Time controlled Cryogenic Zone Compression
(t- CZC) GC-HRMS:
- can we detect attogramm amounts of TCDD?
Dirk Krumwiede & Heinz Mehlmann
Thermo Fisher Bremen
Acknowledgements
Daniela Cavagnino
Brock Chittim
Jef Focant
Don Patterson Jr.
Wayman Turner
2
Wayman Turner
Andreas Sjodin
Jack Cochran
It is all about (instrumental) sensitivity!
3
DFS: magnetic sector HRMS
- Highest selectivity!
- Highest sensitivity!
- Highest……speed?
It is all about (instrumental) sensitivity!
50
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70
75
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Re
lative
Ab
un
da
nce
10.83
ppb
ppt
Picogram - pg (/ul)
20 fg 2378-TCDD,
Femtogram
5
DFS: magnetic sector HRMS
- Highest selectivity!
- Highest sensitivity!
- Highest……speed?
7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5
Time (min)
0
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15
20
25
30
35
40
45
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Re
lative
Ab
un
da
nce
10.91 11.0910.299.09 11.609.268.81 11.268.78 10.04 10.539.749.338.478.137.68 7.92 9.638.407.59
ppt
ppq
20 fg 2378-TCDD,
1 ul injected
Attogram – ag!?
Dried Blood Spot (DBS) analysis for POPs:
• Very small sample (20 - 50 ul Serum)
• Extremely low analyte amounts for TCDD
• Sample extract volume = ca. 5 ul
� Sensitivity must come from instrument
DBS – the challenge: ultra sensitivity for some analytes
7
� Sensitivity must come from instrument
Newborn Screening Programs (NSPs), etc.:
• Very important samples for epidemiological studies
• Population exposure and trends
What can GC do to increase sensitivity?
RT: 21.69 - 22.12
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Rela
tive A
bundance
NL:3.10E4
m/z= 303.9419-303.9723 MS ICIS 5w_cryomix_11
9
21.7 21.8 21.9 22.0 22.1
Time (min)
0
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Rela
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RT: 21.89AA: 10792
What can GC do to increase sensitivity?
RT: 21.69 - 22.12
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Rela
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RT: 21.89AA: 10539
NL:3.10E4
m/z= 303.9419-303.9723 MS ICIS 5w_cryomix_13
10
21.7 21.8 21.9 22.0 22.1
Time (min)
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RT: 21.95AA: 51.20
PCB 77 (13C, IS)
Produce narrow peaks = increase Peak Height
narrower peak
RT: 21.69 - 22.12
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Rela
tive A
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RT: 21.89AA: 10539
NL:3.10E4
m/z= 303.9419-303.9723 MS ICIS 5w_cryomix_11
NL:3.10E4
m/z= 303.9419-303.9723 MS ICIS 5w_cryomix_13
0.6 sec pw (at 10%)
11
= higher peak
= better sensitivity
1/pw = ph
21.7 21.8 21.9 22.0 22.1
Time (min)
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RT: 21.89AA: 10792
RT: 21.95AA: 51.209.6 sec pw (at 10%)
GCxGC – increased peak capacity / separation power
13
Modulation: several fast 2 dim. chromatograms over each first dim. Peak
GCxGC main feature: increased peak capacity
GCxGC secondary feature: increased sensitivity (very narrow 2d dim peaks)
How to maximize the sensitivity effect of GCxGC?
GCxGC
15
- modulation of 1 dim. Peak
- several 2 dim. chromatograms
How to maximize the sensitivity effect of GCxGC?
GCxGC CZC
16
- modulation of 1 dim. Peak
- several 2 dim. chromatograms
- cryofocus complete 1 dim. Peak
- one single 2 dim. chromatogram
„Ion dilution“
Original idea for CZC – magnetic sector HRMS by….see paper
- R&D work at CDC Atlanta:
For Dioxin analysis…
…combine highest sensitive MS
with highest sensitive GC method
���� GCxGC + sector HRMS
17
���� GCxGC + sector HRMS
The entire peak is cryo-trapped
in one single event.
For only 2378 Cl containing…
- blood / serum samples
- dried blood spot analysis
Injector
timed Cryogenic Zone Compression (t-CZC)
- one single standard column (no 2d column, no connection)
- only one jet is used
- jet on/off timed controlled
� Simple & flexible
19
(4m)56m
CO2 Jet
MS
2nd Dimension1st Dimension
Guideline: As complicated as necessary, as simple as possible!
Peak Zone Compression: Cryo Zone Compression (CZC)
20
CO2 Modulator of Thermo Scientific Trace GCxGC ultra
(4m)56m
MS
Injector
2nd Dimension1st Dimension
(t-CZC) Principle
21
CO2 Jet
RTnormaltime
ChromatogramNormal Peak – CZC inactive
(4m)56m
MS
Injector
2nd Dimension1st Dimension
At the time when the peak of interest reaches the Jet…
(t-CZC) Principle
22
CO2 Jet
RTJettime
Chromatogram
(4m)56m
MS
Injector
2nd Dimension1st Dimension
…the Jet is switched on to trap the entire peak of interest
(t-CZC) Principle
23
Jet on
CO2 Jet
RTJettime
Chromatogram
(4m)56m
MS
Injector
2nd Dimension1st Dimension
…and then reinject to the 2 d dimension (Jet off again)
(t-CZC) Principle
24
Jet off
CO2 Jet
RTCZCtime
ChromatogramReinject on 2d Dim.
c:\xcalibur\data\110622_cryo_01\cryo08 6/22/2011 12:00:10 PM CSL X1/10GC2: column: 60m x 0.25 mm x 0.1 um; 120(2)-10-220(0)-3-235(7)-4.6-310(5); He flow 1.0ml/min ; 45eV tcdd precool 23.10-24.3 tcdd trap I: 25.15-25.55 pcdd/f precool 29-29.5 and 30.5-31.07
RT: 19.90 - 50.42
10
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50
60
70
80
90
100R
ela
tive A
bundance
48.42
39.21 45.3338.0425.62 42.9337.81 40.5824.98 30.14 44.6132.01
32.50
NL:2.60E6
TIC - m/z= 314.4000-318.4000 MS Cryo09
„normal“ Dioxin/Furan chromatogram on 60 m col.
(t-CZC) Principle
25
20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50
Time (min)
0
10
20
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40
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Rela
tive A
bundance
0
32.50
25.76
32.08
32.58
48.43
38.02 39.18 45.3242.9240.5837.7924.99 30.1340.02
NL:2.60E6
TIC - m/z= 314.4000-318.4000 MS cryo08
c:\xcalibur\data\110622_cryo_01\cryo08 6/22/2011 12:00:10 PM CSL X1/10GC2: column: 60m x 0.25 mm x 0.1 um; 120(2)-10-220(0)-3-235(7)-4.6-310(5); He flow 1.0ml/min ; 45eV tcdd precool 23.10-24.3 tcdd trap I: 25.15-25.55 pcdd/f precool 29-29.5 and 30.5-31.07
RT: 19.90 - 50.42
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40
50
60
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80
90
100R
ela
tive A
bundance
48.42
39.21 45.3338.0425.62 42.9337.81 40.5824.98 30.14 44.6132.01
32.50
NL:2.60E6
TIC - m/z= 314.4000-318.4000 MS Cryo09
Selection of target analytes to becryofocused
(t-CZC) Principle
26
20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50
Time (min)
0
10
20
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40
50
60
70
80
90
100
Rela
tive A
bundance
0
32.50
25.76
32.08
32.58
48.43
38.02 39.18 45.3242.9240.5837.7924.99 30.1340.02
NL:2.60E6
TIC - m/z= 314.4000-318.4000 MS cryo08
c:\xcalibur\data\110622_cryo_01\cryo08 6/22/2011 12:00:10 PM CSL X1/10GC2: column: 60m x 0.25 mm x 0.1 um; 120(2)-10-220(0)-3-235(7)-4.6-310(5); He flow 1.0ml/min ; 45eV tcdd precool 23.10-24.3 tcdd trap I: 25.15-25.55 pcdd/f precool 29-29.5 and 30.5-31.07
RT: 19.90 - 50.42
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40
50
60
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100R
ela
tive A
bundance
48.42
39.21 45.3338.0425.62 42.9337.81 40.5824.98 30.14 44.6132.01
32.50
NL:2.60E6
TIC - m/z= 314.4000-318.4000 MS Cryo09
Same scaling
(t-CZC) Principle
27
20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
0
32.50
25.76
32.08
32.58
48.43
38.02 39.18 45.3242.9240.5837.7924.99 30.1340.02
NL:2.60E6
TIC - m/z= 314.4000-318.4000 MS cryo08
CZC focused peaks
c:\xcalibur\data\110622_cryo_01\cryo08 6/22/2011 12:00:10 PM CSL X1/10GC2: column: 60m x 0.25 mm x 0.1 um; 120(2)-10-220(0)-3-235(7)-4.6-310(5); He flow 1.0ml/min ; 45eV tcdd precool 23.10-24.3 tcdd trap I: 25.15-25.55 pcdd/f precool 29-29.5 and 30.5-31.07
RT: 19.90 - 50.42
10
20
30
40
50
60
70
80
90
100R
ela
tive A
bundance
48.42
39.21 45.3338.0425.62 42.9337.81 40.5824.98 30.14 44.6132.01
32.50
NL:2.60E6
TIC - m/z= 314.4000-318.4000 MS Cryo09
(t-CZC) Principle
28
20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
0
32.50
25.76
32.08
32.58
48.43
38.02 39.18 45.3242.9240.5837.7924.99 30.1340.02
NL:2.60E6
TIC - m/z= 314.4000-318.4000 MS cryo08
Selected analytes with CZC2378-TCDD
1234-TCDD 12378-PeCDF
12378-PeCDD
23478-PeCDF Only selectedpeaks arerefocussed
standard GC-HRMS vs. CZC GC-HRMSpooled blood sample on DFS (HRMS) – 2378-TCDD ca. 10 fg
RT: 21.00 - 26.31
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100
Re
lative
Ab
un
da
nce
0
20
40
60
80
100
RT: 25.97AA: 630SN: 8RMS
21.1725.3124.8724.3422.8422.51
23.52 24.14 24.5522.1121.77 23.47
RT: 25.99AA: 865SN: 16RMS
25.95
NL:1.87E2
m/z= 319.8810-319.9130 MS ICIS Cryo20
NL:2.58E2
m/z= 321.8779-321.9101 MS ICIS Cryo20
RT: 23.15 - 26.31
60
80
100
Re
lative
Ab
un
da
nce
0
20
40
60
80
100
RT: 26.08AA: 1023SN: 149RMS
25.18
RT: 26.08AA: 1292SN: 273RMS
NL:2.61E3
m/z= 319.8810-319.9130 MS ICIS Cryo18
NL:3.43E3
m/z= 321.8779-321.9101 MS ICIS Cryo18
AA: 866
Height: 258
SignoiseRMS: 16AA: 1292
Height: 3430
Qual mz
Quan mz
29
21 22 23 24 25 26
Time (min)
0
20
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80
100
0
20
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60
Re
lative
Ab
un
da
nce
25.7323.2224.2022.41 24.0621.51 22.19 22.82 25.6324.80
21.94
RT: 25.29AA: 254225SN: 3574
NL:5.68E4
m/z= 333.9173-333.9507 MS ICIS Cryo20
23.5 24.0 24.5 25.0 25.5 26.0
Time (min)
0
20
40
60
80
100
0
20
40
60
Re
lative
Ab
un
da
nce
RT: 26.07AA: 45.44SN: 13RMS
RT: 26.08AA: 260302SN: 70559
RT: 25.26AA: 275184SN: 6397
NL:7.05E5
m/z= 333.9173-333.9507 MS ICIS Cryo18
SignoiseRMS: 16 Height: 3430
SignoiseRMS= 273
non-cryofocused
1234-TCDD
Quan mz
13C IS mz
t-CZC paper at: http://pops.thermo-bremen.com
In Google:
- type „POPs“ & „Thermo“
- first hit is this page
30
Chromatography Today
February / March 2012
Goal: optimize method for as low iLOQs as possible
Wellington attogram standards
32
Simple goal:
Go as low as possible (absolute amount injected)
Thx, Brock!!
Lower & lower & lower …the Patterson LimboRT: 14.00 - 14.91
0
50
100
Rela
tive A
bundance
0
50
100
0
50
100
RT: 14.64
AA: 4727
RT: 14.64
AA: 1740
RT: 14.65
AA: 667
NL:
1.10E4
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_116
NL:
4.02E3
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_115
NL:
1.48E3
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_113
5 fg/ul ?
34
14.0 14.1 14.2 14.3 14.4 14.5 14.6 14.7 14.8 14.9
Time (min)
0
50
100
0
50
100
0
50
100
0Rela
tive A
bundance
RT: 14.65
AA: 300
RT: 14.64
AA: 104
NL:
9.74E2
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_108
NL:
3.36E2
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_104
NL:
2.40E1
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_100
Lower & lower & lower …the Patterson limboRT: 14.00 - 14.91
0
50
100
Rela
tive A
bundance
0
50
100
0
50
100
RT: 14.64
AA: 4727
RT: 14.64
AA: 1740
RT: 14.65
AA: 667
NL:
1.10E4
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_116
NL:
4.02E3
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_115
NL:
1.48E3
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_113
5 fg/ul
2 fg/ul ?
35
14.0 14.1 14.2 14.3 14.4 14.5 14.6 14.7 14.8 14.9
Time (min)
0
50
100
0
50
100
0
50
100
0Rela
tive A
bundance
RT: 14.65
AA: 300
RT: 14.64
AA: 104
NL:
9.74E2
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_108
NL:
3.36E2
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_104
NL:
2.40E1
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_100
Lower & lower & lower …the Patterson limboRT: 14.00 - 14.91
0
50
100
Rela
tive A
bundance
0
50
100
0
50
100
RT: 14.64
AA: 4727
RT: 14.64
AA: 1740
RT: 14.65
AA: 667
NL:
1.10E4
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_116
NL:
4.02E3
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_115
NL:
1.48E3
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_113
5 fg/ul
2 fg/ul
1 fg/ul ?
36
14.0 14.1 14.2 14.3 14.4 14.5 14.6 14.7 14.8 14.9
Time (min)
0
50
100
0
50
100
0
50
100
0Rela
tive A
bundance
RT: 14.65
AA: 300
RT: 14.64
AA: 104
NL:
9.74E2
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_108
NL:
3.36E2
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_104
NL:
2.40E1
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_100
Lower & lower & lower …the Patterson limboRT: 14.00 - 14.91
0
50
100
Rela
tive A
bundance
0
50
100
0
50
100
RT: 14.64
AA: 4727
RT: 14.64
AA: 1740
RT: 14.65
AA: 667
NL:
1.10E4
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_116
NL:
4.02E3
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_115
NL:
1.48E3
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_113
5 fg/ul
2 fg/ul
1 fg/ul
37
14.0 14.1 14.2 14.3 14.4 14.5 14.6 14.7 14.8 14.9
Time (min)
0
50
100
0
50
100
0
50
100
0Rela
tive A
bundance
RT: 14.65
AA: 300
RT: 14.64
AA: 104
NL:
9.74E2
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_108
NL:
3.36E2
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_104
NL:
2.40E1
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_100
500 ag/ul ??
Lower & lower & lower …the Patterson limboRT: 14.00 - 14.91
0
50
100
Rela
tive A
bundance
0
50
100
0
50
100
RT: 14.64
AA: 4727
RT: 14.64
AA: 1740
RT: 14.65
AA: 667
NL:
1.10E4
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_116
NL:
4.02E3
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_115
NL:
1.48E3
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_113
5 fg/ul
2 fg/ul
1 fg/ul
38
14.0 14.1 14.2 14.3 14.4 14.5 14.6 14.7 14.8 14.9
Time (min)
0
50
100
0
50
100
0
50
100
0Rela
tive A
bundance
RT: 14.65
AA: 300
RT: 14.64
AA: 104
NL:
9.74E2
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_108
NL:
3.36E2
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_104
NL:
2.40E1
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_100
500 ag/ul
200 ag/ul ???
Lower & lower & lower …the Patterson limboRT: 14.00 - 14.91
0
50
100
Rela
tive A
bundance
0
50
100
0
50
100
RT: 14.64
AA: 4727
RT: 14.64
AA: 1740
RT: 14.65
AA: 667
NL:
1.10E4
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_116
NL:
4.02E3
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_115
NL:
1.48E3
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_113
5 fg/ul
2 fg/ul
1 fg/ul
39
14.0 14.1 14.2 14.3 14.4 14.5 14.6 14.7 14.8 14.9
Time (min)
0
50
100
0
50
100
0
50
100
0Rela
tive A
bundance
RT: 14.65
AA: 300
RT: 14.64
AA: 104
NL:
9.74E2
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_108
NL:
3.36E2
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_104
NL:
2.40E1
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_100
500 ag/ul
200 ag/ul
????
Lower & lower & lower …the Patterson limboRT: 14.00 - 14.91
0
50
100
Rela
tive A
bundance
0
50
100
0
50
100
RT: 14.64
AA: 4727
RT: 14.64
AA: 1740
RT: 14.65
AA: 667
NL:
1.10E4
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_116
NL:
4.02E3
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_115
NL:
1.48E3
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_113
5 fg/ul
2 fg/ul
1 fg/ul
40
14.0 14.1 14.2 14.3 14.4 14.5 14.6 14.7 14.8 14.9
Time (min)
0
50
100
0
50
100
0
50
100
0Rela
tive A
bundance
RT: 14.65
AA: 300
RT: 14.64
AA: 104
NL:
9.74E2
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_108
NL:
3.36E2
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_104
NL:
2.40E1
m/z=
321.8739-
321.9061 MS
ICIS
1209_CZC_TF_
LL_100
500 ag/ul
200 ag/ul
blank
Wellington TF-LL-CS standards: 200 ag/ul to 5 fg/ul 2378-TCDD
2378-TCDD 5.0 fg/µl
triplicats of low levels
41
� 800 ag absolute amount for lowest level
�4 ul injected500 ag/µl
200 ag/µl
1.0 fg/µl
2.0 fg/µl
triplicats of low levels
Wellington TF-LL-CS standards: 200 ag/ul to 5 fg/ul 23478-PCDF
23478-PCDF 5.0 fg/µl
triplicats of low levels
42
500 ag/µl
1.0 fg/µl
2.0 fg/µl
200 ag/µl [fg / ul]
�4 ul injected
� More difficult than TCDD
triplicats of low levels
800 ag 2378-TCDD (Wel. TF-LL-CS) – repeatability
RT: 13.64 - 14.97
50
60
70
80
90
100
Rela
tive A
bundance
0
10
20
30
40
50
60
70
80
90
100
RT: 14.64AA: 85.11
RT: 14.64AA: 103
NL:1.87E2
m/z= 319.8810-319.9130 MS ICIS 1209_CZC_TF_LL_104
NL:3.36E2
m/z= 321.8779-321.9101 MS ICIS 1209_CZC_TF_LL_104
RT: 13.64 - 14.97
50
60
70
80
90
100
Rela
tive A
bundance
0
10
20
30
40
50
60
70
80
90
100
RT: 14.65AA: 138
14.62
14.71 14.9013.99 14.07 14.3413.69 14.60
RT: 14.64AA: 49.65
NL:2.83E2
m/z= 319.8810-319.9130 MS ICIS 1209_CZC_TF_LL_107
NL:1.38E2
m/z= 321.8779-321.9101 MS ICIS 1209_CZC_TF_LL_107
1.) 800 ag 2.) 800 ag
RM
QM
RT: 13.64 - 14.97
50
60
70
80
90
100
Rela
tive A
bundance
0
10
20
30
40
50
60
70
80
90
100
RT: 14.65AA: 128
RT: 14.65AA: 29.67
NL:2.61E2
m/z= 319.8810-319.9130 MS ICIS 1209_CZC_TF_LL_105
NL:7.60E1
m/z= 321.8779-321.9101 MS ICIS 1209_CZC_TF_LL_105
3.) 800 ag
43
14.0 14.5
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
Rela
tive A
bundance
RT: 14.64AA: 32198
RT: 14.66AA: 480
NL:7.81E4
m/z= 333.9173-333.9507 MS ICIS 1209_CZC_TF_LL_104
14.0 14.5
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50R
ela
tive A
bundance
RT: 14.64AA: 37307
NL:9.26E4
m/z= 333.9173-333.9507 MS ICIS 1209_CZC_TF_LL_107
1.) 800 ag 2.) 800 ag
IS (13C)
14.0 14.5
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
Rela
tive A
bundance
RT: 14.65AA: 32728
RT: 14.66AA: 398
RT: 14.22AA: 90.18
RT: 14.63AA: 150
NL:7.68E4
m/z= 333.9173-333.9507 MS ICIS 1209_CZC_TF_LL_105
3.) 800 ag
4 ul Injection Volume (4 x 200 ag = 800 ag)
RT: 13.64 - 14.97
50
60
70
80
90
100
Rela
tive A
bundance
0
10
20
30
40
50
60
70
80
90
100
RT: 14.64AA: 85.11
RT: 14.64AA: 103
NL:1.87E2
m/z= 319.8810-319.9130 MS ICIS 1209_CZC_TF_LL_104
NL:3.36E2
m/z= 321.8779-321.9101 MS ICIS 1209_CZC_TF_LL_104
RT: 13.64 - 14.97
50
60
70
80
90
100
Rela
tive A
bundance
0
10
20
30
40
50
60
70
80
90
100
RT: 14.65AA: 138
14.62
14.71 14.9013.99 14.07 14.3413.69 14.60
RT: 14.64AA: 49.65
NL:2.83E2
m/z= 319.8810-319.9130 MS ICIS 1209_CZC_TF_LL_107
NL:1.38E2
m/z= 321.8779-321.9101 MS ICIS 1209_CZC_TF_LL_107
1.) 800 ag 2.) 800 ag
RM
QM
RT: 13.64 - 14.97
50
60
70
80
90
100
Rela
tive A
bundance
0
10
20
30
40
50
60
70
80
90
100
RT: 14.65AA: 128
RT: 14.65AA: 29.67
NL:2.61E2
m/z= 319.8810-319.9130 MS ICIS 1209_CZC_TF_LL_105
NL:7.60E1
m/z= 321.8779-321.9101 MS ICIS 1209_CZC_TF_LL_105
3.) 800 ag
Sum Area:
188
Sum Area:
187Sum Area:
158
800 ag 2378-TCDD (Wel. TF-LL-CS) – repeatability
44
14.0 14.5
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
Rela
tive A
bundance
RT: 14.64AA: 32198
RT: 14.66AA: 480
NL:7.81E4
m/z= 333.9173-333.9507 MS ICIS 1209_CZC_TF_LL_104
14.0 14.5
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50R
ela
tive A
bundance
RT: 14.64AA: 37307
NL:9.26E4
m/z= 333.9173-333.9507 MS ICIS 1209_CZC_TF_LL_107
1.) 800 ag 2.) 800 ag
IS (13C)
14.0 14.5
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
Rela
tive A
bundance
RT: 14.65AA: 32728
RT: 14.66AA: 398
RT: 14.22AA: 90.18
RT: 14.63AA: 150
NL:7.68E4
m/z= 333.9173-333.9507 MS ICIS 1209_CZC_TF_LL_105
3.) 800 ag
PW = ca. 0.7 sec
(at 10%)
4 ul Injection Volume (4 x 200 ag = 800 ag)
CZC sensitivity
- We do not see nothing (!) at levels of 800 ag TCDD
- Statistics are getting worse the lower the level – no big surprise, is it?
- This is where we are – is it the optimum?
45
sensitivity
GC peak width MS dwell time
Why „only“ 0.6 – 1 sec wide peaks, why not narrower?
?
�Slow down GC
�Speed up HRMS
Find optimum settings
47
Time [sec]
? Find optimum settings
� Both sides - GC and MS - must
work at their optimum for best results!!
Method development – ion detection rateC:\Xcalibur\...\5w_cryomix_13 7/21/2011 4:25:22 PM CZC: 1 ul 2fg/ul pcb/pcdd/pcdf mix (WEL. TF-LL-CS5)
120(2)-10-220(10)-3-235(7)-4.6-310(8); He flow 1.0ml/min ; 45eV, 3 mass MID (+ L & C): cycle time 0.1 sec, ML ratio 10, eld 6 msec, 24 ms on nat, 12 on IS
RT: 21.80 - 21.98
10
20
30
40
50
60
70
80
90
100
Rela
tive
Abu
nda
nce
RT: 21.89
AA: 10792
NL:
2.66E3
m/z=
303.9419-
303.9723
MS ICIS
5w_cryomix
_11
standard pw >10 s
48
21.80 21.82 21.84 21.86 21.88 21.90 21.92 21.94 21.96 21.98
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0RT: 21.89
AA: 10539
NL:
3.10E4
m/z=
303.9419-
303.9723
MS ICIS
5w_cryomix
_13
CZC pw 0.6-0.8 s
Low level statistics
2378-TCDD
49
����We want to quantify where others hope to have their calculated LODs
RSD
e.g. ion ratios
(or RRFs, etc.)
+15%
Low level statistics – ion statistics
� Very few ions are detected at very low levels
50
amount
-15%
+15%
- Which minimum RSD value is required to quantify ?
How much variation is acceptable ?
CZC sensitivity – authors opionion
Statistics: ion ratios, RSDs, linearity, data points over peak, etc.??
Why do you need your prescription glasses in total darkness?
Before you are good you are not so good.
51
Standardization � Innovation
PCDF: CZC vers. normal peak at 50 fg (CSL 1:10 dil) D:\Xcalibur\...\1209_CZC_pcddf_027 9/13/2012 3:11:07 PM
(used) TR-Dioxin5ms 30x0.25 (0.1), set jet on / off times, determin precool times without distorting non CZC targets, adapt MID times for CZC targets
RT: 16.03 - 17.47 SM: 5G
70
80
90
100
Rela
tive A
bundance
0
10
20
30
40
50
60
70
80
90
100
RT: 17.41
AA: 4439
RT: 16.58
AA: 3891
RT: 17.41
AA: 2656
NL:
8.35E3
m/z=
339.8409-
339.8749
MS ICIS
1209_CZC_
pcddf_027
NL:
5.15E3
m/z=
341.8399-
341.8741
MS ICIS
1209_CZC_
50 fg
Quan mz
- PCDFs have very similar response
One analysis run / same injection:
- 1st PCDF � normal detection
- 2nd PCDF � cryofocussed
- compare directly
12378-PeCDF
23478-PeCDF
53
16.1 16.2 16.3 16.4 16.5 16.6 16.7 16.8 16.9 17.0 17.1 17.2 17.3 17.4
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
Rela
tive A
bundance
RT: 16.57
AA: 2678
RT: 17.40
AA: 766176
RT: 16.56
AA: 819509
RT: 16.73
AA: 11968
1209_CZC_
pcddf_027
NL:
1.32E6
m/z=
351.8824-
351.9176
MS ICIS
1209_CZC_
pcddf_027
Qual mz
13C IS mz
here: 30 m column
10 pg
PCDF: CZC vers. normal peak at 2.5 fg (CSL 1:200 dil) D:\Xcalibur\...\1209_CZC_pcddf_204 9/13/2012 9:17:06 PM
(used) TR-Dioxin5ms 30x0.25 (0.1), set jet on / off times, determin precool times without distorting non CZC targets, adapt MID times for CZC targets
RT: 16.03 - 17.47 SM: 5G
70
80
90
100
Rela
tive A
bundance
0
10
20
30
40
50
60
70
80
90
100
RT: 17.42
AA: 168
RT: 16.58
AA: 18.00
RT: 17.42
AA: 71.28
NL:
2.70E2
m/z=
339.8409-
339.8749
MS ICIS
1209_CZC_
pcddf_204
NL:
1.78E2
m/z=
341.8399-
341.8741
MS
1209_CZC_
2.5 fg
Quan mz
54
16.1 16.2 16.3 16.4 16.5 16.6 16.7 16.8 16.9 17.0 17.1 17.2 17.3 17.4
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
Rela
tive A
bundance
RT: 16.57
MA: 126
RT: 17.42
AA: 26236
RT: 16.57
AA: 27574
1209_CZC_
pcddf_204
NL:
5.01E4
m/z=
351.8824-
351.9176
MS ICIS
1209_CZC_
pcddf_204
here: 30 m column
Qual mz
13C IS mz
500 fg
RT: 23.00 - 28.00 SM: 9G
80
100
0
20
40
60
80
100
0
20
40
60
80
100
RT: 23.96AA: 8378
RT: 26.44AA: 7718
RT: 25.56AA: 7655
RT: 25.99AA: 6939
RT: 27.07AA: 7184
RT: 25.67AA: 8420
RT: 25.96AA: 7725
RT: 26.41AA: 7406
RT: 27.04AA: 7006
RT: 26.11AA: 8740
NL:1.30E4
m/z= 321.7940-321.9940 MS ICIS hm_tczc_trap_01
NL:1.30E4
m/z= 321.7940-321.9940 MS ICIS hm_tczc_trap_02
NL:1.30E4
m/z= 321.7940-321.9940 MS
1378-TCDD
1478-TCDD
1234-TCDD
2378-TCDD
• 1.) No Cryofocusing
• 2.) Only 1378 TCDD focused
Selective focusing of peaks in t-CZC
55
23.0 23.5
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
25.0 25.5 26.0 26.5 27.0 27.5 28.0
Time (min)
RT: 25.57AA: 8813
RT: 26.42AA: 8281
RT: 27.07AA: 8408
RT: 26.54AA: 7486
RT: 25.58AA: 7826
RT: 27.06AA: 7248
RT: 26.02AA: 7369
RT: 27.17AA: 7371
RT: 25.97AA: 7885
RT: 25.55AA: 7543
RT: 26.45AA: 7351
321.9940 MS ICIS hm_tczc_trap_03
NL:1.30E4
m/z= 321.7940-321.9940 MS ICIS hm_tczc_trap_04
NL:1.30E4
m/z= 321.7940-321.9940 MS ICIS hm_tczc_trap_05
• 3.) Only 1478 TCDD focused
• 4.) Only 1234 TCDD focused
• 5.) Only 2378 TCDD focused
PCDFs separated on Dim 1 vers Dim 2
RT: 16.23 - 17.73 SM: 5G
50
60
70
80
90
100
Rela
tive A
bundance
0
10
20
30
40
50
60
70
80
90
100
RT: 17.22AA: 112591RT: 16.47
AA: 111717
RT: 17.22AA: 69028
RT: 16.47AA: 66509
NL:3.62E4
m/z= 339.8409-339.8749 MS ICIS 1207_pcdd_cryo_dk623
NL:2.20E4
m/z= 341.8399-341.8741 MS ICIS 1207_pcdd_cryo_dk623
RT: 16.23 - 17.73 SM: 5G
50
60
70
80
90
100
Rela
tive A
bundance
0
10
20
30
40
50
60
70
80
90
100
RT: 17.64AA: 118578
RT: 17.62AA: 1623
RT: 17.64AA: 70077
NL:2.13E5
m/z= 339.8409-339.8749 MS ICIS 1207_pcdd_cryo_dk624
NL:1.28E5
m/z= 341.8399-341.8741 MS ICIS 1207_pcdd_cryo_dk624
RT: 17.51 - 17.83 SM: 5G
50
60
70
80
90
100
Rela
tive A
bundance
0
10
20
30
40
50
60
70
80
90
100
RT: 17.64AA: 118578
RT: 17.64AA: 70077
NL:2.13E5
m/z= 339.8409-339.8749 MS ICIS 1207_pcdd_cryo_dk624
NL:1.28E5
m/z= 341.8399-341.8741 MS ICIS 1207_pcdd_cryo_dk624
Dim 1 Dim 2 Dim 2
PCDFs trapped
together and reinjected
56
16.5 17.0 17.5
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
Rela
tive A
bundance
RT: 16.46AA: 6520812
RT: 17.21AA: 6193921
NL:1.95E6
m/z= 351.8824-351.9176 MS ICIS 1207_pcdd_cryo_dk623
16.5 17.0 17.5
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50R
ela
tive A
bundance
RT: 17.64AA: 6768311
RT: 17.61AA: 105529
NL:1.22E7
m/z= 351.8824-351.9176 MS ICIS 1207_pcdd_cryo_dk624
17.6 17.7 17.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
Rela
tive A
bundance
RT: 17.64AA: 6768311
17.67
NL:1.22E7
m/z= 351.8824-351.9176 MS ICIS 1207_pcdd_cryo_dk624
together and reinjected
on Dim. 2
• NTC resistor positioned in the cryo jet and connectet to a digital meter
• Jet temperature at the RTJet (peakfront) 195.25°C
Method development – temperature profiles
• Determination of the trapping-temperature
57
Jet on Jet off
Fast GC experiments for narrow peaks – here: 5 m column
RT: 4.34 - 8.16
60
80
100
Re
lative
Ab
un
da
nce
20
40
60
80
100
RT: 5.77AA: 25376796
RT: 6.21AA: 19945764
RT: 6.73AA: 12244407
RT: 7.38AA: 5598786
RT: 5.01AA: 26360860
RT: 5.45AA: 19065901
RT: 5.84
NL:2.90E7
TIC MS ICIS 1204_5m018_pcddf114
NL:3.61E7
TIC MS ICIS 1204_5m018_pcddf117
Ramp 30 ºC/min
Ramp 40 ºC/min
58
4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
Time (min)
20
40
60
80
100
20
40
60
Re
lative
Ab
un
da
nce
RT: 5.84AA: 11185391
RT: 6.63AA: 4432709
RT: 4.91AA: 19089855
RT: 5.33AA: 11012244
RT: 5.67AA: 6168403
_pcddf117
NL:3.62E7
TIC MS ICIS 1204_5m018_pcddf121Ramp 50 ºC/min
Column: Restek RXI 5 Sil MS 5 m x 0.18 mm (0.18 um)
Thx for your help, Jack!
Fast GC – peak width “tuning”
RT: 5.70 - 5.86
20
40
60
80
100
Re
lative
Ab
un
da
nce
RT: 5.77AA: 25443419
RT: 5.77
NL:2.90E7
m/z= 317.8390-318.0390 MS ICIS 1204_5m018_pcddf114
NL:
TCDF Column:
Restek RXI 5 Sil MS
5 m x 0.18 mm (0.18 um)
Ramp 30 ºC/min
PW (at 10 %) = 1.5 sec
59
5.70 5.72 5.74 5.76 5.78 5.80 5.82 5.84 5.86
Time (min)
20
40
60
80
100
RT: 5.77AA: 24060076
NL:3.62E7
m/z= 317.8390-318.0390 MS ICIS 1204_5m018_pcddf121
TCDF
Ramp 50 ºC/min
PW (at 10 %) = 1.08 sec
to overlay:
RT + 1.25
Serum samples from CDC – dilution of reference samples
61
- dilute contaminated serum with blank matrix (bovine serum)
- prepare defined TCDD, PCDF, PCDD serum sample levels this way
Serum sample TCDD - 1ul injectedRT: 14.20 - 14.59
50
60
70
80
90
100
Rela
tive A
bundance
0
10
20
30
40
50
60
70
80
90
100
RT: 14.54MA: 173
14.4414.32
14.3414.2614.23 14.3814.57
14.27 14.4614.39
14.47
RT: 14.54MA: 151
14.53
14.3514.52
14.25
NL:3.71E2
m/z= 319.8810-319.9130 MS 1207_serum_1021
NL:3.94E2
m/z= 321.8779-321.9101 MS 1207_serum_1021
RT: 14.20 - 14.59
50
60
70
80
90
100
Rela
tive A
bundance
0
10
20
30
40
50
60
70
80
90
100
RT: 14.48MA: 387
14.47
14.53
14.27 14.41 14.5114.23 14.4314.29 14.4014.32
14.54
RT: 14.48MA: 474
NL:7.73E2
m/z= 319.8810-319.9130 MS 1207_serum_1022
NL:1.09E3
m/z= 321.8779-321.9101 MS 1207_serum_1022
8A – 1.25 fg/ul 6A – 5 fg/ul
62
14.3 14.4 14.5
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
Rela
tive A
bundance
14.58
14.4114.3214.31
14.3914.46
14.22
RT: 14.54AA: 967532
NL:2.42E6
m/z= 333.9173-333.9507 MS ICIS 1207_serum_1021
14.3 14.4 14.5
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
Rela
tive A
bundance
14.23 14.47
14.46 14.52 14.5314.24 14.35 14.4214.30
RT: 14.48AA: 1087219
RT: 14.50AA: 8814
NL:2.57E6
m/z= 333.9173-333.9507 MS ICIS 1207_serum_1022
Blanks – the real limitation?
Instrumental analysis push to improve
63
Sample preparationpush to improve
Conclusions & Outlook:
In general: - t-CZC = sensitivity „booster“
- basically on any GC, preferable: selective detector
- selected applications / target analytes
Features & Advantages: - simplicity & flexibility
- low consumption of CO2
65
Conclusions & Outlook:
In general: - t-CZC = sensitivity „booster“
- basically on any GC, preferable: selective detector
- selected applications / target analytes
Features & Advantages: - simplicity & flexibility
- low consumption of CO2
Current status (DFS): - sensitivity ca. 700 - 1000 ag (2378-TCDD, absolute amount)
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Next step: - transfer method to customer lab (CDC Atlanta, US)
Conclusions & Outlook:
In general: - t-CZC = sensitivity „booster“
- basically on any GC, preferable: selective detector
- selected applications / target analytes
Features & Advantages: - simplicity & flexibility
- low consumption of CO2
Current status (DFS): - sensitivity ca. 700 - 1000 ag (2378-TCDD, absolute amount)
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Next step: - transfer method to customer lab (CDC Atlanta, US)
…other fields of application?
shortlong
CO2 Jet
MS
Injector
2nd Dimension1st Dimension
GCxGC – switch on/off different modes as needed?
Ability to switch on different modes just
as needed throughout a chromatogram:
1.) „normal“ sections – jet off
2.) CZC for increased sensitivity
3.) CZC for additional sep. on 2d dim
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3.) CZC for additional sep. on 2d dim
4.) full GCxGC sectionsunpolar polar
(low bleed)