hplc 2013 - impact of particle designs for achieving ... · © 2012 sigma-aldrich co. all rights...

© 2012 Sigma-Aldrich Co. All rights reserved.

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Impact of Particle Designs for Achieving Performance in Liquid Separations

David S. BellHPLC 2013


2

Introduction

In recent years, a major driving force in HPLC innovation has been the desire for faster method development and analysis

To achieve fast analysis, resolution must be maintained while other parameters such as flow rate or column length are optimized

Since resolution is, in part, dependent on theoretical plates, or ‘efficiency,’ one major trend in the industry has been to utilize smaller and smaller porous particles

4N

Efficiency SelectivityRetention

R=k’

k’+1-1

. .


3

Column Efficiency Increases with Smaller Particles

Theoretical Curves for Porous Silica

CuuBAH

A-term: relates to the uniformity of the packed bed

B-term: describes molecular diffusion along the column axis

C-term: contains all terms relating to radial mass transfer


4

Particle Size Pressure N ~R

1.8 6000 27,000 1.64

3 2100 18,000 1.34

5 800 10,000 1

10 200 5,000

100 mm column, 3 mm/s linear velocity

pdN 1

2

1

pdP

Something more sophisticated is needed than just continuing to reduce particle size.

Pressure Rises Faster than Efficiency and Resolution when Particle Size is Reduced

Performance of Porous Particles


5

Fused Core Technology

• Another approach to reducing dispersion (increasing efficiency) is shown above• By placing a solid core at the center of a particle, the potential diffusion path length of an analyte molecule is effectively shortened

• A larger particle then can theoretically generate similar efficiency of a smaller totally porous particle without generating high backpressures


Efficiency comparison between fully porous and Fused Core 5 mm columns

250x4.6 mm 5 µm columns. Test compound: benzophenone, mobile phase ACN/H20, k' = 6.2 (isoelutropic conditions)

5

10

15

20

25

0 2 4 6 8

Supelco 5mu fitSupelco 5mu dataAgilent 5mu dataSeries4

u0 (mm/s)

H (µm)

K. Broeckhoven, D. Cabooter, G. Desmet., Kinetic performance comparison of fully and superficially porous particles with sizes ranging between 2.7 µm and 5 µm: Intrinsic evaluation and application to a pharmaceutical test compound, J. Pharm. Anal. (2013)

Fully Porous

Fused-Core

Hmin = 9.9µm

Hmin = 7.2µm


0.1

1.0

10.0

100.0

1000.0

10000 100000 1000000

2.7 mu 600 bar data2.7 mu 600 bar fit5mu supelco data5 mu supelco fit3.5 mu Agilent data3.5 mu Agilent fit5mu Agilent data5mu Agilent fitSupelco 2.7mu dataSupelco 2.7mu fit

Kinetic Plot - BenzophenoneIn addition to fully porous and Fused-Core 5µm particles, a fully porous C18 3.5 µm (4.6x100mm) and a Fused-Core 2.7 µm (2.1x100mm) were tested under the same conditions (isoelutropic) and the kinetic plots constructed for ∆Pmax = 400 bar

=> 5µm Fused-Core outperforms 3.5 and 5µm fully-porous particles over entire range

5µm,FC

2.7µm,FC

5µm3.5µm

2.7µm,FC 600 bar

Benzophenonek' = 6.2

t0 (min)

N

K. Broeckhoven, D. Cabooter, G. Desmet., Kinetic performance comparison of fully and superficially porous particles with sizes ranging between 2.7 µm and 5 µm: Intrinsic evaluation and application to a pharmaceutical test compound, J. Pharm. Anal. (2013)


Desirability of Monodisperse Particles in HPLC

8

• Desmet, et. al. (1) observed a strong trend between narrow silica particle size distribution (PSD) and good column performance.

• Particles with standard deviation range between 5 and 20% PSD showed a near linear relationship between the A-term constant, hmin value, and minimum separation impedance.

• Desmet’s findings supported previous observations that porous-layer particles with very narrow PSD demonstrate superior efficiency and kinetic performance.

• Results suggest that performance of columns with fully porous particles might be significantly improved by very narrow PSD and become the new standard.

• A commercial process called Ecoporous™ was developed to produce silica particles with ca. 6% standard deviation that would not require further sizing.

1. D. Cabooter, A. Fanigliulo, G. Bellazzi, B. Allieri, A. Rottigni, G. Desmet, J. of Chromatography A, 1217 (2010) 7074–7081.


Monodisperse Particle Advantages

During our (and others) studies of Fused-Core particles, there always seemed to be an extra reason beyond the short diffusion path when trying to fully explain the increase in efficiency obtained using Fused-Core particles.

One possible theory was that the inherent monodispersity of the Fused-Core particles may be providing a significant contribution toward efficiency through:

improved packing of the bedlower eddy diffusion due to more uniform paths

To begin to study this more closely, an economical process was developed to produce monodisperse, fully porous particles.


Ecoporous™ Silica Process Creates Very Narrow Particle Size

10

Ds (90/10) = particle size at 0.9 divided by particle size at 0.1; scale units arbitrary.

Ds at 0.1 Ds at 0.9

New Ecoporous process results in very narrow distribution (D90/10 < 1.15) without additional sizing.


0

2

4

6

8

10

12

14

16

0 1 2 3 4 5 6

number

count

(

%)

Particle Size (m)

Particle Size Distribution Data for Silicas

Size Distribution Comparison for Modern Silicas

11

Titan 1.9

Fused-Core 2.7

Fused-Core 5

1.9 m porous (Titan)1.8 m porous2.7 m Fused‐Core3 m porous5 m Fused‐Core5 m porous


A New Line of Monodisperse, Fully PorousSub-2 µm Columns

• A new monomeric C18 chemistry has been constructed using the new particle design and dubbed Titan™.

Particle Size1

µmPore Diameter

ÅSurface area

m2/gPore Volume

cc/g

1.9 80 410 0.76

Titan Porous Silica Characteristics


Particle Size Distribution Comparison – Sub-2 µm Porous Particles

13

0

2

4

6

8

10

12

14

0 0,5 1 1,5 2 2,5 3 3,5 4

incrementalnumber(%)

Particle Size (um)

Titan (1.9um)

Shimadzu (1.6um)

Agilent (1.8um)

Waters (1.7um)

Daiso (1.7um)

Sepax (1.8um)

* Particles removed from commercial columns.

Highlights:‐ Elimination of wasteful silica

sizing process‐ High purity Silica at Lower

cost‐ Potential to improve

performance across HPLC & UHPLC


0

4

8

12

16

20

0 1 2 3 4 5 6

u0 (mm/s)

H (µ

m) Ascentis 2.7µm

Poroshell - Agilent 2.7µmWaters XP 2.5µmTitan - 1.9µmWaters BEH 1.7µm

Plate height measurements

Butyrophenone, k' = 7 (ACN ~ 40%, adjusted for each column), isocratic separation. (New 1.9 monodisperse, 2.7 Fused Core, 1.7 fully porous and 2.5 fully porous)

Improved efficiency over 1.7 FP, lowest H

Data courtesy of Ken Broeckhoven, Vrije Universiteit Brussel


Evaluation of Titan Pressure Drop

15

0

150

300

450

600

750

0 1 2 3 4 5 6

u0 (mm/s)

Pres

sure

(Bar

)

Ascentis Express 2.7 µm Poroshell - Agilent 2.7 µmWaters XP 2.5 µmTitan - 1.9 µmWaters BEH 1.7 µm

Lower P for Titan compared to similar sized particles: Kv0(velocity based permeability ) is 25% higher than Waters BEH and only 7 % lower than2.5 µm XP column



Figure 8: Evaluation of Titan Kinetic Performance Limits

16

0.1

1.0

10.0

100.0

10000 100000 1000000N (/)

t 0(m

in) Ascentis 2. .7 µm (Express)

Waters XP 2.5 µmPoroshell - Agilent 2.7 µmTitan - 1.9 µmWaters BEH 1.7 µm

Kinetic plot limit for Pmax = 600 bar (Ascentis® Express, Poroshell, Waters XP)and Pmax = 1000 bar (Titan and Waters BEH) Butyrophenone, k' = 7 (ACN ~ 40%, adjusted for each column), isocratic separation. (New 1.9 monodisperse, 2.7 Fused Core, 1.7 fully porous and 2.5 fully porous

Titan offers best kinetic performance over entire t0 or N range for Pmax = 1000 bar



Titan C18 1.9µm Column Efficiency and Asymmetry

1.0 2.0

NB= 13,573N/m = 271,460h= 1.90= 1.03Pressure*: 3,100psi

50 x 2.1mm0.4mL/min

= 3.29mm/s

1.0 2.0

NN= 14,710N/m = 294,200h= 1.75= 1.05Pressure*: 4,100psi

50 x 3.0mm0.9mL/min

= 4.07mm/s

2

1.0 2.0Time (min)

NN= 15,220N/m = 304,400h= 1.69= 1.02Pressure*: 6,030psi

50 x 4.6mm2.4mL/min

= 4.50mm/s

1

3

45

Dionex 3000Mobile Phase: 60% AcetonitrileTemp: 35CFlow Rate: As IndicatedDetection: 254nmLow D Connecting TubingInlet: 35cm X 75mOutlet: 35cm X 75m

1.Uracil2.Diazepam3.Toluene4.Naphthalene5.Biphenyl

Titan C18 Performance at Different Column ID

17* Total System Pressure


Waters BEH C18 1.7um

Agilent C18 Eclipse Plus 1.8um

Titan C18 1.9um

Dionex 3000 (Low D tubing)Column: 50 X 3.0mmMobile Phase: 60% AcetonitrileTemp: 35CFlow Rate: 0.9mL/min (4 mm/s)Detection: 254nm

1.Uracil2.Diazepam3.Toluene4.Naphthalene5.Biphenyl

NNap= 9,783Nnap/m= 195,700 Pressure*= 4,900psi

NNap= 9,260NNap/m= 185,200Pressure*= 4,650psi

Titan C18 Performance Comparison in ACN

18

1.0 2.0

NNap= 14,710N/m = 294,200Pressure*= 4,100psi

1.0 2.0

1.0 2.0

1

23

4 5

* Total System Pressure


0 2 4 6 8Time (min)

0.46

3

2.52

3

2.93

1

3.34

93.

473

4.64

14.

847

5.30

6

5.56

5

7.78

8

TolueneD8

H8

Naphthalene

D8

H8

P-Xylene

D10

H10

Dionex 3000 (low D tubing)Column: Titan C18 1.9mmDimension: 100 x 2.1mmMobile Phase: 50% ACNTemperature: 35CFlow Rate: 0.4mL/minDetection: UV / 254nmPressure: 5650 psi/390 bar

Dia

zepa

m

N,N

-Dim

etyl

anili

ne

Bip

heny

l

N = 25,530N/m = 255,000

Titan C18: High Speed and High Resolution

19

Deuterated isomers separation


20

Control Responses for Antineoplastic Analytesfrom Plasma on Titan C18 across 1000 injections

Control concentration of 300ng/mL

050

100150200250300350400

0050

1300

18.d

0050

1300

99.d

0050

1301

83.d

0050

1302

67.d

0050

1303

73.d

0050

1304

44.d

0050

1305

28.d

0050

1306

12.d

0050

1307

08.d

0050

1307

89.d

0050

1308

73.d

0050

1309

57.d

0050

1310

53.d

fluoxetine QC Response

fluoxetine QCResponse

050

100150200250300350400

0050

1300

18.d

0050

1300

99.d

0050

1301

83.d

0050

1302

67.d

0050

1303

73.d

0050

1304

44.d

0050

1305

28.d

0050

1306

12.d

0050

1307

08.d

0050

1307

89.d

0050

1308

73.d

0050

1309

57.d

0050

1310

53.d

endoxifen QC Response

endoxifen QCResponse


Conclusions

21

• A new process called Ecoporous has been developed for making porous silica that matches the narrow size distribution of Fused-Core particles; no extra sizing step is required; no silica is wasted; a new standard has been established.

• Particles with 80 Å pores and 410 m2/g have been prepared in 1.9 µm with a 6% standard deviation in PSD.

• Efficiency matches or exceeds porous particles of 1.7 and 1.8 µm size while pressure drop for the larger Titan particle is lower.

• Uniform Titan particles pack into rugged column beds that are stable over a range of UHPLC flow and pressure conditions.

• Excellent batch reproducibility and robustness is observed.


References and Acknowledgements

22

1. D. Cabooter, A. Fanigliulo, G. Bellazzi, B. Allieri, A. Rottigni, G. Desmet, J. of Chromatography A, 1217 (2010) 7074–7081.

2. M. R. Euerby and P. Petersson, J. of Chromatography A, 994 (2003) 13–36.

The assistance of Ken Broeckhoven, Vrije Universiteit Brussel, Department of Chemical Engineering, Belgium is appreciated for performing a kinetic evaluation of columns with 1.9 µm Titan C18 particles.

William Campbell, Bill Betz, Craig Aurand, Hillel Brandes, Carmen Santasania, Gaurang Parmar, Richard Henry

Ascentis® is a registered trademark of Sigma-Aldrich Co. LLC; Titan™ and Ecoporous™ are trademarks of Sigma-Aldrich Co. LLC; Fused-Core® is a registered trademark of Advanced Materials Technology, Inc.

hplc 2013 - impact of particle designs for achieving ... · © 2012 sigma-aldrich co. all rights...

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