high efficiency multidimensional gas chromatography coupled to isotope ratio mass spectrometry and...
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Un
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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
Multidimensional Gas Chromatography
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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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Multidimensional Gas Chromatography
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)
Multidimensional Gas Chromatography
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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
1.5
2.0
2.5
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
Oct-09 Nov-09 Dec-09 Jan-10 Feb-10 Mar-10
(-)-alpha-Thujene
(-)-alpha-Pinene
(-)-Camphene
(-)-beta-Pinene
(-)-Sabinene
(-)-alpha-Phellandrene
(+)-beta-Phellandrene
(+)-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
Oct-10 Nov-10 Dec-10 Jan-11 Feb-11 Mar-11
(-)-alpha-Thujene
(-)-alpha-Pinene
(-)-Camphene
(-)-beta-Pinene
(-)-Sabinene
(-)-alpha-Phellandrene
(+)-beta-Phellandrene
(+)-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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Multidimensional Gas Chromatography
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
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