pittcon 2002 oral presentation - zirchrom.com · title: pittcon 2002 oral presentation author:...

Characterization of an EfficientNo-Bleed Carbon-Based HPLC Support

for Pharmaceutical Separations

byPeter W. Carr, Clayton V. McNeff,

Dwight R. Stoll, Danielle R. Hawker,Angelos Kyrlidis (Cabot), Greg Gaudet (Cabot)

and Shane Needham (Alturas Analytics).

2002 ZirChrom Separations, Inc.

OUTLINE

• Analytical Advantages of Column Stability.• Production of ZirChrom®-CARB.• Selectivity Comparison of ZirChrom®-

CARB, Hypercarb and Other RP-Zirconias. • MS Column Bleed Comparison: ZirChrom®-

CARB and Leading Silica Columns.• Conclusions (CARB shows ultra-low bleed).

Analytical Advantages of Column Stability

Extraordinary

Chemical Stability

ThermalStability

pHStability

pH > 13pH < 1ThermallyOptimize

Selectivity

LowerPressure

LessOrganicSolvent

EasierMethodDevel-opment

HigherFlowRate: Fast

Analysis

MoreRobustAnalysis

LessWear and Tear

Sanitation/ Depyro-genation

IonSupression

for Amines

Ion Supression

forAcids

Cleaning with

Conc.Acid

NO COLUMN BLEED

Why is Bleed Bad?

At same mass and elution time it can obscure peak of interest and make quantitation difficult, increases L.O.D. and L.O.Q.At same mass and elution time it can cause a chase for “ghost” impurities in sample.At same elution time but different mass can cause ionization problems for analyte of interest.

Proprietary Synthesis of Carbon-Clad Zirconia

Lewis Acid Site

O O OOOOOO OOO

Zr Zr Zr ZrZrZrO O O

O O OHH__ __ __ __ __ __ __

Bulk Zirconia

Comparison of ZirChrom CARB and Hypercarb

92,00041,000N (toluene) a2.77.8k’ (toluene) a

6988Area/Vol. Mobile Phase (m2/g)

0.430.61Particle Porosity

0.140.7Pore Volume (cc/g)

240270Pore Diameter (A)ZirChrom CARBHYPERCARBCHARACTERISTIC

a. 40/60 ACN-water 30 oC

22 Non-ElectrolyteProbe Solutes

B enzene Toluene Ethy lbenzene p-xy lene Propy lbenzene B uty lbenzene

CN N O 2 N O 2Cl

OB romobenzene p-D ichlorobenzene A nisole M ethy lbenzoate N apthalene A cetophenone

B enzonitrile N itrobenzene p -N itrotoluene p-N itrobenzy l Chloride

B enzophenone

OH OH N H H

OH O H

B enzy lalcohol 3-Phenyl Propanol N -B enzyl Formamide Phenol p-Chlorophenol

-1.5000

-1.0000

-0.5000

0.0000

0.5000

1.0000

1.5000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Comparison of Selectivity of ODS,ZirChrom®-PBD &-CARB

ODSZirChrom®-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

Chemical Structure of ZirChrom®-CARB

CHCH2CH3CH3CH2CH

CH3 CH3

OH OH OH

Silica Substrate

CHCH2CH3CH3CH2CH

CH3 CH3

ZirChrom®-CARB

Zirconia Substrate

C C C C C C CC C C C C C C

Minutes2 3 4 5 6 7 8

Dinitroaniline Herbicide Separation (EPA Method 627)

Minutes4 6 8 10 12 14 16

ODS CarbonNO2

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

p-xylene ethylbenzeneαODS=1.03

αC-Zr=1.58

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

General Conditions of M.S. Bleed Study

SCIEX APCI 3000 triple quad in single quad mode at 400 oC.Total ion current 60-600 amu.Spectra is sum of 30-35 scans in time window.Mobile phase gradient 0-100% ACN/buffer in 15 min. plus 14 min. hold at 100%.pH 7 = water only.pH 9 = 2mM NH4Ac + NH4OH.Note pump-column-MS. No injector used.

Gradient Blank at pH 9 and 55 oC8.10e7 cpsTIC of +Q1: from blank1 ��

��

20.125.6 26.8

5 10 15 20 25 30Time, min

8.86e4 cps+Q1: 15.98 min (44 scans) from blank1

98.1 124.1

185.2256.3

371.2391.2

532.5 556.5576.5

2 4 1 .2

1 7 1 .2

8 0 .1

100 150 200 250 300 350 400 450 500 550m/z, amu

M.S. shows typical numerous, ubiquitous phthalate contaminants.Spectra are same from start to finish of gradient.

Return to 0% ACN

ZirChrom-CARB at pH 9 and 55 oC3.42e7 cpsTIC of +Q1: from ZirchromCarb-2 ��

��

10.7 13.014.8 16.6

5 10 15 20 25 30Time, min

6.98e4 cps+Q1: 16.44 min (53 scans) from ZirchromCarb-2

279.1315.2 371.4 391.3

532.3576.5

2 4 1 .3

8 0 .0

1 7 1 .1

100 150 200 250 300 350 400 450 500 550m/z, amu

Total ion chromatogram and mass spectra with ZirChrom CARB nearly identical to blank gradient. No new discernible masses or peaks due to column.

Return to 0% ACN

Blank Gradient at pH 7 & Ambient Temp.1.70e8 cpsTIC of +Q1: from blank1 ��

��

8.4 10.4 12.014.3

15.7 17.2 21.323.2

24.5 25.5 27.2

5 10 15 20 25 30Time, min

2.63e5 cps+Q1: 9.41 min (31 scans) from blank1

298.2 333.1

439.5 471.3 490.3515.4

5 9 9 .2

3 1 5 .32 5 6 .4

100 150 200 250 300 350 400 450 500 550m/z, amu

Total ion current appears cleaner and m.s. looks different thanblank at pH 9 but this is due to changes in ionization (no NH4

M.S. Grade SiO2 (A.) at pH 7 and Ambient Temperature

1.34e8 cpsTIC of +Q1: from HSc18-1��

��

9.3 11.8

19.3 21.9 23.5 25.2 26.6 27.9

5 10 15 20 25 30Time, min

2.37e6 cps+Q1: 1.30 min (27 scans) from HSc18-1

114.2 149.2 172.3 217.1 259.1 285.1 308.1

405.2 445.1 509.4

5 9 9 .2

8 7 .2

100 150 200 250 300 350 400 450 500 550m/z, amu

71.0 124.2149.1

185.3 241.3 267.1

405.1 445.1 490.3 519.3

3 7 0 .4

3 8 3 .4

5 9 9 .2

100 150 200 250 300 350 400 450 500 550m/z, amu

New “humps” in total ion chromatogram and new masses are evident in spectra.

2.50e8 cpsTIC of +Q1: from Xterra-1 ��

��

3.65.8 9.1 10.8 12.4 14.6

15.6 17.1 21.3 24.0 26.0 27.6

5 10 15 20 25 30Time, min

1.18e6 cps+Q1: 12.96 min (48 scans) from Xterra-1

124.2149.1

185.2219.1 241.3 278.3 298.2 371.4 405.2 446.4 471.4 490.3 515.4

551.15 9 9 .2

100 150 200 250 300 350 400 450 500 550m/z, amu

M.S. Grade SiO2 (B.) at pH 7 and Ambient Temperature

Note huge increase in mass 69 (identity unknown).

Blank Gradient at pH 9 and 55 oC5.47e7 cpsTIC of +Q1: from blank1A

��

6.5 8.5 9.3

15.1 16.6 18.3 19.9 23.2 25.5 27.8

5 10 15 20 25 30Time, min

1.35e5 cps+Q1: 15.39 min (46 scans) from blank1A

315.1 369.3 427.2 471.3

515.4 559.3 579.4

2 5 6 .3

8 1 .1

100 150 200 250 300 350 400 450 500 550m/z, amu

M.S. different than blank gradient at pH 7 probably due to ionization differences.

1.36e8 cpsTIC of +Q1: from HSc18-1

��

4.98.1 9.1

17.4 18.7 19.9 20.8 22.1 25.2 27.2

5 10 15 20 25 30Time, min

73.0 97.2127.0

149.1185.3 241.3 283.4

427.4 471.4 490.3 515.4 559.3

3 7 0 .4

3 2 9 .4 3 8 3 .4

100 150 200 250 300 350 400 450 500 550m/z, amu

M.S. Grade SiO2 (A.) at pH 9 and 55 oC

Note big peak at 12-15 min. in total ion chromatogram and new peaks in mass spec.

M.S. Grade SiO2 (B.) at pH 9 and 55 oC

4.99e7 cpsTIC of +Q1: from Xterra-1 ��

��

7.0 8.7 9.510.4

15.418.8 20.6

22.1 23.6 24.6 25.6 28.1

5 10 15 20 25 30Time, min

1.40e6 cps+Q1: 15.01 min (56 scans) from Xterra-1

97.2124.0

148.9185.1

219.4 256.3 315.2 355.0 428.5 459.3 490.3 559.3

100 150 200 250 300 350 400 450 500 550m/z, amu

Note new peak in total ion chromatogram and increase in mass 69.

ZirChrom-CARB at pH 9 and 55 oC3.42e7 cpsTIC of +Q1: from ZirchromCarb-2 ��

��

10.7 13.014.8 16.6

5 10 15 20 25 30Time, min

6.98e4 cps+Q1: 16.44 min (53 scans) from ZirchromCarb-2

279.1315.2 371.4 391.3

532.3576.5

2 4 1 .3

8 0 .0

1 7 1 .1

100 150 200 250 300 350 400 450 500 550m/z, amu

Total ion chromatogram and mass spectra with ZirChrom CARB nearly identical to blank gradient. No new discernible masses or peaks due to column.

Return to 0% ACN

Conclusions

• ZirChrom-CARB is ultra-durable and efficient.

• ZirChrom-CARB is stable at the extremes of pH and at 200 oC.

• ZirChrom-CARB has radically different selectivity.

• ZirChrom-CARB’s durability allows for ultrafast (< 1 min.) separations at elevated temperatures.

• ZirChrom-CARB shows “No-Bleed” by MS under highly aggressive LC conditions(pH 9, 55 oC).

ZirChrom Technical Presentations and Posters

Time Date Room # Title Who Program #

8:50-9:10 am March 18 (Monday) 245

Chromatographic Characterization of an Efficient No-Bleed Carbon-based HPLC Support for Pharmaceutical Separations

Clayton V. McNeff, Dwight Stoll, Peter Carr, Bingwen Yan 160

9:30-9:50 am March 18 (Monday) 245

Development of a Novel Class of Ultra-stable Covalently Bonded Carbon-Based HPLC Phases with Unique Chromatographic Selectivity for Use in Pharmaceutical Analysis

Gregory Gaudet, Peter Carr, Clayton V. McNeff, Dwight Stoll, Danielle Hawker, Angelos Kyrlidis 162

9:10-9:30 am March 19 (Tuesday) 243Development of Carbon Based Chiral Separation HPLC Column

Feng Gu, Angelos Kyrlidis, Clayton V. McNeff, Steve Ponto, Peter W. Carr, John A. Blackwell 523

10:00-12:30 pm March 19 (Tuesday)Aisle 100

Exhibit Floor

A New Generation of Covalently Bonded Stationary Phases on Carbon Clad Nonpourous Zirconia for Fast High Temperature Liquid Chromatography

Clayton V. Mc Neff, Gregory Gaudet, Angelos Kyrlidis, Peter Carr, Bingwen Yan, Alan McCormick 1989P

8:30-8:50 am March 20 (Wednesday) 244Ultra-fast Liquid Chromatography: The Importance of System Temperature Jon D. Thompson, Peter W. Carr 760

8:50-9:10 am March 20 (Wednesday) 244

A Novel Approach to Column Heaters for Application to Ultra-fast High Temperature (UFHTLC) and Thermally Tuned Tandem Column (T3C) Liquid Chromatography.

Peter W. Carr, Jon D. Thompson, Yun Mao, Carl W. Sims, Yuri E. Gerner, Tom Thielen 761

9:30-9:50 am March 21 (Thursday) 235-236

Optimizing the Separations of Basic Compounds in Mixed-Mode Separations by Understanding the Role of Lewis Acidity of Zirconia Based Phases Xiqin Yang, Peter Carr 1089

10:00-12:30 pm March 21 (Thursday)Aisle 100

Exhibit Floor

One-Step Monoclonal Antibody Purification Method Using Preparative Porous Ethylenediamine-N, N'-tetra(methylphosphonic) Acid Modified Zirconia

Clayton V. McNeff, Dwight Stoll, Peter Carr, Danielle Hawker, Anuradha Subramanian 2233P

10:00-12:30 pm March 21 (Thursday)Aisle 100

Exhibit Floor

Stable RPLC Phases: Are They Really Stable? A Comparison of the Stability, Symmetry, Selectivity and Efficiency of Leading Stable Phases at Elevated pH

Clayton V. McNeff, Dwight Stoll, Peter Carr, Danielle Hawker 2323P

Thanks very muchfor listening!

ZirChrom Separations & Cabot Corporation

Partners in Chromatography

For more information and web access to the free Buffer Wizard: www.zirchrom.com

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

Visit Us at Booth 3036

Selectivity Comparison of ZirChrom-CARB

to Other RP Zirconia Phases

LC Conditions: Mobile phase, 40/60 Acetonitrile/50mM phosphate at pH 3.2), Flow rate, 1.0 ml/min., Temperature = 30 oC, Detection at 254nm, 5µl Injection volume. (LC-GC, Vol 13, No. 9, September 1995, 720-726.)Columns Tested: DiamondBondTM-C18, Luna® C18,ZirChrom®-PBD, GammabondTM, XterraTM, Hamilton PLRPTM

Phase Comparison by Covarianceof log k’-log k’ Plots*

ZirChrom-CARB most different from ODS.

* Column names are the trademarks of their respective manufacturers.

CARB DB-C18 PBD RP18 C18 (2) PLRP-S RP-1ZirChrom-CARB 1

DiamondBond-C18 0.80 1ZirChrom-PBD 0.51 0.90 1Xterra RP18 0.53 0.85 0.90 1Luna C18 (2) 0.53 0.86 0.93 0.97 1

PLRP-S 0.60 0.90 0.93 0.92 0.96 1Gammabond RP-1 0.52 0.88 0.96 0.97 0.98 0.95 1

Efficiency Comparison ofLeading HPLC Columns*

20,000

40,000

60,000

80,000

100,000

120,000

140,000Zi

18 †

* Column names are the trademarks of their respective manufacturers.

LC Conditions: Mobile Phase, 65/35 Acetonitrile/50mM Potassium phosphate buffer, pH 3.2; Flow Rate, 1.0 mL/min.; Injection volume, 1 ul; 254 nm detection; Column Temperature, 21°C. Solutes: uracil, phenol, pyridine, 4-butylbenzoic acid, N,N – dimethylaniline, toluene.

† Mobile Phase: 45/5/50Acetonitrile/THF/50mM Potassium phosphate buffer, pH 7.0; Column Temperature: 30°C; all other conditions are identical.

pittcon 2002 oral presentation - zirchrom.com · title: pittcon 2002 oral presentation author:...

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