Applications of Sub-2µm Zirconia-PBD Columns at Elevated ...Idealized van Deemter Plots H = A +...

EAS 2009 1

Applications of Sub-2µm Zirconia-PBD Columns at Elevated pH and

Temperature Dan Nowlan, Bingwen Yan, Clayton V. McNeff and

Richard A. HenryZirChrom Separations, Inc. 617 Pierce St., Anoka, MN

55303

EAS 2009 2

Multi-Mode Behavior of Zirconia

• Zirconia substrate exhibits polar and ionic solute interaction: especially cation-exchange.

• With stable organic coatings, reproducible reversed-phase behavior can be added.

• Extreme resistance to temperature, pH and mechanical stress are unique advantages.

Zr

O

Zr

O

Zr

O

Zr

O

Zr

OO O O O O

H2O OHHO

OH OH2OH2

P- O

O

O

O - NH3+

R

EAS 2009 3

Addition of RP Behavior with Coated Zirconia Phases

• Retention (and selectivity) of ionic analytes modulated by pH, buffer/salt type and concentrations, and temperature.

• Retention of neutral solutes modulated by organic solvent.

ZrO O Zr

O O

Zr

O

Zr

O

Zr

O

Zr

O

Zr

OO O O O O O

O

OHOO

H2O OHHO

OH OH2OH2

P- O

O

O

O -NH3+R

Polymer coating

R R NH3+

R

EAS 2009 4

C18 and Zr-PBD are Orthogonal for Basic Drugs2

Data provided by Sigma-Supelco

R2 = 0.373s.d.=0.592

-0.30

-0.10

0.0

0.3

0.50

0.70

0.90

-1.10 -0.60 -0.10 0.40 0.90 1.40log k' (Discovery Zr-PBD)

log

k' (C

18)

1

2,34

5

67

8

9

10

11

25 mM ammonium phosphate (pH 6): methanol (72:28).

Zr-PBD and Si-C18 have very different selectivity for ionic drugs (especially in phosphate) due to the SCX ZrO2 component.

Solutes 1. Chlordiazepoxide 2. Hydroxyzine3. Buclizine4. Thiothixene5. Doxepin6. Amitriptyline 7. Imipramine8. Perphenazine 9. Nortriptyline

10. Desipramine 11. Thioridazine

C18 (RP) columns separate mainly by hydrophobic forces and Zr-PBD columns separate by a combination of ionic and hydrophobic forces

EAS 2009 5

Cation Retention Observed for Zr-PBD Even in High Organic

02468

101214161820

Pyridin

eLid

ocain

ePyri

lamine

Tripele

neam

ine

Methap

yrilen

e

Chlorph

enira

mineThio

thixe

neDox

epin

Bromph

enira

mineAten

olol

Metopro

lolOxp

renolo

l

Amitrypty

line

Imipr

amine

Alpren

olol

Desipr

amine

Nortryp

tyline

Solutes

K'

Si-C18Zr-PBD

LC Conditions: Machine-mixed 80/20 ACN/10 mM ammonium acetate pH=6.7 without pH adjustment; Flow rate, 1.0 mL/min.; Injection volume 0.1 µL; Temperature, 35 oC; Detection at 254 nm; Columns, Zr-PBD, 50 x 4.6 mm i.d. (3 µm particles); Silica-C18 150 x 4.6 mm i.d., (3.5 µm particles).

Basic Pharmaceutical retention in 80% ACN Excess k due mainly to IE mode

EAS 2009 6

Difficult Compounds for Silica Often Separate on Zirconia

Quaternary amines paraquat and diquat are retained and resolved on Zr-PS (also Zr-PBD or bare ZrO2) due to the cation exchange mechanism; 50% ACN is useful to suppress or regulate retention by RP mode.

column: Discovery® Zr-PS, 7.5 cm x 4.6 mm, 3µmmobile phase: 50% acetonitrile in 25 mM phosphate (pH 7)

flow rate: 3 mL/min.temp.: 65 °C

det.: UV 290 nm

Silica-C18:reversed-phase (silanol tailing)

Zirconia-PS:primarily ion-exchange

0 2 4 6 8 10 12 14Time (min)

0.000

0.010

0.020

0.030

AU

0 2 4 6 8 10Time (min)

0.000

0.010

0.020

0.030

0.040

0.050

AU

Light retention and poor peak shape even in low

organic (5% ACN)

+N N+

+N N+

column: Discovery® C18, 15 cm x 4.6 mm I.D., 3µmmobile phase: 5% acetonitrile in 25 mM phosphate (pH 7)

flow rate: 1 mL/min.temp.: 35 °C

det.: UV 290 nm

Data provided by Sigma-Supelco

EAS 2009 7

Analytical Diameter Porous Zirconia Particles

•Particles produced by a sol-gel process with 1000Å sol

•Pore diameter 250-300Å•Density: 2.6 g/cc (2.5X silica) •Surface area: 25 m2/g•Particle diameters: 3µm and sub-2µm

•Totally porous (porosity: 0.45)

EAS 2009 8

Idealized van Deemter PlotsH = A + B/ν + Cν

Elements of the drawing provided by Sigma-Supelco

Plate Height, H (µm)

Flow velocity, ν (mm/sec)

46

20

10

10µm 5µm

3µm

Sub-2µm

Data plots move lower and become flatter for small particles due to combined effects of the equation terms (esp. A and C).

The B term is important at low flows but drops out at high flows.

EAS 2009 9

Flow Studies on 3μm Zr-PBD: Alkylbenzenes

Plate height based on van Deemter Equation vs linear velocity at various temperatures for retained solutes: Alkylbenzenes, Temperature: 30 ºC, Mobile phase: 55/45 ACN/water, Column: ZirChrom®-PBD, 50 x 4.6mm, Agilent 1100/UV with micro cell (0.007’’i.d. tubing).

2 mL/min test conditions

EAS 2009 10

Flow Studies on Sub-2μm Zr-PBD: Alkylbenzenes

Plate height based on van Deemter Equation vs linear velocity for retained solutes: Alkylbenzenes, Temperature 30 ºC, Mobile phase: 50/50 ACN/water (keep k in the same range as 3µm particles), Column: 50 x 4.6mm, Agilent 1100/UV Micro Cell/0.007’’ i.d. tubing.


EAS 2009 11

Sub-2µm Pressure Drop at Different Temperatures*

30 °C

60 °C

Mobile phase: 50/50 ACN/water50x4.6mm column

* 3µm particles show about half the pressure drop

EAS 2009 12

Optimization and Configuration for Elevated Temperature Operation

EAS 2009 13

Background Pressure Drop Across Agilent 1100 at High Flow Rate

603773

392502

211261

BP (bar)Flow (mL/min)

BP (bar)Flow (mL/min)

100% H2O at 75 oC100% H2O at 30 oC

* Reference point: Waters Acquity (0.005” ID inlet/0.0025” ID outlet), 60/40 ACN/water, 0.5 mL/min, background pressure = 1700 psi (113 bar).

EAS 2009 14

Plate Height Vs. Linear Velocity for a PBD Column

0

2

4

6

8

10

12

14

16

18

20

0.03 0.13 0.23 0.33 0.43 0.53

u (cm/s)

Plat

e He

ight

, H (u

m)

Benzene Toluene Ethylbenzene Propylbenzene Butylbenzene

Flow Studies on Sub-2μm Zr-PBD: Factory Instrument

Plate height based on van Deemter Equation vs linear velocity for retained solutes: Alkylbenzenes, Temperature 30 ºC, Mobile phase: 50/50 ACN/water (keep k’ in the same range as 3µm particles), Column: 50 x 4.6mm, Agilent 1100/UV with Standard Cell and 0.007’’ i.d. tubing.


EAS 2009 15


0

2

4

6

8

10

12

14

16

18

20

0.03 0.13 0.23 0.33 0.43 0.53

u (cm/s)

Plat

e He

ight

, H (u

m)


Flow Studies on Sub-2μm Zr-PBD: Factory + Micro Cell Only

Plate height based on van Deemter Equation vs linear velocity for retained solutes: Alkylbenzenes, Temperature 30 ºC, Mobile phase: 50/50 ACN/water (keep k’ in the same range as 3µm particles), Column: 50 x 4.6mm, Agilent 1100/UV with Micro Cell and 0.007’’i.d. tubing.


EAS 2009 16


0

2

4

6

8

10

12

14

16

18

20

0.03 0.13 0.23 0.33 0.43 0.53

u (cm/s)

Plat

e H

eigh

t, H

(um

)


Flow Studies on Sub-2μm Zr-PBD: Micro Cell + Optimized Tubing

Plate height based on van Deemter Equation vs linear velocity for retained solutes: Alkylbenzenes, Temperature 30 ºC, Mobile phase: 50/50 ACN/water (keep k’ in the same range as 3µm particles), Column: 50 x 4.6mm, Agilent 1100/UV with Micro Cell and optimized 0.005’’ i.d. tubing.

1.6 mL/min test conditions

EAS 2009 17


0

2

4

6

8

10

12

14

16

18

20

0.03 0.13 0.23 0.33 0.43 0.53

u (cm/s)

Plat

e H

eigh

t, H

(um

)


Flow Studies on Sub-2μm Zr-PBD: Heat Exchanger + Fitting + μCell

Plate height based on van Deemter Equation vs linear velocity for retained solutes: Alkylbenzenes, Temperature 30 ºC, Mobile phase: 50/50 ACN/water (keep k’ in the same range as 3µm particles), Column: 50 x 4.6mm, Agilent 1100/UV with Micro Cell, high pressure fitting and passing through heat exchanger.

1.8 mL/min test conditions

EAS 2009 18

Flow Studies on Sub-2μm Zr-PBD: Factory Instrument at Ambient

Plate height vs linear velocity for retained solutes: Alkylbenzenes, Temperature 30 ºC, Mobile phase: 50/50 ACN/water (keep k’ in the same range as 3µm particles), Column: 50 x 4.6mm, Agilent 1100/UV with Standard Cell and 0.007’’ i.d. tubing.

Plates (N) vs Linear Velocity for a sub-2um PBD Column

0

2000

4000

6000

8000

10000

12000

14000

0.03 0.13 0.23 0.33 0.43 0.53

u (cm/s)

Num

ber o

f pla

tes

(N)


N=(L/H)

EAS 2009 19

Flow Studies on Sub-2μm Zr-PBD: Micro Cell

Plate height vs linear velocity for retained solutes: Alkylbenzenes, Temperature 30 ºC, Mobile phase: 50/50 ACN/water (keep k’ in the same range as 3µm particles), Column: 50 x 4.6mm, Agilent 1100/UV with Micro Cell and 0.007’’ i.d. tubing.


0

2000

4000

6000

8000

10000

12000

14000

0.03 0.13 0.23 0.33 0.43 0.53 0.63

u (cm/s)

Num

ber o

f pla

tes

(N)


EAS 2009 20

Flow Studies on Sub-2μm Zr-PBD: Micro Cell + Tubing

Plate height vs linear velocity for retained solutes: Alkylbenzenes, Temperature 30 ºC, Mobile phase: 50/50 ACN/water (keep k’ in the same range as 3µm particles), Column: 50 x 4.6mm, Agilent 1100/UV with Micro Cell and 0.005’’ i.d. tubing.


0

2000

4000

6000

8000

10000

12000

14000

0.03 0.13 0.23 0.33 0.43 0.53

u (cm/s)

Num

ber o

f pla

tes

(N)


EAS 2009 21

Flow Studies on Sub-2μm Zr-PBD: Heat Exchanger + Fitting + μCell

Plate height vs linear velocity for retained solutes: Alkylbenzenes, Temperature 30 ºC, Mobile phase: 50/50 ACN/water (keep k’ in the same range as 3µm particles), Column: 50 x 4.6mm, Agilent 1100/UV with Micro Cell, high pressure fitting and passing through heat exchanger.


0

2000

4000

6000

8000

10000

12000

14000

0.03 0.13 0.23 0.33 0.43 0.53

u (cm/s)

Num

ber o

f pla

tes

(N)


EAS 2009 22

Plates (N) vs Linear Velocity for a 3um PBD Column

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80

u (cm/s)

Num

ber o

f pla

tes

(N)


Flow Studies on 3μm Zr-PBD: Factory Instrument

Plate height based on van Deemter Equation vs linear velocity at various temperatures for retained solutes: Alkylbenzenes, Temperature: 30 ºC, Mobile phase: 50/50 ACN/water, Column: ZirChrom®-PBD, 50 x 4.6mm, Agilent 1100/UV standard cell (0.007’’ i.d. tubing).

EAS 2009 23

Flow Studies on 3μm Zr-PBD: Factory + Micro Cell

Plate height based on van Deemter Equation vs linear velocity at various temperatures for retained solutes: Alkylbenzenes, Temperature: 30 ºC, Mobile phase: 50/50 ACN/water, Column: ZirChrom®-PBD, 50 x 4.6mm, Agilent 1100/UV micro cell (0.007’’ i.d. tubing).

Plates (N) vs Linear Velocity for a 3um PBD Column

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80

u (cm/s)

Num

ber o

f pla

tes

(N)


EAS 2009 24

Alkylbenzylamine Separation on sub-2μm Zr-PBD, 50 oC

LC Conditions: Column: ZirChrom®-PBD, 50 x 4.6 mm i.d., sub-2μm Mobile Phase: 21/79 ACN/20 mM K3PO4 at pH=12; Flow rate: 1.5 mL/min; Temperature: 50 oC; Injection Vol.: 3.0 µL; Detection: UV at 254 nm

min0 0.2 0.4 0.6 0.8 1

Norm.

0

5

10

15

20

25

30

35216 barNH2

NH

N N

EAS 2009 25

N

Cl

N

Antihistamine Separation on sub-2μm Zr-PBD, 80 oC

LC Conditions: Column: ZirChrom®-PBD, 50 x 4.6 mm i.d., sub-2μm Mobile Phase: 28/72 ACN/50 mM TMA-OH at pH=12.2; Flow rate: 2.5 mL/min; Temperature: 80 oC; Injection Vol.: 2.0 µL; Detection: UV at 254 nm

min0 0.2 0.4 0.6 0.8 1 1.2

Norm.

0

5

10

15

20

25

30

35

40

260 bar O

N

N NS

N

NN

N

EAS 2009 26

β-blockers on ZirChrom®-PBD sub-2μm, High Temp

ZirChrom®-PBD50mm x 4.6mm, sub-2μm22/78 ACN/20mM K3PO4 at pH=12F=2.5 mL/minUV=254nmT=75 oC

min0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Norm.

0

20

40

60

80

100

120

163,000 plates/m

Analytes1=Labetalol2=Atenolol3=Acebutolol4=Metoprolol5=Oxprenolol6=Lidocaine7=Quinidine8=Alprenolol9=Propranolol

246 bar (3660 psi)Background pressure: ca. 25 barDetector Response Time (0.5s)

EAS 2009 27

β-blockers on ZirChrom®-PBD sub-2μm, High Temp, Faster sampling

ZirChrom®-PBD50mm x 4.6mm, sub-2μm21/79 ACN/20mM K3PO4 at pH=12F=2.5 mL/minUV=254nmT=75 oC, faster sampling rate

246 bar (3660 psi)Background pressure: ca. 25 barDetector Response Time (<0.12s)

min0 0.2 0.4 0.6 0.8 1

Norm.

0

25

50

75

100

125

150

175

185,000 plates/m

Analytes1=Labetalol2=Atenolol3=Acebutolol4=Metoprolol5=Oxprenolol6=Lidocaine7=Quinidine8=Alprenolol9=Propranolol

EAS 2009 28

Plans for Further Development

• Extend the range of ultra-high speed applications using sub-2µm Zr-PBD, especially at high pH and temperature (“extreme conditions for silica”); develop generic conditions for LC-MS.

• Develop sub-2µm Zr-CARB and compare performance to Zr-PBD under ambient and extreme temperature conditions.

• Study additional advantages of optimizing the IBW of an Agilent Model 1100 HPLC instrument using a high performance (Model 1200) heat exchanger.

EAS 2009 29

Conclusions

• Multi-mode HPLC columns have become popular fordifficult applications where compounds have ionic character and vary widely in chemical nature. Several ZirChrom® phases are ideal and popular for multi-mode applications and are stable over a much wider range of pH and temperature than any silica-based phase.

• Zirconia 3µm HPLC columns are currently available in a wide range of stable coatings and produce efficiencies in excess of 100,000 N/M.

• New sub-2µm zirconia UHPLC columns with very high efficiency in excess of 200,000 N/M with a PBD polymer-coated phase permit higher speed separations with shorter residence time at elevated temperatures.

EAS 2009 30

References and Acknowledgements

1. J. A. Blackwell and P. W. Carr, "Development of an Eluotropic Series for the Chromatography of Lewis Bases on Zirconium Oxide," Anal. Chem. 64, 863-73 (1992).

2. R. A. Henry, H. K. Brandes, D. S. Bell and C. T. Santasania, 30th Annual HPLC Meeting, Oral Presentation, 2006, San Francisco, CA.

3. R. A. Henry and H. K. Brandes, Eastern Analytical Symposium, Oral Presentation, 2006, Somerset, NJ.

4. B. Yan. C. V. McNeff. R. A. Henry and D. Nowlan, Eastern Analytical Symposium, Poster, 2008, Somerset, NJ.

5. B. Yan. C. V. McNeff. R. A. Henry and D. Nowlan, Pittsburgh Analytical Conference, 2009, Oral Paper, Chicago, IL..

6. B. Yan. C. V. McNeff. R. A. Henry and D. Nowlan, Horvath Award Symposium, 2009, Oral Paper, Hartford, CT.

The assistance of Supelco Division of Sigma-Aldrich is gratefully appreciated, including the use of a high-pressure column fitting.

Applications of Sub-2µm Zirconia-PBD Columns at Elevated ...Idealized van Deemter Plots H = A +...

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