unique properties of zirconia phases for …t415154 unique properties of zirconia phases for...

T415154

Unique Properties of Zirconia Phases for Structurally Similar Compounds and Other Difficult HPLC SeparationsKelly S. Johnson1, Merlin Bicking2, and Richard A. Henry3

1 ZirChrom Separations, 617 Pierce St, Anoka, MN 553032 ACCTA, Inc., 3534 Jessie Ct, Saint Paul, MN 55125

3 Consultant, Supelco, Div. of Sigma Aldrich

Why Zirconia? - Bigger Method Development ToolboxUnique Multimodal SelectivityBuffer Type and Concentration can ‘Tune” SelectivityStable Over a Wide Range of pH (1-14)Stable at Temperatures Up to 200 o C

Zirconia Surface Chemistry

Ligand Exchange Interaction

Zr Zr

O

OZr Zr

O

OZr Zr

O

OZr Zr

O

O

O

OO

O

O

O

OHO

OH H2O OH2H2OOH

Weak Brönsted Base: Zr Zr

O

O

O

H++ Zr Zr

O

O

O

HWeak Brönsted Acid: OHZrOOHZrOH 2

Strong Lewis Acid: Zr

O

O

O

O P

O

OO

O

+Zr

O

O

O

O

P

OO O

O -

Choosing Buffer Type

HydroxidePhosphateFluorideCitrateSulfateAcetateFormateChloride

Strongest

Weakest

Interaction StrengthLewis Base Additive (A)

Small Lewis bases with higher electron density and lower polarizability interact more strongly with zirconia.

Retention

More Retention

Less Retention

A stronger interaction strength means a greater

charge on the surface, producing more retention

for cations.

Anions would be affected in the opposite way, and neutrals are not affected.

A stronger interaction strength means a greater

charge on the surface, producing more retention

for cations.

Anions would be affected in the opposite way, and neutrals are not affected.

Effect of Buffer Type & Concentration on Selectivity of ZirChrom-PBD

(5) pKa 9.4 (6) pKa 9.71. Lidocaine2. Norpseudo

ephedrine3. Tryptamine4. Quinidine5. Amitriptyline6. Nortriptyline

20% ACN, 20 mM Lewis base additive, pH 7.5; 0.8 mL/min; 30 °C

0 10 20 30 40 (min)

Acetate

Fluoride

Phosphate

5

6

56

65

14 2,3

1 42 3

1 23

4

N NH

0 10 20 30 40

654

32

1

654

321

65

432

1

6 54321

Effect of pH on Selectivity of ZirChrom-PBD

(min)

pH 4

pH 7.5

pH 9.5

pH 12

RNH2 + H+ <---> RNH3+

5 and 6 switch elution order

Exposure Conditions: Mobile phase, ACN/50 mM Potassium phosphate buffer at indicated pH; Temperature, 30 oC.LC Conditions: Mobile phase, ACN (or THF)/50 mM Potassium phosphate buffer at indicated pH; Flow Rate, 1.0 mL/min.; Temperature, 30 oC; Injection Volume, 5 µL; Detection, 254 nm.

High pH Stability Comparison*

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

0 2000 4000 6000 8000 10000 12000 14000 16000

Column Volumes of Mobile Phase

k' P

ropr

anol

ol

ZirChrom-PBD pH 12.0Gammabond RP-1 pH 12.0Luna C18 (2) pH 10.0Extend C18 Rapid Res. pH 11.5Xterra RP18 pH 12.0DiamondBond-C18 pH 12.5

* Column names are the trademarks of their respective manufacturers.

Elevated Temperature Enables Fast Separation Without Switching Columns or Losing Resolution

LC Conditions: Mobile Phase, 29/71 ACN/50mM Tetramethylammonium hydroxide, pH 12.2; Flow Rate, 1.35 mL/min.; Injection volume, 0.5 µl; 254 nm detection; Column Temperature, 21°C; Pressure drop = 195 bar; Solutes: 1=Doxylamine, 2=Methapyrilene, 3=Chlorpheniramine, 4=Triprolidine, 5=Meclizine 100 x 4.6 ZirChrom-PBD

3

min0 2 4 6 8 10 12 14

mAU

0

10

20

30

40

50

60 1

2

3

4

5min2.4 2.6 2.8 3 3.2

2Temperature: 21 oCFlow rate: 1.35 ml/min.Pressure drop: 195 barResolution (2,3): 2.1Analysis time: 12.5 min.

LC Conditions: Mobile Phase, 26.5/73.5 ACN/50mM Tetramethylammonium hydroxide, pH 12.2; Flow Rate, 3.00 mL/min.; Injection volume, 0.2 µl; 254 nm detection; Column Temperature, 80°C; Pressure drop = 195 bar; Solutes: 1=Doxylamine, 2=Methapyrilene, 3=Chlorpheniramine, 4=Triprolidine, 5=Meclizine 100 x 4.6 ZirChrom-PBD

min0 2 4 6 8 10 12 14

mAU

0

20

40

60

80

Temperature: 80 oCFlow rate: 3.00 ml/min.Pressure drop: 195 barResolution (2,3): 2.1Analysis time: 2.5 min.

3

24

5

1

min0.9 0.95 1 1.05 1.1 1.15 1.2

3

2

ZirChrom®-SAX

ZirChrom®-SAX is a zirconia particle coated with polyethyleneimine polymer and then crosslinked using a hydrophobic crosslinker. The crosslinker modifies the phase with a bit of reversed phase character, giving the ZirChrom-SAX phase a unique, multimodal selectivity.

Water Soluble Vitamins on ZirChrom®-SAX

Column: ZirChrom-SAX, 150 x 4.6 mm i.d., 3 µm Mobile Phase: 50 mM Ammoniumdihydrogenphosphate, pH 4.5Flow rate: 1.0 mL/min.Temperature: 30 oCInjection Vol.: 5.0 µLDetection: UV at 254 nm

1=Thiamine (Vit. B1), 2=Pyridoxine (Vit. B6), 3= Nicotinamide (form of Vit. B3), 4=Riboflavin (Vit. B2), 5=Nicotinic acid (form of Vit. B3), 6=Ascorbic acid (Vit. C)

Six water soluble vitamins are easily separated under isocratic conditions.Six water soluble vitamins are easily separated under isocratic conditions.

These two compounds are difficult to retain under either reversed

phase or HILIC conditions.

These two compounds are difficult to retain under either reversed

phase or HILIC conditions.

“Green” Analysis of Energy Drink on ZirChrom®-SAX

ZirChrom®-CARB

Zirconia Substrate

C C C C C C CC C C C C C C

Zirconia Substrate

-1.5000

-1.0000

-0.5000

0.0000

0.5000

1.0000

1.5000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Log

k'

Comparison of Selectivity on Silica ODS, ZirChrom®-PBD &-CARB

ODS

ZirChrom®-PBD

ZirChrom®-CARB 1. N-benzyl formamide2. Benzylalcohol 3. Phenol4. 3-phenyl propanol 5. p-chlorophenol6. Acetophenone7. Benzonitrile 8. Nitrobenzene (NB)9. Methyl benzoate

10. Anisole 11. Benzene 12. p-chlorotoluene 13. p-nitrobenzyl chloride 14. Toluene15. Benzophenone 16. Bromobenzene 17. Naphthalene 18. Ethylbenzene 19. p-xylene 20. p-dichlorobenzene21. Propylbenzene22. n-butylbenzene

TNB

While a traditional ODS and ZirChrom-PBD have similar selectivities for neutrals, the

ZirChrom-CARB phase is unique.

While a traditional ODS and ZirChrom-PBD have similar selectivities for neutrals, the

ZirChrom-CARB phase is unique.

Column: 4.6 mm x 100 mm, ZirChrom-CARBMobile Phase: Gradient elution from 55/5/40 to 90/5/5

A/B/C from 0 to 4.5 minutes.A: AcetonitrileB: TetrahydrofuranC: 25 mM Ammonium fluoride, 10 mM Ammonium acetate, pH 5.6

Temperature: 80 oCInjection Vol.: 10 µLFlow rate: 3.0 mL/min.Pressure Drop: 195 barDetection: UV at 270 nm

1=Dihydroestradiol sulfate, 2=Dihydroequilin sulfate, 3=Equilin sulfate, 4=Estrone sulfate, 5=Equilenin sulfate, 6=Dihydroequilenin sulfate

This degree of selectivity is not possible on a traditional reversed phase column.

This degree of selectivity is not possible on a traditional reversed phase column.

Conclusions Zirconia-based stationary phases have very different selectivity from silica-based phases. ZirChrom®-SAX has a unique multimodal selectivity – ionic and hydrophobic. Zirconia phases have selectivity that can be tuned by the addition of different buffers.

Zirconia reversed phases, such as ZirChrom®-PBD &-CARB offer excellent chemical and thermal stability: Faster separations at high temperature. Better selectivity of ZirChrom®-CARB for structurally similar compounds.

Acknowledgements☞ The authors extend their appreciation to the following sponsors

who supplied resources for this research.

Structurally Similar Sulfated Steroids on ZirChrom®-CARB

ZirChrom®-PBD

ZirChrom®-PBD is a zirconia particle coated with polybutadiene polymer and then crosslinked. The unique surface chemistry coupled with the polymer coating enables a superior, multi-modal selectivity.

Separation Mode

Cations Slightly Negative

CationExchange

+ RP

Cations NegativeCation

Exchange + RP

Near Neutral

More Negative

CationExchange

+ RP

All Neutral

Most Negative RP

Analyte Charge

Surface Charge

Columns: Leading C18 column, ZirChrom-SAX,100 x 4.6 mm i.d., 3 µm

Mobile Phase: 10 mM NH4H2PO4 + 5 mM NH4CO3

Flow rate: 1.5 mL/minTemperature: 50 oCInjection Vol.: 5 µLDetection: UV at 230 nm

Traditional C18 phases do not readily separate benzoate and sorbate (preservatives in energy drinks). The unique multimodal selectivity of Zirchrom-SAX can take advantage of differences between the ionized forms, producing excellent selectivity and retention, resulting in 6 minute baseline resolution of caffeine (1), sorbate (2), and benzoate (3) .

C18: pH 6, 5% Acetonitrile

C18: pH 2, 5% Acetonitrile

ZirChrom “Green” Mobile Phase

Fast Separation of Vitamin D on ZirChrom®-CARB

Column: 4.6 mm x 50 mm, 3 µm, ZirChrom-CARBMobile Phase: Isocratic elution from 50/50 A/B

A: Acetonitrile/Isopropyl AlcoholB: Tetrahydrofuran

Temperature: 70 oCInjection Vol.: 5 µLFlow rate: 1.5 mL/min.Pressure Drop: 74 barDetection: UV at 275 nm1=Cholecalciferol (vitamin D3), 2= Ergocalciferol (vitamin D2)

Unique selectivity allows separation using an isocratic UV

detection method

Unique selectivity allows separation using an isocratic UV

detection method

1

2