Post on 13-Jul-2020
Characteristics and Advantages of Zirconia-Based Stationary Phases for
Use in Multi-Dimensional HPLC
Dwight Stoll and Clayton V. McNeff
ZirChrom Separations, Inc.
Multi-Dimensional Chromatography Workshop
Eastern Analytical Symposium, 2003
Outline
1. Review of theory and requirements – Why bother with multi-dimensional chromatography?
2. Why use zirconia for two-dimensional chromatography?
3. ZirChrom®-CARB and DiamondBond-C18TM– Very unique phases for RPLC
4. ZirChrom®-PBD, ZirChrom®-EZ and ZirChrom®-MS – Phases with mixed mode retention characteristics for ionizable analytes
5. Selectivity comparisons using ZirChrom®-CARB
6. Selectivity comparisons using ZirChrom®-PBD
7. An example two-dimensional HPLC separation of ten triazine herbicides using ZirChrom®-PBD and ZirChrom®-CARB
8. Conclusions
A Common Problem in HPLC
Sample composed of 20 components with
randomly distributed k’ values
150 mm x 4.6 mm i.d. column
0
0.1
0.2
0.3
0.4
0.5
0.6
0 5 10 15 20 25 30Time (min.)
AU
N = 10,000 plates/columnnc = 38, 12 unresolved
components
0
0.2
0.4
0.6
0.8
1
0 5 10 15 20 25 30Time (min.)
AU
N = 25,000 plates/columnnc = 60, 9 unresolved
components
Even with state-of-the-art HPLC, only
50% of the components in this
sample can be resolved !!!
Requirements and Advantages in Two-Dimensional HPLC
*Two conditions must be met for the technique to be considered “two-dimensional”1. Orthogonality of separation mechanisms – This is a requirement imposed on the
stationary phase chemistry
2. Separation gained in one dimension cannot be diminished by separation in the other dimension
If these two conditions are satisfied, the maximum total peak capacity of the two-dimensional system is:
21 cccTotal nnn ×=
Giddings, J. C. Multidimensional Chromatography: Techniques and Applications; Marcel Dekker: New York, 1990
What Can We Expect From a Two-Dimensional Separation Based on Known One-Dimensional Data?
0.01.0
2.03.0
4.05.06.0
7.08.0
9.010.0
0.0 2.0 4.0 6.0 8.0 10.0
Condition A
Con
ditio
n A'
0.01.02.0
3.04.05.06.07.0
8.09.0
10.0
0.0 2.0 4.0 6.0 8.0 10.0
Condition A
Con
ditio
n B
Condition A’ is the same as Condition A except that the retention has been
varied randomly by 5%
Condition B assumes no relationship to Condition A
This scenario is ineffective in two-dimensional HPLC
This scenario has a higher probability of success in two-dimensional HPLC
Comparison of Variables Affecting Selectivity
logk' (50/50 ACN/H2O)
0.00 1.00
0.00
1.00
2.00
ODS vs. ZirChrom®-PBD
ZirChrom®-CARB vs. ZirChrom®-PBD
30% ACN vs. 50% ACNMeOH vs. THF
2O)
0.00
1.00
2.00
logk' (THF/H0.00 1.00
R2=0.896SD=0.17R2=0.989
SD=0.05
logk' (PBD-ZrO2)-2.00 -1.00 0.00 1.00
0.00
1.00
R2=0.973SD=0.09
logk' (PBD-ZrO2)-1.00 0.00
0.00
1.00
R2=0.385SD=0.42
80oC vs. 30oC
logk' (C18, 30 oC)0 1 2 3
0
1
2 R2=0.995SD=0.03
Stationary phase type can have a very large effect on selectivity
Why Zirconia-Based Phases - Advantages for Multi-Dimensional RPLC
1. Stability - Enables the use of otherwise extreme conditions for adjustment of selectivity
2. Stationary phase chemistry – Allows the user to explore a wide range of chemistry to obtain the largest changes in selectivity
A. Carbon-clad zirconia phasesB. Polymer coated phases with mixed mode charateristics
I. Reversed-phaseII. Ion-exchange
3. Speed – Thermal stability allows for faster multi-dimensional separations
ZirChrom HPLC Columns
Part # Packing ModeDB01 DiamondBond®-C18 Reversed-PhaseEZ01 ZirChrom®-EZ Reversed-Phase (Lewis Acid Deactivated)MS01 ZirChrom®-MS Reversed-Phase (Lewis Acid Deactivated)ZR01 ZirChrom®-CARB Reversed-PhaseZR02 ZirChrom®-PHASE Normal Phase and SECZR03 ZirChrom®-PBD Reversed-PhaseZR04 ZirChrom®-WCX Weak Cation-ExchangerZR05 ZirChrom®-WAX Weak Anion-Exchanger and Sugar AnalysisZR06 ZirChrom®-SAX Strong Anion-ExchangerZR07 ZirChrom®-SHAX Strong Hydrophilic Anion-ExchangerZR08 ZirChrom®-PEZ Cation-Exchanger for ProteinsZR09 ZirChrom®-PS Reversed-Phase
ZirChrom®-CARB and DiamondBond®-C18
Z irc o n ia S u b s t ra t e
C C C C C C CC C C C C C C
Z irc o n ia S u b s t ra t e
C C C C C C CC C C C C C C
Zirconia Substrate
C C C C C C CC C C C C C C
CH3
(CH2)16
CH2
CH3
(CH2)16
CH2
CH3
(CH2)16
CH2
ZirChrom®-CARBDiamondBond-C18TM
-1.50
-1.00
-0.50
0.00
0.50
1.00
1.50
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Log k'
Retention of Different Solutes on ODS,ZirChrom®-PBD and ZirChrom®-CARB
ODS
ZirChrom®-PBD
ZirChrom®-CARB
1. N-benzyl formamide 6. Acetophenone 11. Benzene 16. Bromobenzene 21. Propylbenzene 2. Benzylalcohol 7. Benzonitrile 12. p-chlorotoluene 17. Naphthalene 22. n-butylbenzene3. Phenol 8. Nitrobenzene 13. p-nitrobenzyl chloride 18. Ethylbenzene 4. 3-phenyl propanol 9. methyl benzoate 14. Toluene 19. p-xylene5. p-chlorophenol 10. Anisole 15. Benzophenone 20. p-dichlorobenzene
C-C-C-C-C-C-C-C-C-C-C-C
p-xylene ethylbenzene
αODS=1.03
αC-Zr=1.58
C-C-C-C-C-C-C-C-C-C-C-C
Selectivity and Shape: Isomeric Analytes
Selectivity of ZirChrom®-CARB and SB-C18 for 18 Substituted Phenols
-1.50
-1.00
-0.50
0.00
0.50
1.00
1.50
-0.50 0.00 0.50 1.00Log k' - ZirChrom-CARB
Log
k' -
SB-C
18
LC Conditions: Mobile phase, 45/55 ACN/10mM phosphoric acid, pH 2.4; Flow rate, 2.0 ml/min.; Temperature, 40 oC – Data courtesy of Adam Schellinger
ZirChrom®-PBD and ZirChrom®-PS
~20
CH2 CH CH CH2 CH2 CH CH CH2 CH2 CHCH
CH2 CH CH CH2 CH2 CH
CH2
CH2CH
Z i rc o n ia S u b s t ra te
~20
CH2 CH CH CH2 CH2 CH CH CH2 CH2 CHCH
CH2 CH CH CH2 CH2 CH
CH2
CH2CH
~20
CH2 CH CH CH2 CH2 CH CH CH2 CH2 CHCH
CH2 CH CH CH2 CH2 CH
CH2
CH2CH
Z i rc o n ia S u b s t ra te
Z i rc o n ia S u b s t ra t eZ i rc o n ia S u b s t ra t e
ZirChrom®-PBD
ZirChrom®-PS
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
ZirChrom®-EZ
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
1 Chemisorb Ethylenediamine N,N,N’,N’-tetra(methylenephosphonic)acid (EDTPA) to the zirconia surface.
2 Quaternize amines on the zirconia surface with allyl iodide.
3 Coat polybutadiene (PBD) on the chelator-modified zirconia surface and crosslink PBD with allyl group and PBD itself using dicumyl peroxide as initiator.
ZirChrom®-MS
Allyl iodidequaternization
H OP
O
OP
N
NP
O
O
O H
OP
O H
O O H
O
O
n
EDTPA
zirconia Chemisorptions
PBD/ dicumyl peroxide
Crosslinking n
-OP
O
OP
N
NP
O
O
OH
OP
OH
O O-
O
O
+
+
n
-OP
O
OP
N
NP
O
O
OH
OP
OH
O O-
O
O
PB D
PB D+
+
Selectivity Study of Eleven Antidepressants
Dai, J.; Yang, X.; Carr, P. W. Journal of Chromatography, A 2003, 1005, 63-82
N
N+
Cl
NH
O-
Chlordiazepoxide
N
N N
Desipramine Nortriptyline
O
NN
N
SN
O
O
S
Amitriptyline
Doxepin
Buclizine Hydroxyzine Thioridazine
Imipramine
Perphenazine
N
NN
ClN
N
Cl
NN
OOH
SN
S
N
-1.0-0.8
-0.6-0.4
-0.20.0
0.20.4
0.60.8
1.0
-1.00 -0.50 0.00 0.50 1.00 1.50
log k' - Silica Brand B
log
k' -
Silic
a B
rand
AR2 = 0.84
Std. Dev. = 0.15
LC Conditions: Mobile phase, 72/28 MeOH/25 mM ammonium phosphate, pH 6.0; Flow rate, 1.0 mL/min; Temperature, 35 °C; UV detection at 254 nm.
Selectivity for Antidepressant Compounds on ODS Brand A vs. Brand B
Selectivity for Antidepressant Compounds on ZirChrom®-PBD vs. ODS
LC Conditions: Mobile phase, 72/28 MeOH/25 mM ammonium phosphate, pH 6.0; Flow rate, 1.0 mL/min; Temperature, 35 °C; UV detection at 254 nm.
-1.0
-0.8
-0.6
-0.4
-0.2
0.00.2
0.4
0.6
0.8
1.0
-1.00 -0.50 0.00 0.50 1.00 1.50
log k' - ZirChrom-PBD
log
k' -
Silic
a B
rand
AR2 = 0.09
Std. Dev. = 0.66
0%
20%
40%
60%
80%
100%
15mM 25mM 35mM 50mM
[NH4+mM]
k'IE
X/k'
of p
-pro
pylb
enzy
lam
ine Ace Alltima PBD-ZrO2
15 25 35 50
Significantly Higher Ion-Exchange Retention of Amines on ZirChrom®-PBD
Leads To Selectivity Differences
RPIEX kkk ''' −=
OZr
OZr
OZr
OZr
OZr
OZr
OZr
OZr
O
OOHO-
H2O OH OHH2O H2OO-P OHOO-
PBD Coating — Reversed-Phase (RP) Moieties
Lewis Base Anions — Ion-Exchange (IEX) Sites
Zr-L-:X+ + A+ = Zr-L-:A+ + X+
Zr-O-:X+ + A+ = Zr-O-:A+ + X+
A+: analyte cation, X+: counterion, L-: adsorbed Lewis base anion.
Retention of Basic Analytes on ZirChrom®-PBD
PBD coatingA+
A+
A+
A+Adsorbed Lewis base
anion
The very large s.d. for ZirChrom®-PBD vs. all other phases indicates a dramatic difference in selectivity from ODS (Antidepressant solute set)
ZirChrom®-PBD is Very Different Compared to All ODS Phases
ACEIN
ER
EX
EC
RX
SBALLTIM
A
INER
EC
SB
PBD-ZrO20.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8S
tand
ard
Dev
iatio
n
ZirChrom®-MS Compared to ODS
LC Conditions: Mobile Phase, 72/28 MeOH/25mM Ammonium phosphate, pH 6.0; Flow Rate, 1.0 ml/min.; Temperature, 35 oC; Injection Volume, 5 µl; Detection by UV at 254 nm; Solutes from left to right: Methapyrilene,Pyrilamine, Tripelennamine, Brompheniramine, Desipramine, Nortryptyline, Doxepin, and Amitryptyline.
R2 = 0.340
1.00
1.50
2.00
2.50
3.00
3.50
4.00
-1.50 -1.00 -0.50 0.00 0.50 1.00
ln k' Silica C18
lnk'
ZirC
hrom
-MS Basic Compounds
are much more retained on ZirChrom-MS than on Silica C18 and have very different chromatographic selectivity.
An Example 2DLC Separation - TenTriazine Herbicides
N N
NHNH3C
HN CH3
Cl
CH3
Atrazine
N N
NHN CH3
HNH3C
Cl
Simazine
N N
NHN CH3
CH3
Cl
HNH3C
CH3
Propazine
N N
NH3CO
HN
HN CH3
CH3
CH3
CH3
Prometon
CH3
NO2
H3C
2-nitroxylene
N
N
N
O
CH3
NH
CH3
NH
CH3
H3C
Atraton
N N
NCl
NH
CH3
HN
CH3
CH3
CH3
Terbuthylazine
N N
NCl
NH
CH3
HN
CNCH3
CH3
Cyanazine
N N
NHN CH3
HNH3C
SCH3
Simetryne
N N
NHNH3C
HN CH3
S
CH3
CH3
Ametryne
N N
NHN CH3
CH3
S
HNH3C
CH3
CH3
Prometryne
N N
NS
NH
CH3
HN
CH3
CH3
CH3
CH3
Terbutyrne
2DLC Separation of Ten Triazine Herbicides
0
2 0
4 0
6 0
8 0
1 0 0
0 5 1 0 1 5 2 0T im e (m in .)
mA
U
1st Dimension Conditions: Column, 50 mm x 2.1 mm i.d. ZirChrom®-PBD; Mobile phase, 20/80 ACN/Water; Flow rate, 0.08 ml/min.; Injection volume, 20 µl; Temperature, 40 oC
N N
NHN CH 3
HNH3C
ClSimazine
2nd Dimension Conditions: Column, 50 mm x 2.1 mm i.d. ZirChrom®-CARB; Mobile phase, 20/80 ACN/Water; Flow rate, 7.0 ml/min.; Injection volume, 15 µl; Temperature, 150 oC; 1st dimension sampling frequency, 0.1 Hz
Conclusions
1. The importance of differences in selectivity between conditions selected for different dimensions in multi-dimensional chromatography cannot be emphasized enough.
2. The most dramatic changes in selectivity are most easily brought about by changing the stationary phase.
3. Zirconia-based reversed phases (there are 5 of them) offer dramatically different selectivity relative to conventional silica-based phases for several classes of analytes
Acknowledgements
ZirChrom Separations, Inc.Bingwen Yan
University of MinnesotaXiqin Yang
Jun Dai
Adam Schellinger