Post on 14-Jul-2020
Hernan J. Cortes. CPAC Summer Institute, 2011
Hernan J. Cortes Hernan J. Cortes Consulting, LLC.
Midland, MI. USA University of Tasmania, Australia
hjcortes1@gmail.com
Synge, Nobel Lecture 12/12/1952 Hernan J. Cortes. CPAC Summer
Institute, 2011
Synge, Nobel Lecture 12/12/1952 Hernan J. Cortes. CPAC Summer
Institute, 2011
Synge, Nobel Lecture 12/12/1952 Hernan J. Cortes. CPAC Summer
Institute, 2011
Synge, Nobel Lecture 12/12/1952 Hernan J. Cortes. CPAC Summer
Institute, 2011
Synge, Nobel Lecture 12/12/1952 Hernan J. Cortes. CPAC Summer
Institute, 2011
real-world samples are normally very complex mixtures, containing hundreds and sometimes thousands of 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 Hernan J. Cortes. CPAC Summer
Institute, 2011
Hernan J. Cortes. CPAC Summer Institute, 2011
Peak Capacity
n = 1 + N ½ ln 1 + k’
r
N = theoretical plates
r = standard deviations equaling peak width (4)
k’ = capacity factor of the last peak in a series
J.C. Giddings, Anal. Chem. 53 (1983) 418
P= n α e-α Number of visible peaks P = 0.37 Number of single component peaks S = 0.19
Hernan J. Cortes. CPAC Summer Institute, 2011
Hernan J. Cortes. CPAC Summer Institute, 2011
Comprehensive Two Dimension Gas Chromatography
vs traditional heartcutting
Comprehensive First Dimension
Transfer Device
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
1st Col (min)
2nd Col(sec)
Constant temperature bath (GC) and valves Column arrangement inside bath (GC)
Simmons and Snyder, Anal. Chem., Vol. 30, No. 1, January 1958, pp 32-35 Hernan J. Cortes. CPAC Summer
Institute, 2011
Hernan J. Cortes. CPAC Summer Institute, 2011
Key benefits: 1. Suitable for fast moving molecules 2. Doesn’t require cryogen 3. In-Oven with no moving parts
Hernan J. Cortes. CPAC Summer Institute, 2011
Augsburg aerosol sample
Vogt, L., Groeger, T., Zimmermann, R., J. Chromatogr. A 2007, 1150, 2 – 12.
Hernan J. Cortes. CPAC Summer Institute, 2011
commercial perfume
d'Acampora Zellner, B., Casilli, A., Dugo, P., Dugo, G., Mondello, L., J. Chromatogr. A 2007, 1141, 279 – 286
fast LC has become a major research area in academia and industry
governed by progress in column design (sub 2 µm particles) and availability of higher pressure pumps (>> 400 bar)
Analysis times for many applications could be reduced by factor 5-10
Also, with the availability of longer columns packed with smaller diameter particles, it is possible to do high-resolution LC experiments
More resolution is needed for very complex samples, such as oligomers or biological samples
Hernan J. Cortes. CPAC Summer Institute, 2011
Hernan J. Cortes. CPAC Summer Institute, 2011
Ddkf
C p2)(
=
CuuBAH ++= /Sub-2 µm particle (dp)
Fused core (dp)
Monolith (C)
Temperature (D)
Hernan J. Cortes. CPAC Summer Institute, 2011
High-speed separation of inhibitors at 80C (ZorbaxTM C18 XDB, 50x4.6 µm 1.8 mm)
First two peaks are broadened – solvent effect (injection of MeCN solution in MeCN/H2O 80/20 ZorbaxTM is a registered trademark of Agilent Technolgies
min0 0.2 0.4 0.6 0.8 1
OH
OH
CH3
OH
OH
CH3
O
OH
CH3O
OH
CH3
OH
OH
OH
OH
OH
R2
R1
OH
R2
R1
NH
S
NH
S
Hernan J. Cortes. CPAC Summer Institute, 2011
min0.3 0.4 0.5 0.6 0.7 0.8 0.9
1
3
4
5
6
7
8
2
9 10
1112
Resin 4Resin 3
Resin 2
Resin 1
Separations executed on 1.8 µm silica column (ZorbaxTM Rx-Sil, 4.6x100 mm)
Critical LC is powerful to monitor functionality type distribution (epoxy / epoxy-, epoxy / phenolic- end groups, branching, etc.)
12 species separated in less than 1 min run time – ideal for LCxLC
O O 3 O O 3
O O H
O H 12 O
O H
O H 12
6 O
O H
6 O
O H
O
O H
• H. J. Cortes, L. Mondello, D. West, S. Maynard, et al. “Anal Chem. 81 (2009) 4271-4279
Hernan J. Cortes. CPAC Summer Institute, 2011
Separation of NIST SRM 869a executed on 1.8 µm ZorbaxTM SB C18 (15 cm length)
Good separation efficiency and peak shape
Sub-2 µm columns have received significant competition from other column manufacturers (partially porous silica, other 2-3 um columns with low backpressure)
In few cases column plugging can be observed which results in an increase of column back-pressure
Fused Core (partially porous) columns thus far have shown excellent performance
Such columns are less susceptible to back-pressure increase/plugging
Hernan J. Cortes. CPAC Summer Institute, 2011
Hernan J. Cortes. CPAC Summer Institute, 2011
From Joseph J. Kirkland and Timothy J. Langlois, US patent 2007,0189944 A1
2.7-µm fused-core
1.8-µm totally porous
2.7-µm fused-core
1.8-µm totally porous
Particle size 2.7 µm - 1.7 µm solid core- 0.5 µm porous layer
Reduced mass transfer path length, reduced resistance of mass transfer
Hernan J. Cortes. CPAC Summer Institute, 2011
min1 2 3 4 5 6 min1 2 3 4 5 6
NH
NH
O
O
NH
NH
O
O
CH3CH3CH3CH3
O
O
OH
OH
O
O
OH
OH
N CH3CH3
N CH3CH3
SRM870 separation on Ascentis ExpressTM C18 (4.6 x 150 mm)
very low metal content, very low silanol activity
good retention properties (T/EB) Ascentis ExpressTM is a registered trademark from Supelco
Hernan J. Cortes. CPAC Summer Institute, 2011
min 0 2 4 6 8 10 12 14
mAU
0
10
20
30
40
50
60
70
Ascentis ExpressTM C18 2.7 µm (4.6x150 mm)
p = 180 bar (starting), 60C
ZorbaxTM C18-SB 1.8 µm (4.6x100+50mm)
p = 250 bar (starting), 80C
n = 0
n = 1
n = 2
O O O
OOO
n
p,p o,p
o,o
flow= 1.4 ml/min
Hernan J. Cortes. CPAC Summer Institute, 2011
min 0 2 4 6 8 10
2
1
4
3 5
6 7 8
9
10 11 12
13 14 15
N/m = 150000
33 minutes 12 minutes 33 minutes 12 minutes
Ascentis ExpressTM C18 (4.6 x 100 mm)
Hernan J. Cortes. CPAC Summer Institute, 2011
Batch 49
Batch 63
Batch 52
Batch 68
min 0 2 4 6 8 10
mAU
0
10
20
30
40
50
DAD1 A, Sig=250,4 Ref=360,100 (N:\HPCHEM\1\DATA\JAN08\SPIN-C51.D)
0.4
01
0.4
55
0.5
44
0.7
08
0.7
77
0.9
60
1.1
59
1.2
13
1.3
13
1.5
12
1.5
90
1.8
20
1.9
32
2.1
29
2.7
81
3.4
35
3.7
10
3.8
26
4.0
08 4.3
41
5.1
29
5.3
64
7.3
88
10.
076
min 0 2 4 6 8 10
mAU
0
10
20
30
40
50
60
70
DAD1 A, Sig=250,4 Ref=360,100 (N:\HPCHEM\1\DATA\DEC07\SPIN-C41.D)
0.3
98
0.4
44
0.5
25
0.7
26
0.7
63
0.8
15
0.9
11
1.0
18
1.0
98
1.1
51
1.1
93
1.2
48
1.3
03
1.3
62
1.4
33
1.5
51
1.6
63
1.8
30
2.2
50
2.9
69
3.4
63
3.7
79
3.8
82
4.1
41
4.6
74
5.2
22
5.3
80
7.7
85
10.
726
min 0 2 4 6 8 10
mAU
0
20
40
60
80
100
120
140
160
DAD1 A, Sig=250,4 Ref=360,100 (N:\HPCHEM\1\DATA\OCT07\SPIN-C31.D)
0.4
09
0.5
35
0.5
87
0.6
45
0.6
89
0.7
78
0.9
26
0.9
96
1.1
17
1.1
67
1.2
57
1.3
20
1.3
81
1.4
49
1.5
40
1.7
39
1.8
50
1.9
77
2.0
61
2.2
62
2.6
93
2.9
62
3.3
18
3.6
12
3.7
06
3.9
40
4.2
89
4.9
57
5.0
88
5.2
96
6.6
81
7.3
10
10.
013
min 0 2 4 6 8 10
mAU
0
20
40
60
80
DAD1 A, Sig=250,4 Ref=360,100 (N:\HPCHEM\1\DATA\OCT07\SPREP313.D)
0.3
98
0.5
26
0.6
48
0.7
69
0.9
23
0.9
94
1.1
12
1.1
96
1.2
72
1.3
29
1.3
85
1.4
57
1.5
82
1.7
75
1.8
70
2.0
13
2.1
30
2.8
02
3.4
08
3.7
26
3.8
28
4.1
15
4.5
10
5.1
51
5.3
48
5.5
69
7.7
46
10.
680
12+13
3+4
Batch 49
Batch 63
Batch 52
Batch 68
min 0 2 4 6 8 10
mAU
0
10
20
30
40
50
DAD1 A, Sig=250,4 Ref=360,100 (N:\HPCHEM\1\DATA\JAN08\SPIN-C51.D)
0.4
01
0.4
55
0.5
44
0.7
08
0.7
77
0.9
60
1.1
59
1.2
13
1.3
13
1.5
12
1.5
90
1.8
20
1.9
32
2.1
29
2.7
81
3.4
35
3.7
10
3.8
26
4.0
08 4.3
41
5.1
29
5.3
64
7.3
88
10.
076
min 0 2 4 6 8 10
mAU
0
10
20
30
40
50
60
70
DAD1 A, Sig=250,4 Ref=360,100 (N:\HPCHEM\1\DATA\DEC07\SPIN-C41.D)
0.3
98
0.4
44
0.5
25
0.7
26
0.7
63
0.8
15
0.9
11
1.0
18
1.0
98
1.1
51
1.1
93
1.2
48
1.3
03
1.3
62
1.4
33
1.5
51
1.6
63
1.8
30
2.2
50
2.9
69
3.4
63
3.7
79
3.8
82
4.1
41
4.6
74
5.2
22
5.3
80
7.7
85
10.
726
min 0 2 4 6 8 10
mAU
0
20
40
60
80
100
120
140
160
DAD1 A, Sig=250,4 Ref=360,100 (N:\HPCHEM\1\DATA\OCT07\SPIN-C31.D)
0.4
09
0.5
35
0.5
87
0.6
45
0.6
89
0.7
78
0.9
26
0.9
96
1.1
17
1.1
67
1.2
57
1.3
20
1.3
81
1.4
49
1.5
40
1.7
39
1.8
50
1.9
77
2.0
61
2.2
62
2.6
93
2.9
62
3.3
18
3.6
12
3.7
06
3.9
40
4.2
89
4.9
57
5.0
88
5.2
96
6.6
81
7.3
10
10.
013
min 0 2 4 6 8 10
mAU
0
20
40
60
80
DAD1 A, Sig=250,4 Ref=360,100 (N:\HPCHEM\1\DATA\OCT07\SPREP313.D)
0.3
98
0.5
26
0.6
48
0.7
69
0.9
23
0.9
94
1.1
12
1.1
96
1.2
72
1.3
29
1.3
85
1.4
57
1.5
82
1.7
75
1.8
70
2.0
13
2.1
30
2.8
02
3.4
08
3.7
26
3.8
28
4.1
15
4.5
10
5.1
51
5.3
48
5.5
69
7.7
46
10.
680
12+13
3+4
min 0 2 4 6 8 10
mAU
0
10
20
30
40
50
DAD1 A, Sig=250,4 Ref=360,100 (N:\HPCHEM\1\DATA\JAN08\SPIN-C51.D)
0.4
01
0.4
55
0.5
44
0.7
08
0.7
77
0.9
60
1.1
59
1.2
13
1.3
13
1.5
12
1.5
90
1.8
20
1.9
32
2.1
29
2.7
81
3.4
35
3.7
10
3.8
26
4.0
08 4.3
41
5.1
29
5.3
64
7.3
88
10.
076
min 0 2 4 6 8 10
mAU
0
10
20
30
40
50
60
70
DAD1 A, Sig=250,4 Ref=360,100 (N:\HPCHEM\1\DATA\DEC07\SPIN-C41.D)
0.3
98
0.4
44
0.5
25
0.7
26
0.7
63
0.8
15
0.9
11
1.0
18
1.0
98
1.1
51
1.1
93
1.2
48
1.3
03
1.3
62
1.4
33
1.5
51
1.6
63
1.8
30
2.2
50
2.9
69
3.4
63
3.7
79
3.8
82
4.1
41
4.6
74
5.2
22
5.3
80
7.7
85
10.
726
min 0 2 4 6 8 10
mAU
0
20
40
60
80
100
120
140
160
DAD1 A, Sig=250,4 Ref=360,100 (N:\HPCHEM\1\DATA\OCT07\SPIN-C31.D)
0.4
09
0.5
35
0.5
87
0.6
45
0.6
89
0.7
78
0.9
26
0.9
96
1.1
17
1.1
67
1.2
57
1.3
20
1.3
81
1.4
49
1.5
40
1.7
39
1.8
50
1.9
77
2.0
61
2.2
62
2.6
93
2.9
62
3.3
18
3.6
12
3.7
06
3.9
40
4.2
89
4.9
57
5.0
88
5.2
96
6.6
81
7.3
10
10.
013
min 0 2 4 6 8 10
mAU
0
20
40
60
80
DAD1 A, Sig=250,4 Ref=360,100 (N:\HPCHEM\1\DATA\OCT07\SPREP313.D)
0.3
98
0.5
26
0.6
48
0.7
69
0.9
23
0.9
94
1.1
12
1.1
96
1.2
72
1.3
29
1.3
85
1.4
57
1.5
82
1.7
75
1.8
70
2.0
13
2.1
30
2.8
02
3.4
08
3.7
26
3.8
28
4.1
15
4.5
10
5.1
51
5.3
48
5.5
69
7.7
46
10.
680
12+13
3+4
With a few exceptions (high-throughput or 2nd dimension in 2D LC), the desired analysis times for LC for most applications are on the order of 3-5 min
Introduction of new LC columns with reduced backpressure such as partially porous silica may delay the need for ultra-high pressure (1000+ bar) but column reproducibility still an issue.
However, the trend toward higher pressure instrumentation will continue
Hernan J. Cortes. CPAC Summer Institute, 2011
GC oven (massive) Resistive wire heating
in a small “oven” which has very low thermal mass.
Luong, J.; Gras, R.; Mustacich, R.; Cortes, H. J. Chromatogr. Sci. 2006, 44, 253-261.
Hernan J. Cortes. CPAC Summer Institute, 2011
Ideal attributes for fast GC Low power consumption Rapid cooling Fast heating
Hernan J. Cortes. CPAC Summer Institute, 2011
Fast temperature gradients (> 50°C/min) were never studied in LC
Apply LTM concept to LC separations
Requires use of capillary columns (e.g., i.d. of 200 – 300 um, compared to 2.1-4.6 mm for standard columns)
Hernan J. Cortes. CPAC Summer Institute, 2011
Capillary column 0.25 mm vs 4.6 mm i.d. (300 time lower in mass)
Housing and end-fittings
LTM assembly
Hernan J. Cortes. CPAC Summer Institute, 2011
aluminum tube, i.d. of 0.50 mm, o.d. of 0.55 mm
Resistive wire, RTD, insulation fiber, controlled by LTM A68
B. Gu, H. J. Cortes, M. Pursch, J. Luong, P. Eckerle, R. Mustacich. Anal. Chem. 2009. 81 (4), 1488–1495
Hernan J. Cortes. CPAC Summer Institute, 2011
Inlet frit
Micro-column
LTM assembly
Ending frit
Hernan J. Cortes. CPAC Summer Institute, 2011
heated tubing (~40 cm length)
250 µm i.d. fused silica column
from injector
to DAD (300 mm x 50 µm)
Hernan J. Cortes. CPAC Summer Institute, 2011
Wolcott et al. J. Chromatogr. A 2000, 869, 211-230.
Hernan J. Cortes. CPAC Summer Institute, 2011
0 4 8 12 16 20
400
800
1200
1600Re
spon
se (m
V)
Retention time (min)
A
B
C
D
E
F
25 oC
150 oC
125 oC
100 oC
75 oC
50 oC
Column: 250 um x 25 cm; Restek Pinnacle II C18, 5 um
Mobile phase: 60% ACN/0.1% TFA
Column flow rate: 3.0 uL/min
UV: 220 nm
Analytes: neutral and acidic
Hernan J. Cortes. CPAC Summer Institute, 2011
Column efficiency (plates) with the same column flow rate (3 µL/min). 25 oC 50 oC 75 oC 100 oC 125 oC 150 oC
Uracil 12300 13200 14900 12500 11500 9100
Benzoic acid 9300 10000 11400 9800 8100 9200
2,4-D 7800 9000 8900 8500 7000 6100
4-phenylphenol 10000 9800 9100 9000 7700 6700
Ethylbenzoate 11300 11500 10900 9700 7400 6300
Benzophenone 11300 11700 10900 10000 7900 6300
Naphthalene 9700 8800 7500 6600 4600 4000
4-hexylbenzoic acid 8600 8300 7200 5300 3800 3500
Hernan J. Cortes. CPAC Summer Institute, 2011
6 oC/min
12 oC/min
18 oC/min
24 oC/min
50 oC/min
1800 oC/min
100 oC/min
0 4 8 12 16
400
800
1200
1600
Resp
onse
(mV)
Retention time (min)
A
B
C
D
E
F
G 200 oC/min
100 oC/min
6 oC/min
12 oC/min
18 oC/min
24 oC/min
50 oC/min
0 2 4 6 8 10200
400
600
800
1000
1200
1400
1600
1800
Resp
onse
(mV)
Retention time (min)
A
B
C
D
E
F
G
Hernan J. Cortes. CPAC Summer Institute, 2011
0 4 8 12 16 20200
300
400
500
600Re
spon
se (m
V)
Retention time (min)
A
B
C
D
1 2
3+4 5
6 7+8
1 234 5
6 7 8
1234
5
67 8
12
3+4 5
678
Column: 250 um x 25 cm; Zorbax SB C18, 5 um
Mobile phase: 45/55% v/v ACN/40 mM phosphate, pH (2.30)
UV: 220 nm
Analytes: neutral, acidic and basic
100 oC
75 oC
50 oC
25 oC
uracil (1), diphenhydramine (2), benzoic acid (3), nortriptyline (4), dimethylphthalate (5), sulfinpyrazone (6), 4-phenylphenol (7), and terfenadine (8)
Hernan J. Cortes. CPAC Summer Institute, 2011
50 oC for 4.5 min, then ramped to 100 oC at a rate of 12 oC/min, and hold at 100 oC for 3.5 min
0 2 4 6 8 10 12
240
280
320
360
400Re
spon
se (m
V)
Retention time (min)
1 2
3 45
6 7 8
1 23 4
5
6 7 8
100 to 25 oC at a rate of 25 oC/min was used, followed by 1 min hold at 25 oC and then ramped to 100 oC at 25 oC/min
Hernan J. Cortes. CPAC Summer Institute, 2011
0.0 0.5 1.0 1.5
400
500
600
700
800
Resp
onse
(mV)
Retention time (min)
A
B
C
D
E 100 oC/min
75 oC/min
40 oC/min
20 oC/min
25 oC isothermal
Hernan J. Cortes. CPAC Summer Institute, 2011
min 0 1 2 3 4 5 6 7
mAU
0
100
200
300
400
500
600
700
800
Chromolith CapRod RP-18e HR (150 mm x 0.2 mm) from Merck (Darmstadt, Germany) was used
Terphenyl Biphenyl
Uracil
Phthalates
Data:
2.75 uL/min, MeCN/H2O 65/35, 0.10 uL inj.
N Terphenyl: ~25000 (160,000/m)
Very competitive column performance - comparable to ~2.5 um silica particle column
Hernan J. Cortes. CPAC Summer Institute, 2011
Optimum column efficiency is at lower temperature for non-polar compounds Application of fast temperature gradients (increasing & decreasing) should not affect
efficiency dramatically
Terphenyl
0.00050
0.00060
0.00070
0.00080
0.00090
0.00100
0.00110
0.00120
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50
Flow rate [uL/min]
H [c
m]
25C
50C
70C
Flow vs. pressure
0.0
20.0
40.0
60.0
80.0
100.0
120.0
140.0
160.0
180.0
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50
Flow rate [uL/min]pr
essu
re [b
ar]
25C50C70CLinear (25C)Linear (50C)Linear (70C)
Hernan J. Cortes. CPAC Summer Institute, 2011
ambient
50°C
70°C
70°C to ambient at -5°C/min
• Complex sample mixture, containing more than 30 components • Selectivity & resolution needed – T gradients can provide this • Separation efficiency apparently better at higher T for current sample (relatively polar)
• mobile phase gradient (MeCN/buffer) applied throughout all separations
Hernan J. Cortes. CPAC Summer Institute, 2011
25°C isothermal
30°C - 100°C – 30°C at 200 °C/min
• Application of two targeted heat pulses (200°C/min) during a gradient LC separation provides improved separation of selected components
• Facilitates selectivity tuning Hernan J. Cortes. CPAC Summer
Institute, 2011
• Series of four consecutive separations. Good reproducibility for retention times is observed.
bar
Hernan J. Cortes. CPAC Summer Institute, 2011
min 7 7.5 8 8.5 9 9.5 10 10.5
100
200
300
400
500
Norm.
200°C/min (heating)
~ -60°C/min (passive air cooling)
• Cooling rate lags the heating rate significantly
• Still fairly rapid considering passive cooling with surrounding ambient temperature.
Hernan J. Cortes. CPAC Summer Institute, 2011
LTMLC provides reliable heating and cooling capability. Very fast temperature gradients (both increasing and decreasing)
can be applied. Resolution improvement via selected thermal pulses Use of monolithic columns Stationary phase stability LTMLC is a very powerful tool to provide additional selectivity &
speed for LC separations Allows inclusion of ultra-fast temperature gradients for tailored
separations of complex samples Use as second dimension in Comprehensive Multidimensional LC.
Hernan J. Cortes. CPAC Summer Institute, 2011
Matthias Pursch, Patric Eckerle , Binghe Gu, and Jim Luong. TDCC
Robert Shellie, Emily Hilder, Tim Causon. UTAS
Hernan J. Cortes. CPAC Summer Institute, 2011
Hernan J. Cortes. CPAC Summer Institute, 2011