applications of sub-2µm zirconia-pbd columns at elevated ...idealized van deemter plots h = a +...
TRANSCRIPT
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.
2 mL/min test conditions
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.
2 mL/min test conditions
EAS 2009 15
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 + 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.
2 mL/min test conditions
EAS 2009 16
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 H
eigh
t, H
(um
)
Benzene Toluene Ethylbenzene Propylbenzene Butylbenzene
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
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 H
eigh
t, H
(um
)
Benzene Toluene Ethylbenzene Propylbenzene Butylbenzene
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)
Benzene Toluene Ethylbenzene Propylbenzene Butylbenzene
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.
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 0.63
u (cm/s)
Num
ber o
f pla
tes
(N)
Benzene Toluene Ethylbenzene Propylbenzene Butylbenzene
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.
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)
Benzene Toluene Ethylbenzene Propylbenzene Butylbenzene
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.
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)
Benzene Toluene Ethylbenzene Propylbenzene Butylbenzene
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)
Benzene Toluene Ethylbenzene Propylbenzene Butylbenzene
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)
Benzene Toluene Ethylbenzene Propylbenzene Butylbenzene
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.