Synthesis of a New Lewis Acid Deactivated Reversed-Phase Zirconia Stationary Phase for HPLC
Dwight Stoll, Clayton V. McNeff, Bingwen Yan, Peter W. Carr
ZirChrom Separations, Inc.
Pittcon 20031-866-STABLE-1
www.zirchrom.com
Outline
• Background – Advantages and surface chemistry of zirconia-based supports for HPLC
• Synthesis of Lewis acid deactivated reversed-phase support
• Chromatographic characterization • Chromatography of Lewis-base analytes
• Reversed-phase characteristics
• Ion-exchange characteristics
• Chemical stability testing
• LC/MS column bleed study
• Pharmaceutical applications
• Conclusion – ZirChrom®-EZ allows the use of LC/MS compatible mobile phases for the analysis of both acidic and basic analytes not previously possible on other zirconia-based reversed-phases
Pore structureParticle sizeChemical flexibilitySurface homogeneityMechanical stabilitySwellibilityChemical stabilityThermal stabilityColumn efficiency
Silica+ ++ ++ +- -+ ++ +- -+
++
Zirconia+ ++ ++- -+ ++ ++ ++ +++
Polymericphase
++ +++ +- -+ +- -- -
++ excellent; + good; -- fair.
Advantages of Zirconia-Based Supports for HPLC
Surface Chemistry of Zirconia-Based Supports for HPLC
Zr Zr
O
OZr Zr
O
OZr Zr
O
OZr Zr
O
O
O
OO
O
O
O
OHO
OH H2O OH2H2OOH
Brönsted Base: Zr Zr
O
O
O
H++ ⇔Zr Zr
O
O
O
H
Brönsted Acid: OHZrOOHZrOH 2+⇔+ −−
Lewis Acid: Zr
O
O
O
O P
O
OO
O
⇔+Zr
O
O
O
O
P
OO O
O
Zr Zr
O
OZr Zr
O
OZr Zr
O
OZr Zr
O
O
O
OO
O
O
O
OHO
OH H2O OH2H2OOH
3. Reflux PBD-ZrO2 in Ethylenediamine-N,N,N’,N’-tetra(methylenephosphonic)acid (EDTPA) solution
4. Wash to remove residual EDTPA
PBD
CH2
H2C N
N
H2C
CH2
CH2
H2C
P
P
P
P
O-
OHHO
O
-O
-O
O
O--O
O
-O
O
EDTPA
Synthesis of a Lewis-Acid Deactivated Reversed-Phase Support
1. Coat bare zirconia with polybutadiene(PBD)1
2. Crosslink PBD chains together using dicumyl peroxide as initiator
1) Li, J. W.; Reeder, D. H.; McCormick, A. V.; Carr, P. W. Journal of Chromatography A 1998, 791, 45-52
0102030405060708090
100
0 5 10 15 20 25 30Time (min.)
mA
U
Chromatographic Characterization –Chromatography of Lewis Base Analytes
LC Conditions: Mobile phase, 40/60 ACN/Water; Flow rate, 1.0 ml/min.; Temperature, 30 oC; Injection volume, 1 µl; Detection at 254 nm; Solutes: 1=methoxybenzoic acid, 2=ethoxybenzoicacid, 3=propoxybenzoic acid, 4=butoxybenzoic acid; Column, 50 mm x 4.6 mm i.d. ZirChrom®-EZ
These analytes are irreversibly adsorbed on
unmodified zirconia under these conditions
0
10
20
30
40
50
60
70
80
0 0.5 1 1.5 2Time (min.)
mA
U
1
32
4
OH
O
O
H3C
Reversed-Phase Characteristics
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
0.25 0.35 0.45 0.55φ
Log
Ret
entio
n Fa
ctor
AcetophenonePropiophenoneButyrophenoneValerophenone
LC Conditions: Mobile phase, indicated composition of ACN/Water; Flow rate, 2.0 ml/min.; Temperature, 35 oC; Injection volume, 5 µl; Detection at 254 nm; Column, 50 mm x 4.6 mm i.d. ZirChrom®-EZ
Solute Slope R2
Acetophenone -2.67 0.999Propiophenone -3.06 0.999Butyrophenone -3.51 0.998Valerophenone -4.03 0.997
φSk logk' log RP −= w
Ion-Exchange Characteristics
0.0
0.3
0.5
0.8
1.0
1.3
1.5
-1.70 -1.50 -1.30 -1.10 -0.90Log [NH4
+]
Log
Ret
entio
n Fa
ctor
N,N-dimethylbenzylamine N-methylbenzylamine
Benzylamine Benzene
Slope R2
Benzylamine -0.846 0.999N-methylbenzyamine -0.847 0.999
N,N-dimethylbenzylamine -0.840 0.999
N
LC Conditions: Mobile phase, 15/85 ACN/5mM MES, 25-100mM Ammonium acetate, pH 6.0; Flow rate, 2.0 ml/min.; Temperature, 35 oC; Injection volume, 5 µl; Detection at 254 nm; Column, 50 mm x 4.6 mm i.d. ZirChrom®-EZ
mIEX ]log[CConstantlogk' +−=
Chemical Stability
0.0
5.0
10.0
15.0
20.0
25.0
30.0
0 1000 2000 3000 4000 5000Column Volumes
Ret
entio
n Fa
ctor
(k')
Hydroxybenzoic acid Ethoxybenzoic acid
Propoxybenzoic acid Butoxybenzoic acid
Exposure and Evaluation Conditions: Mobile phase, 15/85 ACN/0.1M Nitric acid, pH 1.0, or 0.1M Ammonium hydroxide, pH 10.0; Flow rate, 1.0 ml/min.; Temperature, 30 oC; Injection volume, 5 µl; Detection at 254 nm; Column, 50 mm x 4.6 mm i.d. ZirChrom®-EZ
0.0
5.0
10.0
15.0
0 1000 2000 3000 4000 5000Column Volumes
Ret
entio
n Fa
ctor
(k')
4-octyloxybenzoic acid
pH 1.0 pH 10.0
LC/MS Bleed Study – TIC’s From Gradient Elution
LC Conditions: Mobile phase, 0-100% ACN from 0-30 minutes; Flow rate, 0.80 ml/min.; Temperature, 25 oC; Detection by ESI-MS.
Blank gradient – No column
installed
Gradient #1
Gradient #2
Extracted Spectra
Gradient #1
Gradient #2
TIC
TIC
Scan
Scan
Separation of Acidic Drugs with LC/MS-Friendly Mobile Phase
0
200
400
600
800
1000
1200
1400
1600
0 2 4 6 8 10
Time (minutes)
Abs
orba
nce
(mA
U)
5
2
4
3
1
LC Conditions: Column, 150 mm x 4.6 mm i.d. ZirChrom®-EZ; Mobile phase, A = 20mM ammonium acetate, pH 5.0, B = ACN; Flow rate, 1.0 ml/min.; Temperature, 35 oC; Injection volume, 10 µl; Detection at 254 nm.; Solutes: 1=Acetaminophen, 2=Naproxen, 3=Ketoprofen, 4=Fenoprofen, 5=Indomethacin
Time (min.) %A %B0 90 10
10 10 90
O
OH
O
ZirChrom®-PBD vs. ZirChrom®-EZ for Metanephrine by LC-MS
A: 20mM Ammonium acetate, pH 6.0
B: Acetonitrile
0
1
2
3
Metanephrine Normetanephrine
Gra
dien
t Ret
entio
n Ti
me
(min
.) ZirChrom-EZZirChrom-PBD
Time (min.) %A %B0 90 105 10 90
0
10
20
30
40
50
60
0 2 4 6 8 10Time (min.)
mA
U
LC Conditions: Column, 50 mm x 4.6 mm i.d. ZirChrom®-EZ; Mobile phase, 25/75 ACN/20mM ammonium acetate, pH 6.0; Flow rate, 1.20 ml/min.; Temperature, 35 oC; Injection volume, 10 µl; Detection at 254 nm.
Advantages of ZirChrom®-EZ Over Silica-and Other Zirconia-Based Phases
Advantages over silica reversed-phases…
Stable from pH 1-10, with similar temperature stability
Increased retention and loading for cationic compounds
Very different selectivity, particularly for cationic compounds
Advantages over other zirconia reversed-phases…
Does not require non-volatile buffers for Lewis base analytes
Increased retention for cationic compounds, particularly at low pH
Conclusion – ZirChrom®-EZ allows the use of LC/MS compatible mobile phases for the analysis of both acidic and basic analytes not previously possible on other zirconia-based reversed-phases
Acknowledgements
Cabot Corporation
LSER Comparison of ZirChrom-PBD and ZirChrom-EZ
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
N-benzy
lform
amide
Benzy
lalco
holPhe
nol
3-phen
ylprop
anol
p-chlorophe
nol
Acetop
henon
e
Benzo
nitrile
Nitroben
zene
Methylb
enzo
ateAnis
ole
p-chlorotoluen
e
p-nitrobe
nzyl c
hlorid
e
Benzo
phenone
Bromobenze
neNap
thalen
ep-xy
lene
p-dichloroben
zene
Benze
neToluen
e
Ethylben
zene
Propylben
zene
Butylben
zene
log
(k' S
olut
e/k'
ben
zene
)
ZirChrom-EZZirChrom-PBD
-2.5
-1.5
-0.5
0.5
1.5
2.5
m s a bLSER Coefficient
ZirChrom-PBD
ZirChrom-EZ
logk’=logk’0 + mVx + sπ*2 +a Σ α2 + b Σ β2
LC Conditions: Mobile phase, 40/60 ACN/Water; Flow rate, 1.0 ml/min.; Temperature, 30 oC; Injection volume, 5 µl; Detection at 254 nm; where mVx represents cavity formation and dispersion interactions, sπ*2 represents polar and dipolar interactions, a Σ α2 represents hydrogen bond acidity, b Σ β2 represents hydrogen bond basicity, and logk’0 is the intercept term.
Chemical Stability – pH 1.0
0.0
5.0
10.0
15.0
20.0
25.0
30.0
0 1000 2000 3000 4000 5000Column Volumes
Ret
entio
n Fa
ctor
(k')
Hydroxybenzoic acid Ethoxybenzoic acid
Propoxybenzoic acid Butoxybenzoic acid
0
2000
4000
6000
8000
10000
0 2500 5000 7500Column Volumes
Plat
e C
ount
(N, t
olue
ne)
Exposure and Evaluation Conditions: Mobile phase, 15/85 ACN/0.1M Nitric acid, pH 1.0; Flow rate, 1.0 ml/min.; Temperature, 30 oC; Injection volume, 5 µl; Detection at 254 nm; Column, 50 mm x 4.6 mm i.d. ZirChrom-EZ
Chemical Stability – pH 10.0
Exposure and Evaluation Conditions: Mobile phase, 15/85 ACN/0.1M Ammonium hydroxide, pH 10.0; Flow rate, 1.0 ml/min.; Temperature, 30 oC; Injection volume, 5 µl; Detection at 254 nm; Column, 50 mm x 4.6 mm i.d. ZirChrom-EZ
0
2000
4000
6000
8000
10000
0 2500 5000 7500Column Volumes
Plat
e C
ount
(N, t
olue
ne)
0.0
5.0
10.0
15.0
0 1000 2000 3000 4000 5000Column Volumes
Ret
entio
n Fa
ctor
(k')
4-octyloxybenzoic acid
min0 5 10 15 20 25
mAU
0
2.5
5
7.5
10
12.5
15
17.5
min0 5 10 15 20 25
mAU
0
2.5
5
7.5
10
12.5
15
17.5
“Fresh” Column
6,700 Column Volumes of pH 10 (TMAH)
7,000 Column Volumes of pH 1 (HNO3)
6,700 Column Volumes of pH 12 (TMAH)
ZirChrom®-PBD
Flushing Conditions: Mobile phase, 15/85 ACN/indicated buffer; Temperature, 30 oC; Evaluation Conditions: Mobile phase, 15/85 ACN/20 mM ammonium acetate, pH 4.0; Temperature, 30 oC. Injection volume, 5 µl; Detection at 254 nm; Solute: Hydroxybenzoic acid.
Summary of Stability Testing
HO
O
OH
Probe Solute =
Pharmaceutical Applications –Opioid Isomers
MorphineM.W. 285.33
HydromorphoneM.W. 285.33
O
NH
H
HO
H
HO
H
H
O
NH
H
HO
H
HO
CodeineM.W. 299.36
O
NH
H
HO
H
O
H
H
H 3C
O
NH
H
HO
H
OH 3C
HydrocodoneM.W. 299.36
0
20
40
60
80
100
120
140
160
0 1 2 3 4 5Time (minutes)
Abs
orba
nce
(mA
U)
1
2
3
4
Time (min.) %A %B0 90 105 10 90
LC Conditions: Column, 50 mm x 4.6 mm i.d. ZirChrom-EZ; Mobile phase, A = 20mM ammonium acetate, pH 6.0, B = ACN; Flow rate, 2.00 ml/min.; Temperature, 35 oC; Injection volume, 10 µl; Detection at 254 nm.; Solutes: 1=Morphine, 2=Hydromorphone, 3=Codeine, 4=Hydrocodone
