

O

HO

H

(R)-(-)-Linalool

floral, fresh, woody, lavender note

Odor Threshold = 9-11 ppb in air

(S)-(+)-Linalool

herbaceous, musty green, odor

reminiscent of petitgrain

Odor Threshold = 35- 40 ppb in air

(4S)-(+)-Carvone

herbaceous, dill and caraway seeds

Odor Threshold = 85-130 ppb in air

(4R)-(-)-Carvone

herbaceous, spearmint

Odor Threshold = 2 ppb in air

(1S,3S,4R)-(+)-Menthol

Sweet, fresh, minty, strong

(1R,3R,4S)-(-)-Menthol

Dusty, vegetable,

less minty, less fresh

H

OH

OH HO

Why is the Olfactive Investigation of Enantiomers important?

• Continuous demand for new synthetic compounds evoking unusual odour sensations or reproducing those elicited by natural fragrances;

• To establish odour-structure relationship requirement;

• Increasing environmental and health risks associated with massive usage of fragrances in fine and functional perfumery;

• Compounds added to food and beverages are subjected to strict regulations choose the ‘best aromatic’ isomer;

• To optimise the analytical methods to assess the authenticity of natural materials.

The identification of the isomer present as the main odour vector has become an unavoidable task.

Chiral Chromatography in Olfactive Enantiomer Evaluation

Chiral chromatography enables to the separation of enantiomeric compounds.

Common liquid stationary phases do not possess adequate selectivity for enantiomeric separation.

Application of stationary phases with added derivatized cyclodextrin (CD) macromolecules leads to the separation of several enantiomers.

CDs derive from enzymatic degradation of starch polysaccharide by CD glycosiltransferase from Klebsiella pneumonia or Bacillus macerans.

Olfactive Investigation of Enantiomers through GC-O

Gas Chromatography- Olfactometry

• Standard technique to assess odour-active components in

natural products and complex mixtures.

• Unique analytical technique which associates resolution

power of capillary GC with the selectivity and sensitivity of

the human nose.

GC-Olfactometry

GC-2010

Sniffer - Phaser

PHASER – GC-O

ATAS GL Internatiobal BV

Glass Nose Cone

Heated Transfer

Line (300oC)

Dimensions: 35mm

(diameter) x 60cm

(length)

Water Reservoir with bubbling

filter

Aux. Gas: He or N2

Controller

Power, Heater, Temperature Controller, Air Flow

Flexible Arm

GC inserting

part

Split Manager

Major Drawbacks of Olfactometry

Evaluation by more than one panelist

Normalisation of the descriptive olfactive language

Involves time-consuming methods

Obtained results are based on detection thresholds,

not on real intensities

Goal: optimize data obtained in any evaluation

Software

Odour Evaluation Software

Quality Description of Olfactive Impressions

• Voice Recognition Software

• Touchscreen

Intensity Description of Olfactive Impressions

• Magnitude Estimation using a variable resistor with a

pointer moving according to the pressure exerted by the

thumb along a category scale

• Finger Span Method

Considering Pros & Contras Software Prototype

Application: Enantiomeric Evaluation

Rosewood oil x Ho oil Linalool is one of the most important substances for F & F Industries

(R)-(-)-Linalool Licareol

floral, fresh, woody, lavender note Odor Threshold = 9-11 ppb in air

(S)-(+)-Linalool Coriandrol

herbaceous, musty green, odor reminiscent of petitgrain

Odor Threshold = 35- 40 ppb in air

Rosewood oil (Aniba roseodora Ducke)

rich in (S)-(+)-Linalool

Ho oil (Cinnamomum camphora)

rich in (R)-(-)-Linalool

differences in optical properties and odour

Application: Enantiomeric Evaluation of Rosewood oil

Distilled Leaves Oil

Column: DETTBSBTA - l: 25m ; i.d.: 0.25 mm; df: 0.25μm (MEGA, Milano) Inj. temp.: 250°C; mode: split, ratio: 1/50; Det.: FID, temp.: 250°C; Temp. progr.: 45°C- 2°C/min - 200°C

5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 min

0.0

1.0

2.0

3.0

4.0

uV(x10,000)Chromatogram

(+) Linalool (-) Linalool

Application: Enantiomeric Evaluation of Ho oil


Linalool extracted from Ho oil

10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 min

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

uV(x10,000)

0

50

100

150

200

250

300

350

400

CChromatogram

(-) Linalool

(+) Linalool

Application: Enantiomeric Evaluation of Rosewood oil

Distilled Leaves Oil

3- (+)- Terpinen-4-ol

4- (-)- Terpinen-4-ol

5- (-)- a- Terpineol

6- (+)- a- Terpineol


20.0 22.5 25.0 27.5 min

0.0

0.5

1.0

1.5

2.0


30.0 32.5 35.0 37.5 40.0 42.5 min

2.5

5.0

7.5

10.0


1- (-)- Limonene

2- (+)- Limonene

1

2

3

4

5

6

Application: Enantiomeric Evaluation of Rosemary oil

Column: DETTBSBTA - l: 25m ; i.d.: 0.25 mm; df: 0.25μm (MEGA, Milano)

Inj. temp.: 250°C; mode: split, ratio: 1/50; Det.: FID, temp.: 250°C; Temp. progr.: 45°C- 2°C/min - 200°C

5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 min

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

uV(x10,000)

0

50

100

150

200

250

300

350

400

CChromatogram

(+) Linalool: herbaceous, green, petitgrain

(-) Linalool: floral, fresh, lavender

(+) Limonene: fresh, citrus,

orange-like

(-) Limonene: harsh,

turpentine-like

(+) Terpinen-4-ol: musty, dusty

(-) Terpinen-4-ol: musty

http://webdb.uni-graz.at/~katzer/germ/spice_photo.html#rosm_off

“Il est des parfums frais comme des chairs d’enfants,

Doux comme les hautbois, verts comme les prairies,

et d’autres, corrumpus, riches et triomphants,

ayant l’expansion des choses infinies

Comme l’ambre, le musc, le benjoin et l’encens,

Qui chantent les transports de l’esprit et des sens”

Les Fleurs du Mal

C. Baudelaire

http:/pharma.unime.it/foodchem

[email protected]

Phaser – Hardware Conclusion

MDGCMS - Phaser – Hardware Conclusion

• No cold spot possibility to sniff higher boiling point

compounds • Maximum 300°C on transfer line • Protects your nose from drying by adding moisture air to sniffing port • Easy to connect capillary column to splitter • Sitting down or standing up while sniffing • Compact and easy to use •MDGCMS – detailed separation and identification



85

Some more info on www.shimadzu.eu

Shimadzu Handels GmbH - org.složka

Ocelářská 35, 190 00 Praha

[email protected]

tel.+420 284080221

fax +420 284080225

Thank you for your attention !

http://www.shimadzu.eu/








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