Specialists in High Efficiency, Ultra-Stable Phases for HPLC
Zirconia as a Versatile Substrate for Chiral Stationary Phases
Richard A. Henry1 , Clayton V. McNeff1, Bingwen Yan1
Shengxiang Ji2, Daniel Nowlan2, Thomas R. Hoye2
1. ZirChrom Separations, Inc. 617 Pierce St., Anoka, MN 55303,2. University of Minnesota, 207 Pleasant Street SE, Minneapolis, MN 55455.
2
Surface Chemistry of Zirconia
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
OH2H2O
Lewis Acid: Zr4+: H2O + RPO32- Zr4+: RPO3
2- + H2O
Other Lewis base examples: PO43-, RCO2
- , Catechol
Zirconia chemistry is dominated by Lewis acid-base reactions
3
A Novel Approach to Attaching Chiral Selectors1 to Zirconia2
1. William H. Pirkle, et. al., J. Chromatogr., 316 (1984) 585.
2. 2. Phase I SBIR Grant (NIH).
= ZrO2 = Lewis Base (Anchor) =Chiral Selector
CSP
Step 1 Step 2
4
Interaction Strength of Lewis Bases with Zirconia1
HydroxidePhosphateFluorideCitrateSulfateAcetateFormateNitrateChlorideWater
Strongest
Weakest
Interaction Strength Lewis Base (L)
Small Lewis bases with high electron density and low polarizability interact more strongly with Zr atoms.
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).
5
A Bidentate Phosphonate Anchor–the Key to Improved Stability1
1. Phase II SBIR (NIH).
Pamidronic acid (PDA)1
P
O
HOOH
NH2Aminopropylphosphonic
acid (APPA)
(Phase II Anchor)
Bidentate anchor
NH2
P
P
HO
HOHO
HO
O
OOH
NH2
P
P
HO
HOHO
HO
O
OOH
6
Zirconia CSP 2-Step Synthesis with Bidentate Anchor (PDA)
Lewis acid-base reaction
EEDQ coupling reaction
(PDA)
NH2
P
P
HO
HOHO
HO
O
OOH NH2
P
P
O
OO
O
O
OOH
CS-COOH
CS = Chiral Selector
NH
P
P
O
OO
O
O
OOH
O
CSCS
7
Chiral Selectors Evaluated1
HN H
O
OH
NO2
O2NO
(S)-DNB-L-Phenylglycine [(S)-PG]
(S)-N-[1-(1-naphthyl)ethyl]succinamic acid [(S)-NESA]
CH3NH
HO
O
HO
1. Phase II SBIR (NIH)
(S)-DNB-L-Leucine
[(S)-Leu]
O
HN H
O
OH
NO2
O2N
Point of Attachmentto Anchor Group
8
Chiral Separation on Zr (S)-Leu(π-acceptor phase)
min2 4 6 8 10 12 14 16 18
mAU
010203040
min2 4 6 8 10 12 14 16 18
mAU
020406080
100
min2 4 6 8 10 12 14 16 18
mAU
02468
Trifluoranthryl Ethanol
Conditions: 99/1 Hexane/IPA, Flow=1
1-Naphthyl-Leucine Ester
Conditions: 99/1 Hexane/IPA, Flow=1
Napropamide
Conditions: 99/1 Hexane/IPA, Flow=1
α = 1.15
α = 16.8
α = 1.47
* *
*
**
*
9
Chiral Separation on Zr (R)-PG(Another π-acceptor phase)
Trifluoranthryl Ethanol
Conditions: 99/1 Hexane/IPA, Flow=1, 30 °C
1-Naphthyl-Leucine Ester
Conditions: 99/1 Hexane/IPA, Flow=1, 30 °C
Napropamide
Conditions: 99/1 Hexane/IPA, Flow=1, 30 °Cmin2 4 6 8 10 12 14
mAU
05
1015202530
min2.5 5 7.5 10 12.5 15 17.5 20 22.5-5-2.52.57.5
12.517.5
min0 2 4 6 8 10 120
10203040
α = 1.18
α = 1.14
α = 2.98
* *
* *
**
10
Chiral Separations on Zr (S)-NESA(π- donor phase)
(R/S)-3,5-Dinitro-N-(1-phenylethyl)benzamide.
Conditions: Pre-mixed 88.9/11/0.1 Hexane/IPA/TFA, F=1 ml/min, 30 °C
0 2 4 6 8 10 12 14 16 18
mAU
050
100150200
0 2 4 6 8 10 12 14 16 18
mAU
010203040
min0 2 4 6 8 10 12 14 16
mAU
05
10152025303540
(R/S)-N-3,5-dintrobenzoyl-a-amino-2,2-dimethyl-4-pentenyl dimethyl phosphonate.
Conditions: Pre-mixed 88.9/11/0.1 Hexane/IPA/TFA, F=1 ml/min, 30 °C
(R/S)-(3,5-dinitrobenzoyl)-phenylglycine
Conditions: Machine mixed 15/85 (99.9/0.1 MeOH/TFA) / (89/11 Hexane/IPA), F=1 ml/min, 30 °C
α = 1.65
α = 1.28
α = 2.18
11
Methanol Effect on Zr (S)-NESA
Conditions: 89/11 Hexane/IPA, F=1 ml/min, 30 °C.
Conditions: 80 / 10 / 10 (99/1 Hexane/IPA) / MeOH / (70/30 Hexane/IPA), F=1 ml/min, 30 °C
Conditions: 90 / 2 / 8 (99/1 Hexane/IPA) / MeOH / (70/30 Hexane/IPA), F=1 ml/min, 30 °C
α = 1.59N2=971
α = 1.25N2=13,315
α = 1.42N2=6,425
min0 1 2 3 4 5 6 7 8 9
mAU
01020304050
min0 1 2 3 4 5 6 7 8 90
20406080
100
min0 1 2 3 4 5 6 7 8 90
50100150200250
Sample: (R/S)-N-3,5-dintrobenzoyl-a-amino-2,2-dimethyl-4-pentenyl dimethyl phosphonate
12
Stability of Zr-(S)-NESA at pH 2
Column ID: ZrCSP051605C, Mobile phase: 15/85 ACN/0.01 mM TFA pH 2, Temperature: 30 oC. Injection volume: 5 ul, Wavelength: 254 nm. Probe solutes:(R/S)-3,5-dinitro-N-(1-phenylethyl)benzamide.
min0 2 4 6 8 10
Norm.
0
2
4
6
8
10
12
14
Initial
After 7000 column volume flush
Zirconia CSPs are compatible with reversed phase conditions
13
Stability of Zr-(S)-NESA at pH 2
0.0
1.0
2.0
3.0
4.0
5.0
0 2000 4000 6000
Column Volumes
k'
k‘ (Less Retained)
k‘ (More Retained)
1.00
1.50
2.00
0 2000 4000 6000Column Volumes
Sel
ectiv
ityColumn ID: ZrCSP051605C, Mobile phase: 15/85 ACN/0.01 mM TFA pH 2, Temperature: 30 oC. Injection volume: 5 ul, Wavelength: 254 nm. Probe solutes:(R/S)-3,5-dinitro-N-(1-phenylethyl)benzamide.
14
Stability of Zr-(S)-DNB-Leu at pH 8
Column ID: ZrCSP032805A, Mobile phase: 15/85 ACN/5 mM ammonium hydrogencarbonate pH 8.0, Temperature: 30 oC. Injection volume: 5 ul, Wavelength: 254 nm. Probe solutes:(R/S)-2, 2, 2-trifluoro-1-(9-anthryl)ethanol
0.0
1.0
2.0
3.0
4.0
5.0
0 2000 4000 6000
Column Volumes
k'
k'(Less Retained)
k'(More Retained)
1.00
1.10
1.20
1.30
0 2000 4000 6000
Column Volumes
Sele
ctiv
ity
15
Production Scale-up of Zr (S)-NESA
min2.5 5 7.5 10 12.5 15 17.5
Norm.
0
20
40
60
80
100
120
DAD1 A, Sig=254,4 Ref=450,80 (O:\HPCHEM\DATA\R051605C\05240504.D)
DAD1 A, Sig=254,4 Ref=450,80 (O:\HPCHEM\DATA\R062805E\05062806.D)
7.5 g zirconia per batch used for coating4X scale-up 30 g zirconia per batch used for coating
NapropamideConditions: 90/10 Hexane/IPA, Flow=1
16
2-Step Synthesis of Zirconia CSPs for Chiral Selector Screening
= ZrO 2 = Lewis Base (Anchor) =Chiral Selector
Anchor adsorption
CS bonding
Chromatographic Test
CSP Stripping
Zirconia
17
2-Step Online Zirconia CSP Synthesis for Chiral Screening
1) Lewis Acid-base Anchor Attachment (10 mg/mL aq. PDA, recycle for 16 hrs at 1
mL/min and 60°C)2) EEDQ Coupling Reaction
(CS reagent in THF overnight (16 hrs) at 30°C )
(Pamidronic acid)
NH2
P
P
HO
HOHO
HO
O
OOH NH2
P
P
O
OO
O
O
OOH
CS (Chiral Selector)
NH
P
P
O
OO
O
O
OOH
O
CS
ZrO
2-PD
A-C
S
ZrO
2-PD
A
ZrO
2
ZrO2-PDA-CS
3) Chromatographic Test
4) CSP Column Stripping (1M NaOH
for 2 hrs at 60°C, water and 1M HNO3
finish)
Process not optimized for
time
18
Changing Chiral Selectors
HN H
O
OH
NO2
O2NO
(S)-DNB-L-Phenylglycine (S-PG) (R)-DNB-L-Phenylglycine (R-PG)
NH
O
OH
NO2
O2NO
H
Pamidronic acid derivatives
19
Stripping Experiment: (S)-PG CSP
Pre-mixed 98/0.5/1.5 Hexane/TFA/IPA, flow rate=1 ml/min, ambient temperature, 254 nm, Column: ZirChrom PDA-(S)-PG, S/N SPG122005D (100 × 4.6 mm, 3 µm, Running HPLC coated on PHASE110805A, batch#: 52-132). Solute: (1) 1,3,5-Tri-t-butyl-benzene, (2) (S)-2,2,2-Trifluoro-1-(9-anthryl) ethanol , (3) (R)-2,2,2-Trifluoro-1-(9-anthryl) ethanol 5 µl injection.
min0 1 2 3 4 5 6
Norm.
0
50
100
150
200
After coating.
After stripping
(1)(2+3)
(2)(3)
20
Changes During (S)-PG Stripping
0.00
0.501.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
0 1 2 3 4 5
k' (R)-2,2,2-Trifluoro-1-(9-anthryl) EtOHk' (S)-2,2,2-Trifluoro-1-(9-anthryl) EtOH
1- Original column.2- Column flushed with 15/85 ACN/pH 12 NH4OH for 10 column volumes, then 10 column
volumes of water, 10 column volumes of 1.0 M nitric acid, and 10 column volumes of water.
3- Column then flushed with 50 column volumes of 20/80 ACN/ 1 M NaOH, then 10 column volumes of water, 10 column volumes of 1 M nitric acid and 10 column volumes of water.
4- Column then flushed with 20/80 ACN/ 1 M NaOH for 50 column volumes at 60 oC, then flushed with 10 column volumes of water, 10 column volumes of 1 M nitric acid, and 10 column volumes of water.
Ret
entio
n Fa
ctor
21
Changing (S) to (R)-Phenylglycine CSP on Same Zr Column
Pre-mixed 98/0.5/1.5 Hexane/TFA/IPA, F=1 ml/min, rm °C, 254 nm, Column: ZirChrom PDA-(S)-PG, S/N SPG122005D and ZirChrom PDA-(R)-PG, S/N RPG020806A (100 × 4.6 mm, 3 µm, Running HPLC coated on PHASE110805A, batch#: 52-132). Solute: 1,3,5-Tri-t-butyl-benzene, (R orS)-2,2,2-Trifluoro-1-(9-anthryl) EtOH. 5 µl injection.
2-Step Load (S)-PG CSk’(less) = 2.84k’(more) = 3.81α = 1.34
Strip (S)-PG CS No separation.
2-Step Load (R)-PG CSk’(less) = 2.92k’(more) = 3.83α = 1.34
mAU
0204060
mAU
050
100150200
min0 1 2 3 4 5 6
mAU
020
4060
R S
R
S
22
1-Step Synthesis of Zirconia CSPs for Fast Chiral Screening
= ZrO2 = Chiral Selector withAnchor Group
CS with Anchor Attached
Chromatographic TestStripping-off
Zirconia
23
Example 1-Step Attachment and Detachment Cycle
• Pass a solution of 20 mM N-(4-nitrobenzoyl)-L-glutamic acid (CSP) in tetrahydrofuran for 10 minutes at a column temperature of 60°C and a flow rate of 1 mL/min .
• Flush column with 100% THF for 10 minutes at 2 mL/min at ambient temperature.
• Separate a racemic solution of (±)-2,2,2-trifluoro-1-(9-anthyl)ethanol.
• Strip the CSP by flushing the column with a 50 mM solution of tetramethylammonium hydroxide solution (pH 12) for 20 minutes at 60°C using a flow rate of 1 mL/min.
• Repeat procedure using the same CSP
O O
OHHOHHN
O
NO2
N-(4-nitrobenzoyl)-L-glutamic acid
24
Glutamic Acid Proof of Concept
min2 4 6 8 10
mAU
020406080
100Initial Run
After Stripping and 1-Step Reattachment of the CSP
Comparison between the initial and final separation of (±)-2,2,2-trifluoro-1-(9-anthyl)ethanol leucine ester during a single CSP screening cycle.Chromatographic conditions: mobile phase: 99/1 hexane/IPA; flow rate: 1 ml/min; temperature: 30 oC, solute concentration = 1mg/mL, 5 μL injection.
25
Development of a New Class of Regenerable Cellulosic Coated
Zirconia Stationary Phases
26
1-Step Synthesis of Zirconia CSPs for Fast Chiral Screening
= ZrO2 = Chiral Selector withAnchor Group
CS with Anchor Attached
Chromatographic TestStripping-off
Zirconia
27
Phosphonate Modified Cellulose Based CSP on Zirconia
Carbamate
Anchor
OO
OO
O
O
ONHAr
O
NHAr ONHAr
OO
O
OO
O NHAr OArHN
O
NAr
PO
OAllOAll
OO
OO
O
ONHAr
O
NHAr ONHAr
12
Br P12
OOAll
OAll
1) NaH, DMF
2)
OO
OO
O
O
ONHAr
O
NHAr ONHAr
OO
O
OO
O NHAr OArHN
O
NAr
PO
OHOH12
DimedonePd(PPh3)4THF
Cellulose
ZrO2
ZrO2
28
Cellulose Surface Chemistry
Chiral interactions
Stable supportd-orbital interactions
OO
OO
O
O
ONHAr
O
NHAr ONHAr
OO
O
OO
O NHAr OArHN
O
NAr
PO
OO
ZrO
OZr O
ZrO
ZrO
O
O
H2O
O OO
29
Cellulose – pH 2 Stability
0.000
1.000
2.000
3.000
4.000
5.000
6.000
0 500 1000 1500 2000 2500 3000 3500
Column Volumes
k'
1.00
1.05
1.10
1.15
1.20
1.25
1.30
1.35
1.40
0 500 1000 1500 2000 2500 3000 3500
Column Volumes
alph
a
Column ID: R020907W, Mobile phase: 20/80 ACN/0.01 M TFA pH 2, Temperature: 30 oC. Injection volume: 5 ul, Wavelength: 254 nm. Probe solutes: Benzoin.
30
Cellulose – pH 8 Stability
1.20
1.25
1.30
1.35
1.40
1.45
1.50
0 500 1000 1500 2000 2500 3000 3500 4000
Column Volumes
Alp
ha0.000
1.000
2.000
3.000
4.000
5.000
6.000
0 500 1000 1500 2000 2500 3000 3500 4000
Column Volumes
k'
Column ID: R031607W, Mobile phase: 35/65 ACN/5mM NH4HCO3 pH 8, Temperature: 30 oC. Injection volume: 5 ul, Wavelength: 254 nm. Probe solute: (RS)-trifluroanthrylethanol.
31
Retention Comparison of Hexanoic Acid Modified and Undecylphosphonic Acid
Zirconia Based Cellulosic CSPs
(1) Generally k’ on the new CSP is lower than previous published work (Carr, et al., Anal. Chem., 71 (1999) 3013-3021).
(2) Batch C54 is slightly less retentive than C47.
Carr Ref. 1.36% C
41-C47, 1.44% C - (COOH)
41-C54, 1.13% C – (PO3H2)
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
20.0
k'
trans
-stilb
ene O
xide
Trifluo
ranthr
yl Etha
nol
1-phe
nyl-1
-prop
anol
1-phe
nyl-2
-prop
anol
r-(2-n
aphth
yl)-r-
butyr
olacto
ne
3-phe
nyl-1
-butan
ol
a-(trif
luorom
ethyl)
benz
yl alc
ohol
trans
-stilb
ene O
xide
Burke O
2
3,5-di
nitro-
N-(1-ph
enyle
thyl)b
enza
mide
a-meth
ylben
zyl c
yanid
e
Naprop
amide
α-Meth
yl-1-n
aphth
anlen
s meth
anol
32
Selectivity Comparison of Previous and New Zirconia Based Cellulosic CSPs
*Data of α from Carr, et al., Anal. Chem., 71 (1999) 3013-3021
0.9
1.1
1.3
1.5
1.7
1.9
2.1
2.3
2.5
2.7
trans
-stilb
ene Oxid
e
Trifluo
ranthr
ylEtha
nol
1-phe
nyl-1
-prop
anol
1-phe
nyl-2
-prop
anol
r-(2-n
aphth
yl)-r-
butyr
olacto
ne
3-phe
nyl-1
-butan
ol
a-(trif
luorom
ethyl)
benz
yl alc
ohol
trans
-stilb
ene Oxid
eBurk
e O2
3,5-di
nitro-
N-(1-ph
enyle
thyl)b
enza
mide
a-meth
ylben
zyl c
yanid
eNap
ropam
ide
α
α-Meth
yl-1-n
aphth
anlen
s meth
anol
α=1
*Carr Ref. 1.36% C
41-C47, 1.44% C - (C5-COOH)
41-C54, 1.13% C – (C11-PO3H2)
a a b b a b b b a a a a a
a: 90/10 hexane/IPAb: 98/2 hexane/IPA
33
Retention Comparison Between Alkylphenyl Modified Cellulosic CSPs and
Commercial Silica CSPs
41-C54Commercial silica-based column
41-C54, J04-175, 3,5-dimethylphenyl, -C11H22PO3HCommercial Silica CSP column
α-Meth
yl-1-n
aphth
anlen
s meth
anol
0.02.04.06.08.0
10.012.014.016.018.020.0
trans
-stilb
ene O
xide
Trifluo
ranthr
yl Etha
nol
1-phe
nyl-1
-prop
anol
1-phe
nyl-2
-prop
anol
r-(2-n
aphth
yl)-r-
butyr
olacto
ne
3-phe
nyl-1
-butan
ol
a-(trif
luorom
ethyl)
benz
yl alc
ohol
trans
-stilb
ene O
xide
Burke O
2
3,5-di
nitro-
N-(1-ph
enyle
thyl)b
enza
mide
a-meth
ylben
zyl c
yanid
e
Naprop
amide
k'
New phase has less retention than commercial Silica-based column likely due to lower loading of CSP and anchor group.
a a b b a b b b a a a a a
a: 90/10 hexane/IPAb: 98/2 hexane/IPA
34
Selectivity Comparison Between Dimethylphenyl Carbamate Modified
Cellulosic CSPs and Commercial Silica CSPs
trans
-stilb
ene O
xide
41-C54, J04-175, 3,5-dimethylphenyl, -C11H22PO3H2Commercial Silica CSP column
α-Meth
yl-1-n
aphth
anlen
s meth
anol
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Trifluo
ranthr
yl Etha
nol
1-phe
nyl-1
-prop
anol
1-phe
nyl-2
-prop
anol
r-(2-n
aphth
yl)-r-
butyr
olacto
ne
3-phe
nyl-1
-butan
ol
a-(trif
luorom
ethyl)
benz
yl alc
ohol
trans
-stilb
ene O
xide
Burke O
2
3,5-di
nitro-
N-(1-ph
enyle
thyl)b
enza
mide
a-meth
ylben
zyl c
yanid
eNap
ropam
ide41-C54Commercial silica-based column
Undecylphenylcarbamate modified cellulosic CSP has good selectivity compared to a commercial silica column.
α
a a b b a b b b a a a a a
a: 90/10 hexane/IPAb: 98/2 hexane/IPA
35
Effect of Ionic Strength on the Separation of Basic Chiral Phamaceuticals on Undecylphosphonic Acid
Modified Cellulosic CSPs
41-C54, J04-175, 3,5-dimethylphenyl, -C11H22PO3H
Increasing ammonium acetate increases enantio-selectivity.
200 100 80 40Pindolol 2.87 2.10 1.79 1.30
Propranolol 1.55 1.53 1.35 1.10Atenolol 1.26 1.12 1.09 1.00Nadolol 1.00 1.00 1.00 1.00
Ion Strength/ Selectivity
Ammonium Acetate in IPA (mM)
LC Conditions: Agilent 1100 with chemstation, flow rate 0.5 mL/min., UV 254, mobile phase = 100% IPA with specified concentration of ammonium acetate, Temperature = ambient, column dimension 10 cm x 4.6 mm id, 3 micron particles.
36
Effect of Ionic Strength on Undecylphosphonic Acid Modified
Cellulosic CSPs
min2 4 6 8 10 12 14
Norm.
-202468
1012
(O:\HPCHEM\DATA\R022006A\PINDOL07.D)
min2 4 6 8 10 12
Norm.
020406080
100120140
(O:\HPCHEM\DATA\R022006A\PINDOL49.D)
80 mM 40 mM
min0 1 2 3 4 5 6 7
Norm.
0102030405060
(O:\HPCHEM\DATA\R022006A\PINDL033.D)
100 mM
min0 1 2 3 4 5 6
Norm.
020406080
100120
(O:\HPCHEM\DATA\R022006A\PINDOL58.D)
200 mM
Increasing ammonium acetate increases the selectivity and decreases retention and improves peak shape for Pindolol. This is likely due to supression of cation-exchange retention mechanism that occurs for basic molecules.
37
Comparison of Silica and Zirconia Cellulosic Phases
Columns, (A) CelluloZeTM (Celu022006A), 100 × 4.6 mm, 3 µm Zirconia, (B) Silica-based column, 150 × 4.6 mm, 5 µm Silica, Solute (RS)-(±)-2,2,2-Trifluoro-1-(9-anthryl) EtOH, Mobile phase90 / 10 Hexane / IPA, Flow Rate, 1 mL/min, Column temperature, ambient.
min2 4 6 8 10 12 14 16
Norm.
0
50
100
150
200
250
DAD1 A, Sig=254,4 Ref =450,80 (O:\HPCHEM\DATA\ODRHCD\9222TR01.D)
min1 2 3 4 5 6
Norm.
0
20
40
60
80
100
120
140
160
DAD1 A, Sig=254,4 Ref =450,80 (O:\HPCHEM\DATA\R022006A\9222TR03.D)
A B
38
min0.5 1 1.5 2 2.5 3
Norm.
0
10
20
30
40
50
DAD1 A, Sig=254,4 Ref =450,80 (O:\HPCHEM\DATA\R022006A\9NAPRP02.D)
min1 2 3 4 5 6
Norm.
0
25
50
75
100
125
150
175
200
DAD1 A, Sig=254,4 Ref =450,80 (O:\HPCHEM\DATA\ODRHCD\9PROP002.D)
Columns, (A) CelluloZeTM (Celu022006A), 100 × 4.6 mm, 3 µm Zirconia, (B) Silica-based column, 150 × 4.6 mm, 5 µm Silica, Solute Napropamide, Mobile phase 90 / 10 Hexane / IPA, Flow Rate, 1 mL/min, Column temperature, ambient.
A B
Comparison of Silica and Zirconia Cellulosic Phases
39
Column, CelluloZeTM (Celu022006A), 100 × 4.6 mm, 3 µm Zirconia, Mobile phase, = 50/50 Heptane/IPA (100 mM NH4OAc in IPA), Flow Rate, 1 mL/min, Column temperature, ambient.
Separation of Basic Drugs on Phosphonated Cellulose Zirconia
min0.5 1 1.5 2 2.5
Norm.
0
5
10
15
20
DAD1 A, Sig=254,4 Ref =450,80 (O:\HPCHEM\DATA\R022006A\PROPRA25.D)
min0 0.5 1 1.5 2 2.5 3 3.5 4
Norm.
0
10
20
30
40
50
60
70
DAD1 A, Sig=254,4 Ref =450,80 (O:\HPCHEM\DATA\R022006A\PINDL040.D)
PindololPropranolol
min0 1 2 3 4 5
Norm.
0
2
4
6
8
10
12
14
DAD1 A, Sig=254,4 Ref =450,80 (O:\HPCHEM\DATA\R022006A\ATENOL21.D)
Atenolol
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Conclusions
• Brush-type CSPs were attached to zirconia using multi-dentate pamidronic acid (PDA).
• Zirconia-based CSPs were shown to be reproducible, stable and have comparable chromatographic performance to commercial silica-based Brush-type CSPs for a range of chiral compounds.
• Zirconia-based CSPs offer the user the potential to regenerate the chiral stationary phase online.
• The new zirconia-based cellulosic CSPs showed similar resolving power to commercial silica-based cellulosic CSPs for selected chiral compounds; increased ionic strength improved resolution of basic chiral compounds.
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References
1. C. B. Castells and P. W. Carr, Anal. Chem., 1999, 71, 3013-3021.
2. C. B. Castells and P. W. Carr, Chromatographia, Vol. 52, No. 9/10, November 2000, 535-542.
3. C. B. Castells and P. W. Carr, J. of Chromatogr. A (2000) 904, 17-33.
Acknowledgement: National Institutes of Health Grant(Phase II SBIR) 2R44HL070334-02A2.
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