New Stable Chemically Bonded CarbonStationary Phases for HPLCAngelos Kyrlidis, Lynn Toomey, and Elena Khmelnitskaia,

Cabot CorporationDwight Stoll, Clayton V. McNeff, and Peter W. Carr

ZirChrom Separations, Inc.

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Outline

• Advantages of Stable Phases• Development of a New Type of Reversed Phase Column:

DiamondBond®-C18• Selectivity Comparison of DiamondBond®-C18 and Other

DiamondBond Phases with ODS Silica• Applications• Summary

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Why Stable Phases?

Advantages of ExtraordinaryChemical Stability

pH < 1

pH Stability

pH > 13

Clean withconc. Acids

Suppress Ionsfor Acids

Suppress Ionsfor Amines

SanitationDepyrogenation

LowerPressure Drop

Thermal Stability

Less OrganicSolvent

ThermallyOptimize

Selectivity

Less Wearand Tear

Higher FlowFast Analysis

More RobustAnalysis

Easier MethodDevelopment

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Stable Stationary Phases have advantages in terms ofSelectivity, Column Lifetime, and Analysis Time

Improving the Stability of HPLC Phases: History

l Pure organic polymersl Silica “hardened” by coating with alumina or zirconial Pure carbonl Silica improvements

– Sterically bulk & bidentate ligand– Polymer coated silica– Hybrid organic-inorganic silicaceous composite phase

l Polymer coated porous alumina and zirconial Carbon coated zirconial Chemically bonded carbon-coated zirconia

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ZirChrom® Particle Properties

Characteristic PropertySurface Area (m2/g) 22Pore Volume (cc/g) 0.13Pore Diameter (Å) 250-300Porosity 0.45Density (g/cc) 5.8 (2.5x silica)Particle Diameter (µ) 3.0

ZirChrom®-Carb particlesare prepared by coating basezirconia particles with a thinlayer of carbon using achemical vapor depositionprocess NH2 Y + 2 HA + NaNO2 = AN≡N Y + 2 H2O + NaA

Diazonium SaltCarbon Clad Zirconia Modified Carbon Clad Zirconia

+ N+

N Y

Bonding Reaction on Carbon Clad Zirconia

- C18

DiamondBond®: A New Family of Stable Phases

ZrO2DB-C18

ZrO2DB-C8

ZrO2

NHO

NHO

NHO

DB-Amide18

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DiamondBond-C18 Stabilityk'

0

1

2

3

4

5

6

7

8

0 250 500 750 1000 1250 2000

Column Volumes

k' Butylbenzene (0.5M Nitric Acid)

k' Butylbenzene (1.0M NaOH)

k' Butylbenzene (200 oC)

LC Conditions: Base Stability—DiamondBondTM Phase A, 30 x 4.6 mm id; Mobile phase, 50/50 ACN/Water; Flow rate, 1.0 ml/min.;Temperature, 30 oC; Injection volume, 5ul; Detection at 254nm.Acid Stability—DiamondBondTM Phase A, 50 x 4.6 mm id; Mobile phase, 50/50 ACN/Water; Flow rate, 1.0 ml/min.;Temperature, 30 oC; Injection volume, 5ul; Detection at 254nm.Temperature Stability-- DiamondBondTM Phase B, 50 x 4.6 mm id; Mobile phase, 50/50 ACN/Water; Flow rate, 1.0ml/min.; Temperature, 30 oC; Injection volume, 5ul; Detection at 254nm.

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0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

1

3

1

2

2

4

4

3

DiamondBond-C18

ZirChrom-Carb

Analytes

1: Acetone2: Benzonitrile3: Ethylbenzene4: Methylbenzoate

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Test Chromatogram on DB-C18

LC Conditions: Column dimension, 50 x 4.6 mm id.; Mobilephase, 37.5/5/57.5 ACN/THF/Water; Temperature, 60 oC; Flowrate, 1.0 ml/min.; Injection volume, 5 µl; Detection at 254 nm.

>100000 plates/m

22 Non-Ionizable Solutes

Mobile phase, 40/60 Acetonitrile/Water; Flow rate, 1.0 ml/min.;Temperature, 30 oC; Detection at 254nm; 5 ul Injection volume.

NonpolarBenzene Toluene Ethylbenzene p-xylene Propylbenzene Butylbenzene

PolarBr Cl

Cl

OO

O

CN NO2 NO2Cl

NO2

OBromobenzene p-Dichlorobenzene Anisole Methylbenzoate Napthalene Acetophenone

Benzonitrile Nitrobenzene p-Nitrotoluene p-Nitrobenzyl Chloride

O

Benzophenone

HB DonorOH OH NH H

OOH OH

Cl

Benzylalcohol 3-Phenyl Propanol N-Benzyl Formamide Phenol p-Chlorophenol

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Selectivity Comparison

-1.5

-1

-0.5

0

0.5

1

1.5

2

Diamondbond-C18

ODS-1

Unmodified Carbon coated zirconia

Effect of Polar Embedded Amide

0.1

1

10

100

0.1 1 10 100Absolute Retention on Silica C18

Abs

olut

e R

eten

tion

on S

ilica

RP

Am

ide

Phenol

p-Chlorophenol

Benzylalcohol

N-Benzylformamide3-Phenylpropanol

0.1

1

10

100

0.1 1 10 100Absolute Retention on Carbon C18

Abs

olut

e R

eten

tion

on C

arbo

n R

PA

mid

e

p-Chlorophenol

Phenol

N-Benzylformamide

Benzylalcohol

3-Phenylpropanol

Silica Amidevs. C18

Carbon Amide vs.C18

RPAmide shows increased retention of HB Donorson silica and carbon-based phases

Why are Bonded Carbons Unique?

l Carbon surfaces have π-electrons which increaseretention of certain types ofanalytes:– fused polyaromatics (e.g.

naphthalene, etc)– polar molecules (e.g. amides,

ketones, alcohols, etc)l Surface modified carbon surfaces

combine some of theseinteractions with interactionsspecific to the bonded surfacegroups.

l Bonded Carbons maintain thehigh pH and thermal stability thatis inherent in the C-C bond.

π - electrons

Bonded Carbon

Carbon

Shape Makes a Difference

p-xylene

C-C-C-C-C-C-C-C-C-C-C-C C-C-C-C-C-C-C-C-C-C-C-C

ethylbenzene

α ODS=1.03

α CARB=1.58

α DB-C18=1.22

Shape Selectivity: Anabolic Steroids

min0 1 2 3 4 5

mAU

0

1

2

3

4

5

6

7

VWD1 A, Wavelength=215 nm (G:\HPCHEM\DATA\MDARCHIV\08220275.D)

14

3

2

LC Conditions: Column, 150 x 4.6 DiamondBond-C18; Mobile phase, 60/40 ACN/Water; Flow rate, 2.0 ml/min.; Temperature, 100 oC; Injection volume, 10ul; Detection at 215nm; Solutes: 1=Epietiocholanolone, 2=Etiocholanolone, 3=Androsterone, 4=Epiandrosterone

Anabolic Steroids Separationin less than 3 minutes!

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High pH Stability - Beta Blockers

min0 0.5 1 1.5 2 2.5 3 3.5

Analytes1 - Atenolol2 - Metoprolol3 - Oxprenolol4 - Alprenolol

LC Conditions:20/20/60 ACN/THF/200 mM TMAH and 200 mM NaCl, pH 13.3Flow Rate: 1 ml/min. Temperature: 75 oC. Injection Volume: 5 ulDetection: 254 nm.

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High Temperature Stability - Speed

min0 10 20 30 40

4

1

53

2

6

43

Resolution (min; 3,4) =2.2Temperature = 21 oCFlow Rate = 1.4 ml/min.ACN/Buffer = 18.5/81.5Analysis Time = 43 min.

Resolution (min; 3,4) = 2.2Temperature = 80 oCFlow Rate = 3.2 ml/min.ACN/Buffer = 8.8/91.2Analysis Time = 21 min.

min0 10 20 30 40

3

12

4

5

6

34

Analytes:1 = Barbital2 = Butabarbital3 = Pentobarbital4 = Carbromal5 = Secobarbital6 = Methohexital

Mobile Phase: ACN/5mM Ammonium phosphate, pH 7.0

Pressure drop =195 bar

Application in LC/MS/MS

THC in Saliva by LC/MS/MS=

l Blue – THC (tetrahydrocannabinol parent drug)l Red – D3 THC (Internal Standard)l Column – 50mm X 4.6mm DBC18

– 80o C @ 1.5 mL/min– Solvent A – 20mM NH4CH3CO2 in

70% Acetonitrile, 30% aqueous(0.1% acetic acid, pH 4.5)

– Solvent B – Acetonitrilel Isocratic 35% A, 65%B – 25 ul injection

= Data Courtesy R. Clouette - Clinical Reference Laboratories

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

Time, min

0

1000

2000

3000

4000

5000

6000

7000

8000

Inte

nsi

ty, c

ps

0.86

XIC of +MRM(2 pairs):315.2/193.2 amu

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Summary

l The effect of ligand type on retention for bonded carbon phasesis similar to that for bonded silicas– Unique surface chemistry enables unique separations

l The carbon-carbon attachment bond is extremely stable– Low pH and High pH applications– High Temperature / Fast HPLC

l Both “normal” and high pH, high temperature applications arepossible on these new materials– LC/MS pharmaceutical applications enabled by this

technology

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