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