Hernan J. Cortes Hernan J. Cortes Consulting, LLC. Midland...

Hernan J. Cortes. CPAC Summer Institute, 2011

Hernan J. Cortes Hernan J. Cortes Consulting, LLC.

Midland, MI. USA University of Tasmania, Australia

[email protected]

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


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



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


Key benefits: 1. Suitable for fast moving molecules 2. Doesn’t require cryogen 3. In-Oven with no moving parts


Augsburg aerosol sample

Vogt, L., Groeger, T., Zimmermann, R., J. Chromatogr. A 2007, 1150, 2 – 12.


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



Ddkf

C p2)(

=

CuuBAH ++= /Sub-2 µm particle (dp)

Fused core (dp)

Monolith (C)

Temperature (D)


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


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


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



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


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


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


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)


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


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


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


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


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


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


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


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


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


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.


Ideal attributes for fast GC Low power consumption Rapid cooling Fast heating


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)


Capillary column 0.25 mm vs 4.6 mm i.d. (300 time lower in mass)

Housing and end-fittings

LTM assembly


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


Inlet frit

Micro-column

LTM assembly

Ending frit


heated tubing (~40 cm length)

250 µm i.d. fused silica column

from injector

to DAD (300 mm x 50 µm)


Wolcott et al. J. Chromatogr. A 2000, 869, 211-230.


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


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


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)


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)


A

B

C

D

E

F

G


0 4 8 12 16 20200

300

400

500

600Re

spon

se (m

V)


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)


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)


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


0.0 0.5 1.0 1.5

400

500

600

700

800

Resp

onse

(mV)


A

B

C

D

E 100 oC/min

75 oC/min

40 oC/min

20 oC/min

25 oC isothermal

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


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)


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


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


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.


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.


Matthias Pursch, Patric Eckerle , Binghe Gu, and Jim Luong. TDCC

Robert Shellie, Emily Hilder, Tim Causon. UTAS


Hernan J. Cortes Hernan J. Cortes Consulting, LLC. Midland...

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