High Efficiency Multidimensional Gas Chromatography Coupled to Isotope Ratio Mass Spectrometry and...

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Danilo Sciarrone1, Antonino Schepis1 and Luigi Mondello1,2,3

1University of Messina, Italy

2Chromaleont Srl, Messina, Italy

3Campus Bio-Medico, Rome, Italy

High Efficiency Multidimensional Gas Chromatography Coupled to

Isotope Ratio Mass Spectrometry and Quadrupole Mass Spectrometry

Simultaneous Detection

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Multidimensional Gas Chromatography

The Need for Multidimensionality

real-world samples are normally very complex mixtures,

containing hundreds and sometimes thousands of volatile

components

the total separation of such matrices on a single capillary

column is a difficult, if not impossible, task

a great increase in resolving power can be achieved through

the coupling of two columns with different separation

mechanisms through a specific transfer system

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1° Dim.

5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 min

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0uV(x10,000)

2° Dim.

5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 min

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2.0

uV (x10,000)

InJ.1 InJ.2FID1 FID2

I dimension separation

II dimension separation


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Conventional & Multidimensional Gas Chromatography

Conventional GC

5.0 10.0 15.0 20.0 25.0 30.0 min0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0uV (x10,000)

Peak selected for further separation

1D peak capacity = n1

2D

pea

k c

apac

ity =

n2

MDGC peak capacity = n1 + n2

0.0

2.5

5.0

7.5

10

.01

2.5

min

0.0

1.0

2.0

3.0

4.0

5.0 u

V(x

10

,00

0)

Multidimensional GC

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Peak capacity consideration

1D polar / apolar column

30 m x 0.25 mm x 0.25 mm

2D

ap

ola

r /

po

lar

colu

mn

30

m x

0.2

5 m

m x

0.2

5 m

m

About 120000 theoretical plates

Ab

ou

t 12

00

00

th

eore

tica

l p

late

s

5 CUTS

nMDGC = 120000 + ( 5 x 120000) = 720000

Det 1

Det 2

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higher peak capacity: the resulting peak capacity is approximately equal to

that of the primary column plus that of the product between the number of cuts

and the peak capacity of the secondary column:

Advantages

n1 + (n2 x num. of cuts)

the technique is particularly suitable when only the 2D separation of

specific parts of the sample are required (i.e. chiral compounds)


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MULTIDIMENSIONAL GAS CHROMATOGRAPH (MDGC)

GC-1:

Transfer system

Injector Split/Splitless

FID

GC-2:

Injector Split/Splitless

FID/MS

Transfer

line

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GC-1 AND TRANSFER SYSTEM

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TRANSFER SYSTEM DIMENSION

mm

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0.53 mm I.D. 0.53 mm I.D.

Retetion gap

FID1

1D2D

Auxiliary

pressure

Restrictor

TRANSFER SYSTEM CONNECTIONS

Auxiliary

pressure

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MDGC Switching System Scheme

First Version

1D2DR2

FID1

APC1 APC2

Cut position:

APC1 = P kPa (es. 250)

APC2 = P + 1kPa (es. 251)

LIMIT:

APC Pressure rate

(400 kPa/min)

Stand-by position:

APC1 = P kPa (es. 250)

APC2 = (P – 1) kPa (es. 249)

249250 251250

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MDGC Switching System Scheme

First Modification

APC

PS

Carrier Gas

V

1D2D

R1

R2

FID1

NO NC

No APC Pressure rate limit

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OVEN GC-1FID

APC

V

1D

2D

Transfer System Connections and Gas Supply/Control

APC: Auxiliary Pressure

Controll

V: Three Way Valve

R: Restrictor

Pf: Press fit

Rg: Retention Gap

1D: First Column

2D: Second Column

Pf Pf

Rg

Rg

R

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OVEN GC-1FID

APC

V

1D 2D

NONC

Pf Pf

Rg

Rg

R

MDGC Transfer System

STAND-BY Position

P

P

P

P-DP

P-DP

DPP

P

PP-DP PP

Pressure

1D eluted fraction

to FID1

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OVEN GC-1FID

APC

V

1D 2D

NONC

Pf Pf

Rg

Rg

R

MDGC Transfer System

CUT Position

P

P

P

P

P

DPP-DP

P-DP

P-DPP P-DPPressure

1D eluted fraction

to 2D

P-DP

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CONVENTIONAL MDGC ANALYSIS OF

ESSENTIAL OILS

GC-1:

Column: MEGA SE-52 25m x 0.25mm x 0.25mm

Temperature Program: from 50°C to 280°C (3°C/min)

Carrier gas: Helium. Pressure: 347 kPa at constant pressure

APC: Helium. 250 kPa

Detector: Flame Ionization Detector (FID) (290°C). H2: 50 ml/min, Air: 400 ml/min.

Volume Injected: 1. 0m L (250°C). Split ratio 1:100.

GC-2:

Column : MEGA DetTBuSililBeta 25m x 0.25mm x 0.25mm

Temperature Program : from 45°C (12.00 min) to 180°C (2°C/min)

Detector : Flame Ionization Detector (FID) (210°C). H2: 50 ml/min, Air: 400 ml/min. Make-

up: 50 ml/min (He).

Experimental Conditions

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1.0

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3.0

3.5

4.0

4.5

5.0uV(x10,000)

MDGC Conventional Analysis of Bergamot Oil

1

2 3 4 5

6 7

8 Chiral

Compounds

1 - a-pinene

2 - sabinene

3 - b-pinene

4 - limonene

5 - linalool

6 - terpinen-4-

ol

7 - a-terpineol

8 - linalyl

acetate

data15

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0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0uV(x10,000)

1 2/ 3 4 5 6 7 8

data16I

Transfered

Chiral

Compounds

1 - a-pinene

2 - sabinene

3 - b-pinene

4 - limonene

5 - linalool

6 - terpinen-4-ol

7 - a-terpineol

8 - linalyl

acetate

1D Conventional MDGC Chromatogram of

Bergamot Oil

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5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 min0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50uV(x10,000)Chromatogram

Data16II

1 – (+/-)-a-pinene

2 – (+)-b-pinene

3 – (-)-b-pinene

4 – (+)-sabinene

5 – (-)-sabinene

6 – (-)-limonene

7 – (+)-limonene

8 – (-)-linalool

9 – (+)-linalool

10 – (-)- linanyl

acetate

11 – (+)-linanyl

acetate

12 – (-)-terpinen-4-ol

13 – (+)-terpinen-4-ol

14 – (-)-a-terpineol

15 – (+)-a-terpineol

12

3

4

5

6

7 8

9

10

11

12

1314

15

2D Conventional MDGC Chromatogram of

Bergamot Oil

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Lemon Bergamot Mandarin Key Lime Persian Lime Sweet Orange

Italy Italy Italy Mexico Mexico Italy

(92) (8) (124) (3) (3) (2) (17)

Type A Type B

2 (-)-a-thujene 97-99 97-98 99 98 97-100 99 24-80

3 99-82

4 (+)-a-pinene

(-)-a-pinene 37-49 35-40 55-56 53-55 40-43

5 72-85 76-79 86-86 84-89 77-79

6

(-)-camphene

(+)- 3-11

7 76-98

8 (+)-b-pinene

(-)- 86-91 81-83 91-93 91-93 79-81 -30-89

9 43-63 80-95

10

(+)-sabinene

(-)- 70-75 66-68 69 69 61-63

11 (-)-a-phellandrene -6-5 -1-8 -11-10 2-7 4-17 10-12

13 95-99 28-31 37-47 9-10

14 (-)-b-phellandrene

(+) -8-38 45-33 97-99

16 (+)-limonene 97 96-97 96-97 94 94 95 99

18 (-)-camphor 16-49 27-66

19 4-49 99 37-47 30-32 27-28

20

(-)-linalool

(+)- 59-74 64-84

21 69-90 44 46-62 57-61

22

(-)-citronellal

(+)- 82-92 -5-25

23 (-)-linalyl acetate 99-100

26 (-)-terpinen-4-ol 47-76 57-70 63-81 42 42-58 59-61

27 33-65 35-53 68-79 59-68 55-56

28 (-)-a-terpineol

(+)- -8-58 67-90

Enantiomeric excesses in some Citrus oils

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Enantiomeric ratios are subject to seasonal and annual variationEE 2008-2009

-20,0

0,0

20,0

40,0

60,0

80,0

100,0

Oct-08 Nov-08 Dec-08 Jan-09 Feb-08 Mar-08

(-)-alpha-Thujene

(-)-alpha-Pinene

(-)-Camphene

(-)-beta-Pinene

(-)-Sabinene

(-)-alpha-Phellandrene

(+)-beta-Phellandrene

(+)-Limonene

(-)-Linalool

(-)-Linalyl acetate

(-)-Terpinen-4-ol

(+)-alpha-Terpineol

EE 2009-2010

-20,0

0,0

20,0

40,0

60,0

80,0

100,0


(-)-alpha-Thujene

(-)-alpha-Pinene

(-)-Camphene

(-)-beta-Pinene

(-)-Sabinene



(+)-Limonene

(-)-Linalool

(-)-Linalyl acetate

(-)-Terpinen-4-ol

(+)-alpha-Terpineol

Bergamot oil

EE 2010-2011

-40,0

-20,0

0,0

20,0

40,0

60,0

80,0

100,0


(-)-alpha-Thujene

(-)-alpha-Pinene

(-)-Camphene

(-)-beta-Pinene

(-)-Sabinene



(+)-Limonene

(-)-Linalool

(-)-Linalyl acetate

(-)-Terpinen-4-ol

(+)-alpha-Terpineol

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-9

-7

-5

-3

-1

1

3

alph

a-th

ujen

e

alph

a-pine

ne

beta-p

inen

e

myr

cene

limon

ene

gamm

a-te

rpinen

e

linaloo

l

linalyl ace

tate

alph

a-te

rpinyl a

ceta

te

neryl a

ceta

te

gera

nyl a

ceta

te

(E)-ca

ryop

hylle

ne

trans

-alpha

-Ber

gam

oten

e

beta-b

isab

olen

e

norb

orna

nol*

cam

pher

enol

alph

a-bisa

bolol

nootka

tone

Min cold-

pressed i-STD

myrcene

Max cold-

pressed i-STD

myrcene

A8%L i-STD

myrcene

A2%L i-STD

myrcene

-34

-32

-30

-28

-26

-24

-22

alph

a-th

ujen

e

alph

a-pine

ne

beta-p

inen

e

myr

cene

limon

ene

gamm

a-te

rpinen

e

linaloo

l

linalyl ace

tate

alph

a-te

rpinyl a

ceta

te

neryl a

ceta

te

gera

nyl a

ceta

te

(E)-ca

ryop

hylle

ne

trans

-alpha

-Ber

gam

oten

e

beta-b

isab

olen

e

norb

orna

nol*

cam

pher

enol

alph

a-bisa

bolol

nootka

tone

Min cold-

pressed

Max cold-

pressed

A8%L

A2%L

Self-adulterated bergamot

oil samples with different

synthetic linalool

percentage (A8%L,

A2%L)

d13C

Myr

cene

Internal strandard

(Myrcene) role for

adulterated samples

d13C

VP

DB

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MDGC-IRMS-qMS Instrument

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MDGC-IRMS-qMS Instrument

GC-GC-MSD

Shimadzu

GC interface-IRMS

Isoprime/Elementar

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MDGC-enantio-IRMS-qMS Bergamot EO

IRMS

qMS

(-)-

sabin

ene

(+)-

sab

inen

e

(-)-b

-pin

ene

(+)-b

-pin

ene

(+)-a

-ter

pin

eol

(-)-a

-ter

pin

eol

(-)-

terp

inen

-4-o

l(+

)-te

rpin

en-4

-ol

(+)-

terp

inen

-4-o

l

(-)-

terp

inen

-4-o

l

(-)-a

-ter

pin

eol

(+)-a

-ter

pin

eol

(-)-b-pinene

(+)-b

-pin

ene

(-)-

sabin

ene

(+)-

sabin

ene

Sabinene

b-pinene

Terpinen-4-ol

a-terpineol

INJECTION

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MDGC-enantio-IRMS-qMS Instrument

IRMS

b-phellandrene

Co-eluted with limonene in the first dimension

b-phellandrene

Limonene

Linalool

Linalyl acetate

INJECTION

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Compound d13 C n=9

(+)-a-thujene -24.04

(-)-a-thujene -27.02

(+)-b-pinene -28.79

(-)-b-pinene -26.88

(+)-sabinene -25.99

(-)-sabinene -25.07

(-)-b-phellandrene -25.07

(+)-b-

phellandrene

-26.92

(-)-limonene -27.20

(+)-limonene -27.70

(-)-linalool -27.14

(+)-linalool -27.19

(-)-linalyl acetate -28.03

(+)-linalyl acetate -28.09

(+)-terpinen-4-ol -26.21

(-)-terpinen-4-ol -28.07

(-)-a-terpineol -27.60

(+)-a-terpineol -28.21

Genuine Bergamot

cold-pressed Essential Oil

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CONCLUSION

The MDGC approach has proved to be powerful and versatile.

This approach, which may be generally applied for target

compounds analysis in complex samples, is characterized by

an increased resolving power and high analytical rapidity.

MDGC Enantio separation is the most sophisticated approach

for accurate and precise analysis of chiral components for

quali/quantitative analysis and also very powerful in the

characterization of the isotopic ratio of chiral components.

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Acknoledgements

• My Research Group

• Shimadzu Corporation

• Isoprime/Elementar Corporations

• Supelco/Sigma-Aldrich Corporation

High Efficiency Multidimensional Gas Chromatography Coupled to Isotope Ratio Mass Spectrometry and...

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