Synthesis of a New Lewis Acid Deactivated …Synthesis of a New Lewis Acid Deactivated Reversed-Phase Zirconia Stationary Phase for HPLC Bingwen Yan 1, Clayton V. McNeff 1, Danielle

Synthesis of a New Lewis Acid Deactivated Reversed-Phase Zirconia Stationary Phase for HPLC

Bingwen Yan 1, Clayton V. McNeff 1, Danielle Hawker 1,

Peter W. Carr 2

1 ZirChrom Separations, Inc.2 University of Minnesota

1-866-STABLE-1www.zirchrom.com

Pittcon 2004

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

2

Pore structureParticle sizeChemical flexibilitySurface homogeneityMechanical stabilitySwellibilityChemical stabilityThermal stabilityColumn efficiency

Silica+ ++ ++ +- -+ ++ +- -+

++

Zirconia+ ++ ++- -+ ++ ++ ++ +++

Polymericphase

++ +++ +- -+ +- -- -

++ excellent; + good; -- fair.

Advantages of Zirconia-Based Supports for HPLC

3

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

4

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, 45-525

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

6

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

ylpro

panol

p-chloro

phenol

Acetop

henon

e

Benzo

nitrile

Nitrob

enze

ne

Methylb

enzo

ateAnis

ole

p-chloro

toluene

p-nitrobe

nzyl c

hlorid

e

Benzo

phenone

Bromobe

nzene

Naptha

lene

p-xylen

e

p-dichloro

benze

neBen

zene

Toluene

Ethylben

zene

Propy

lbenze

ne

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 b

LSER 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. 7

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 0.55

φ

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

8

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' +−=

9

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

10

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

11

Extracted Spectra

Gradient #1

Gradient #2

TIC

TIC

Scan

Scan

12

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

13

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.

14

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

H3C

O

NH

H

HO

H

OH3C

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 15

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 pHConclusion – 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 16