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

Hernan J. Cortes. CPAC SI. July, 2010

Hernan J. CortesHernan J. Cortes Consulting, LLC.

Midland, MI. [email protected]

fast LC has become a major research area in academia and industry

governed by progress in column design (sub 2 m 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 m 1.8 mm)

First two peaks are broadened – solvent effect (injection of MeCN solution in MeCN/H2 O 80/20ZorbaxTM 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


Separation of Styrene-Acrylonitrile copolymers with different monomer ratios on ZorbaxTM C18 XDB Eclipse (4.6 x 50 mm, 1.8 um)

A mobile phase gradient from acetonitrile to dichloromethane was used and excellent linearity was observed. Some carry-over is visible.

min0.5 1 1.5 2 2.5 3 3.5

37.5% AN32% AN

25% AN15% AN

0% AN (=PS)

R2 = 0.9994

05

10152025303540

1 1.5 2 2.5 3 3.5

retention time (min)

% A

N in

SA

N


Separation of NIST SRM 869a executed on 1.8 m ZorbaxTM SB C18 (15 cm length)

Good separation efficiency and peak shape

N (TBN) = 37000

~ 250 000/m

TBNPhPh

BaP

min1 2 3 4 5 6 7


High-resolution chromatograms were generated in Pat Sandra‘s group using a series of 5 m columns with efficiencies of 100,000-200,000 plates

Coupling concept has been applied to 1.8 m particle columns (ZorbaxTM C18 SB).

As expected column efficiency of coupled columns is below theoretical values

Still, 70000 plates are very powerful for difficult separations such as oligomers, formulations, or bioanalytical applications

0

20000

40000

60000

80000

100000

120000

0 100 200 300 400 500

total column length (mm)

Plat

e nu

mbe

r (N

)Theoretical data

Experimental data

Sub-2 m 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


min0 2 4 6 8 10 12 14

mAU

0

10

20

30

40

50

60

70

Ascentis ExpressTM C18 2.7 m (4.6x150 mm)

p = 180 bar (starting), 60C

ZorbaxTM C18-SB 1.8 m (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


min0 2 4 6 8 10

2

1

4

35

6 78

9

1011 12

13 1415

N/m = 150000

33 minutes 12 minutes33 minutes 12 minutes

Ascentis ExpressTM C18 (4.6 x 100 mm)


aromatic polyamines (5% in MeCN)

2 x 150mm Ascentis ExpressTM

columns

backpressure 320 bar at 60C and 1.2 ml/min

about 55000 plates – still need for better resolution to determine select compounds at 10-50 ppm levels

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 will delay the need for ultra-high pressure (1000+ bar)

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



Comparison of Throughput Between Conventional GC (CGC) and LTMGCLTMGC: 2 metre, 0.1 mm id, 0.12 micron DB-1 Column Technology

LTMGCEXT: Extended run time to remove wax

0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

CGC LTMGCF LTMGCEXT

GC Technology

Tim

e (m

inut

es)

Cool down timeRun timeautosampler

•

Ultra fast temperature programming: rapid heating (up to 1800°C/min and cool-down times (up to 400°C/min) provide extremely short cycle times (3-4 min).

• Breakthrough technology for gas chromatography.


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)



Ddkf

C p2)(

CuuBAH /Sub-2 µm particle (dp

)

Fused core (dp)

Monolith (C)

Temperature (D)

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


solvent delivery

LTM module with embedded LC capillary

injectorUV detector


Inlet frit

Micro-column

LTM assembly

Ending frit


heated tubing (~40 cm length)

250 m i.d. fused silica column

from injector

to DAD (300 mm x 50 m)


min0 2 4 6 8 10 12 14 16 18

grad. 40-150°C (30°C/min)

grad. 40-90°C (25°C/min)

40°C isothermal

25°C isothermal

C1

C4

C6 C8


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

.


0 4 8 12 16 20

400

800

1200

1600

Res

pons

e (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

y = 819.44x - 3.4064R2 = 0.9976

y = 1327.5x - 2.5299R2 = 0.9974

-2.00

-1.50-1.00

-0.500.00

0.50

1.001.50

2.00

0.00220 0.00270 0.00320 0.00370

1/T (1/K)

lnk

ln1ln

RS

TRHk



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

Res

pons

e (m

V)


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

Res

pons

e (m

V)


A

B

C

D

E

F

G


0 4 8 12 16 20200

300

400

500

600R

espo

nse

(mV

)


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

0 2 4 6 8 10 12 14 16200

250

300

350

400

450

500

25oC

100oC100oC

100oC

50oC

C

B

Resp

onse

(mV

)


A 1 2

3+45

6 7+8

1 23 4

5

67 8

1 23 4

5

6 78

25oC

25 °C

50 °C

75 °C

100 °C

0 4 8 12 16

300

400

500

600

Resp

onse

(mV

)


A

B

C

D

1 23 4

56 8 7

12+3 4

56

78

12+3

45

67 8

12+3

45

6+78

25 °C

50 °C

75 °C

100 °C

• Differences in selectivity are observed for sulfinpyrazone

(6), 4-phenylphenol (7), and terfenadine

(8)

• Oscillating gradients (T increas/decrease) can be used to fine tune a separation



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

400R

espo

nse

(mV

)


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

Res

pons

e (m

V)


A

B

C

D

E 100 oC/min

75 oC/min

40 oC/min

20 oC/min

25 oC

isothermal


0.0 0.5 1.0 1.5

340

360

380

400

Res

pons

e (m

V)


100 oC/min

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


LTMLC was realized by the combined use of a mini-assembly and a Micro LC column, both of which have low thermal masses.

LTMLC provides reliable heating and cooling capability.

Very fast temperature gradients (both increasing and decreasing)

can be applied.

Oscillated temperature gradient was demonstrated for fine tuning

separation.

Ultra-fast and reproducible LTMLC was also demonstrated.

Utility as 2nd

dimension in LCxLC, monoliths, fused-core particles.

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


M. Pursch, B. Gu, P. Eckerle, J. Luong.

TDCC


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

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