Unique Properties of Zirconia Phases for …T415154 Unique Properties of Zirconia Phases for...
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T415154 Unique Properties of Zirconia Phases for Structurally Similar Compounds and Other Difficult HPLC Separations Kelly S. Johnson 1 , Merlin Bicking 2 , and Richard A. Henry 3 1 ZirChrom Separations, 617 Pierce St, Anoka, MN 55303 2 ACCTA, Inc., 3534 Jessie Ct, Saint Paul, MN 55125 3 Consultant, Supelco, Div. of Sigma Aldrich Why Zirconia? - Bigger Method Development Toolbox Unique Multimodal Selectivity Buffer Type and Concentration can ‘Tune” Selectivity Stable 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 O Zr Zr O O Zr Zr O O Zr Zr O O O O O O O O OH O OH H 2 O OH 2 H 2 O OH Weak Brönsted Base: Zr Zr O O O H + + Zr Zr O O O H Weak Brönsted Acid: O H ZrO OH ZrOH 2 Strong Lewis Acid: Zr O O O O P O O O O + Zr O O O O P O O O O - Choosing Buffer Type Hydroxide Phosphate Fluoride Citrate Sulfate Acetate Formate Chloride Strongest Weakest Interaction Strength Lewis 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) pK a 9.4 (6) pK a 9.7 1. Lidocaine 2. Norpseudo ephedrine 3. Tryptamine 4. Quinidine 5. Amitriptyline 6. 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 5 6 6 5 1 4 2,3 1 4 2 3 1 2 3 4 N NH 0 10 20 30 40 6 5 4 3 2 1 6 5 4 3 2 1 6 5 4 3 2 1 6 5 4 3 2 1 Effect of pH on Selectivity of ZirChrom-PBD (min) pH 4 pH 7.5 pH 9.5 pH 12 RNH 2 + H + <---> RNH 3 + 5 and 6 switch elution order Exposure Conditions: Mobile phase, ACN/50 mM Potassium phosphate buffer at indicated pH; Temperature, 30 o C. LC Conditions: Mobile phase, ACN (or THF)/50 mM Potassium phosphate buffer at indicated pH; Flow Rate, 1.0 mL/min.; Temperature, 30 o C; 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' Propranolol ZirChrom-PBD pH 12.0 Gammabond RP-1 pH 12.0 Luna C18 (2) pH 10.0 Extend C18 Rapid Res. pH 11.5 Xterra RP18 pH 12.0 DiamondBond-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 min 0 2 4 6 8 10 12 14 mAU 0 10 20 30 40 50 60 1 2 3 4 5 min 2.4 2.6 2.8 3 3.2 2 Temperature: 21 o C Flow rate: 1.35 ml/min. Pressure drop: 195 bar Resolution (2,3): 2.1 Analysis 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 min 0 2 4 6 8 10 12 14 mAU 0 20 40 60 80 Temperature: 80 o C Flow rate: 3.00 ml/min. Pressure drop: 195 bar Resolution (2,3): 2.1 Analysis time: 2.5 min. 3 2 4 5 1 min 0.9 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 4.5 Flow rate: 1.0 mL/min. Temperature: 30 o C Injection Vol.: 5.0 μL Detection: UV at 254 nm 1=Thiamine (Vit. B 1 ), 2=Pyridoxine (Vit. B 6 ), 3= Nicotinamide (form of Vit. B 3 ), 4=Riboflavin (Vit. B 2 ), 5=Nicotinic acid (form of Vit. B 3 ), 6=Ascorbic acid (Vit. C) Six water soluble vitamins are easily separated under isocratic 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 C C C C C C C C Zirconia Substrate -1.5000 -1.0000 -0.5000 0.0000 0.5000 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 formamide 2. Benzylalcohol 3. Phenol 4. 3-phenyl propanol 5. p-chlorophenol 6. Acetophenone 7. Benzonitrile 8. Nitrobenzene (NB) 9. Methyl benzoate 10. Anisole 11. Benzene 12. p-chlorotoluene 13. p-nitrobenzyl chloride 14. Toluene 15. Benzophenone 16. Bromobenzene 17. Naphthalene 18. Ethylbenzene 19. p-xylene 20. p-dichlorobenzene 21. Propylbenzene 22. n-butylbenzene T NB 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-CARB Mobile Phase: Gradient elution from 55/5/40 to 90/5/5 A/B/C from 0 to 4.5 minutes. A: Acetonitrile B: Tetrahydrofuran C: 25 mM Ammonium fluoride, 10 mM Ammonium acetate, pH 5.6 Temperature: 80 o C Injection Vol.: 10 μL Flow rate: 3.0 mL/min. Pressure Drop: 195 bar Detection: 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. 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 Cation Exchange + RP Cations Negative Cation Exchange + RP Near Neutral More Negative Cation Exchange + 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 NH 4 H 2 PO 4 + 5 mM NH 4 CO 3 Flow rate: 1.5 mL/min Temperature: 50 o C Injection Vol.: 5 μL Detection: 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-CARB Mobile Phase: Isocratic elution from 50/50 A/B A: Acetonitrile/Isopropyl Alcohol B: Tetrahydrofuran Temperature: 70 o C Injection Vol.: 5 μL Flow rate: 1.5 mL/min. Pressure Drop: 74 bar Detection: UV at 275 nm 1=Cholecalciferol (vitamin D 3 ), 2= Ergocalciferol (vitamin D 2 ) Unique selectivity allows separation using an isocratic UV detection method 1 2
Transcript of 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
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