31015 waeghe 1045 am systematic method development with novel inert solid-core

Systematic Method Development with Novel, Inert Solid-Core Bonded Phases

Thomas J. Waeghe, Ph.D., Geoffrey Faden, Carl L. Zimmerman, Alan P. McKeown*

MAC-MOD Analytical, Inc.103 Commons Court

Chadds Ford, PA 19317

*Advanced Chromatography Technologies1 Berry Street, Aberdeen, AB25 1HF, Scotland

Systematic Method Development with Novel, Inert Solid-Core Bonded Phases

Encapsulated bonding technology (EBT) is a new approach to stationary phase endcapping that provides exceptional inertness and excellent phase stability across a broad pH range from 1.5 to 11.0. This technology has previously been successfully applied to 2, 3 and 5 micron totally porous (non-core) packing materials, but has more recently been implemented for 2.5 and 5 micron solid-core packings.

The ability to use stationary phase chemistry (C18, phenyl-hexyl), organic modifier choice, and pH as variables in a systematic approach is a significant advantage for UHPLC and HPLC method development. The usefulness of such a method development strategy will be described and demonstrated with an appropriate example.

23/10/2015 Pittcon 2015 New Orleans

1.00 1.05 1.10 1.15 1.20 1.250.0

0.5

1.0

1.5

2.0

2.5

3.0

0 5000 10000 15000 20000 25000

0 5 10 15 20 25

N

k

Nk

Res

olut

ion

(Rs)

Zhao, J.H. and P.W. Carr. Analytical Chemistry, (1999) 71, 2623-2632

Selectivity: the most powerful variable for increasing resolution

EfficiencySelectivity

Retention

Rs = k1+k

4

√ N

3

0

0

tttk R

1

2

kk

)(16base

r

wtN

2

Selectivity has greatest impact on Rs

Rs changes most rapidly

with


4

Which LC Parameters Affect Selectivity Most? 1,2

Isocratic Separations

1. Mobile phase pH (for ionised analytes only)

2. Column Stationary Phase

3. Organic modifier

4. % Organic modifier

5. Column temperature

• Buffer choice

• Buffer concentration

• Additive concentration

Gradient Separations• All parameters for isocratic

separations and,• Gradient steepness• k* (that is tG, F, , VM, MW)• Delay volume• Column dimensions

1 Adapted from ‘Introduction to Modern Liquid Chromatography”, 3rd Edition, Snyder, Kirkland, and Dolan, 2010, p.29, Wiley & Sons

SVFtk

m

G

85*

MOSTInfluence

LEASTInfluence

Relative Impact of Different Changes in RPLC Parameters on Selectivity2

Parameter Change inParameter

Maximum| |

pH 5 pH units 0.70

Organic Modifier CH3CNCH3OH 0.20

Gradient Time (tG) 10-fold 0.20

Orthogonal Column Fs ≥ ~65 0.19

% Organic Modifier 10% (v/v) 0.08

Column Temperature 20 C 0.07

Buffer Concentration 2-fold 0.02


2 Journal of Chromatography A, 1101 (2006) 122–135, “Orthogonal” separations for reversed-phase liquid chromatography, L.R. Snyder et al.

ACE UltraCore Solid-Core Columns

ACE UltraCore 2.5 m:Total particle diameter = 2.5 µmShell thickness = 0.45 µm

ACE UltraCore 5 m:Total particle diameter = 5 µmShell thickness = 0.7 µm

3/10/2015 Pittcon 2015 New Orleans 5

UltraCore SuperC18 and SuperPhenylHexyl Columns

• Alternate selectivities: hydrophobic and - interactions

• stable to 1000 bar (14,500 psi)• 2 m frits for improved ruggedness

and uptime• 20,000 column volume lifetime

minimum (≤ 40 C, pH 811.0)

Advantages of Encapsulated Bonding Technology (EBT™)

Encapsulated Bonding Technology

• Uniquely developed for ACE UltraCore SuperC18 and UltraCore SuperPhenylHexyl

• EBT bonding and endcapping – dramatically higher ligand coverage– effectively eliminates the negative

effects of unbonded silanol groups

Benefits of EBT

• Inertness—superb peak shape – for bases, acids, and neutrals

(pH 1.5–11.0)• Stability

– silica protected from eluent at mid and high pH

– use with volatile buffers for max. stability, ideal for LC and LC-MS (NH4OH, NH4OAc, etc.)

• Versatility – no memory effects from switching

among eluents at different pHs



xx

xxt r

column 1

tr column 2

R2 = 0.9987

Uwe Neue Selectivity Descriptor, S, as measure of orthogonality

√For collection of representative, diverse analytes,

plot analyte gradient retention times (obtained under the same conditions) for different stationary phases, or you can plot gradient RTs for different combinations of analysis conditions and columns) versus R2 is coefficient of determination (measure of how close

the data are to the fitted regression line.

Neue Selectivity Factor


UltraCore Phases: Orthogonality at low pH

UltraCore SuperPhenylHexyl at low pH vs. SuperC18 at low pH

CH3OHLow pH

S = 19

S ~10 or higher is effective for adjusting

SuperPhenylHexyl Orthogonality: High pH vs. Low pH


UltraCore SuperPhenylHexyl (high pH) vs. SuperPhenylHexyl (low pH)

SuperC18

SuperC18

pH 10.7

SuperPhHexylS = 12

SuperPhHexylS = 11

S =

25

S = 22

MeOH

MeCNNice pattern...

CH3OHHigh pH

S = 83

S values between phases and organic modifiers

at high pH

Objective: Explore performance of ACE UltraCore columns for basic analytes

Parameters– Stationary phase

• ACE UltraCore SuperC18 and SuperPhenylHexyl

– Organic modifier• CH3CN, CH3OH

– pH • 2.8, 3.8, 8.2, 9.2, 10.2; 9.7 and 10.7

Samples– 9 Appetite Suppressants– 16 Drugs of Abuse


Sample 1: Appetite Suppressants

1. methamphetamine2. amphetamine3. ephedrine4. fluoxetine5. caffeine6. phentermine7. fenfluramine8. chlordiazepoxide9. phenylpropanolamine

11

Approach

Columns• UltraCore SuperC18 and

SuperPhenylHexyl, 2.1 x 50 mm, 2 m

Organic modifiers– ACN– MeOH– ACN/MeOH (1:1)

Aqueous buffers– ammonium formate, pH 2.7– ammonium acetate/NH4OH

pH 8.7, 9.2, 10.2– Gradients from 5 to 95%

organic in


More Complex Appetite Suppressant Mixture: optimum pH from DryLab®

0 10 20Time (min)

0.00

E+00

2.00

E+08

4.00

E+08

6.00

E+08

Inte

nsity

caf

ephed

phent unk

phenolphthalein

chlordiazepoxidelorcaserin

fenfluramine

fluoxetine

unk3

DEP

sertraline

DidesSib

Rimonabant

NDesSib

sibutramine

orlistat

unk4

12

predicted optimum pH based on DryLab using only pH not gradient time or temperature

2.1 x 100 mm, 2 m ACE Excel SuperC180.5 mL/min, 25C, 10–90% CH3CN/20 mM pH 9.3 buffer in 20 min.MS Detection Data courtesy of Phyllis Wilson, U.S. FDA


Sample: Appetite Suppressants


Compound pKa log P pI

RT ACN

pH 10.22‐70%9.71 min

0.6 mL/minphenylpropanolamine 9.37 0.89 11.63 2.61caffeine 14.0 -0.55 none 2.66ephedrine 9.52 1.32 11.71 3.44amphetamine 10.10 1.8 none 4.31phentermine 10.25 2.08 none 4.85methamphetamine 9.9 2.24 none 4.96chlordiazepoxide 6.43 3.05 12.47 5.88fenfluramine 10.22 3.47 none 7.18fluoxetine 9.8 4.17 none 8.55

UltraCore SuperC18: Examples with CH3CN and CH3OH at pH 3.8 and pH 10.2


0 2 4 6 8 10Time (min)

0.00

00.

002

0.00

40.

006

0.00

80.

010

Volts

1.1501.849

2.366

2.782

3.154

3.296

6.350

7.354

9.800

0 2 4 6 8 10Time (min)

0.00

0.02

Volts

1.981 2.053

3.047 4.029

4.6724.762

5.692

7.306

8.365

UltraCore SuperC18CH3CN/pH 3.82-50% in 13.7 min.k* = 14.30.6 mL/min, 30 C for all 4

UltraCore SuperC18CH3CN/pH 10.25-70% in 10 min.k* = 15.5

0 2 4 6 8 10 12 14 16Time (min)

0.00

00.

002

0.00

40.

006

0.00

8Vo

lts

1.283

2.1732.656

3.516

4.2155.008

9.145

14.456

15.258

2 4 6 8 10 12Time (min)

0.00

00.

010

0.02

0Vo

lts2.266

2.345

3.517 4.959

6.309

6.455

9.610

10.337

11.785

UltraCore SuperPhenHexCH3OH/pH 3.82-50% in 13.7 min.k* = 14.3

UltraCore SuperPhenHexCH3OH/pH 10.25-70% in 10 min.k* = 15.5

2.2 2.4Time (min)

Volts

2.266 2.345

1.8 2.0Time (min)

Volts

1.981

2.053

Sample: 16 Drugs of Abuse


MW Compound Name pKa log P pI

RT SPH2‐50%pH 2.8

271.31 Normorphine 10.12 0.26 10.12 0.707285.34 Morphine 9.66 0.90 9.66 1.051301.34 Oxymorphone 9.14 0.78 9.14 1.405285.34 Hydromorphone 9.34 1.62 9.34 1.789301.38 Dihydrocodeine 9.33 1.55 11.74 2.432299.36 Codeine 9.19 1.34 11.49 2.519301.34 Noroxycodone 9.14 0.78 9.14 2.663315.36 Oxycodone 8.21 1.03 10.89 2.807327.37 6‐Acetylmorphine 8.42 1.09 9.25 2.874299.36 Hydrocodone 8.61 1.96 13.30 2.989289.33 Benzoylecgonine 9.54 ‐0.59 6.49 3.268193.24 MDMA 10.14 1.86 NA 3.518369.41 Heroin 9.10 1.55 NA 3.966303.35 Cocaine 8.85 2.28 NA 4.051243.39 Phencyclidine (PCP) 10.64 4.49 NA 4.616

271.40 Dextromethorphan 9.85 3.49 NA 4.793

Isobaric compounds highlighted in same colorRTs for SuperPhenylHexyl 2-50% gradient

UltraCore SuperC18, 2.1 x 50 mm, 2.5 m4 pHs with CH3CN

0 2 4 6 8Time (min)

0.00

00.

002

0.00

4V

olts

0.649

0.881

1.187

1.697

2.613

2.7293.023

3.1183.227

3.402

4.021

6.547

6.9587.188

7.399

2 4 6 8Time (min)

0.00

00.

002

0.00

40.

006

0.00

80.

010

0.01

20.

014

Vol

ts

1.972

3.305

3.476

3.731

3.997

4.147

4.274

4.505

4.994

5.180 5.622

6.211

2 4 6 8 10Time (min)

0.00

00.

002

0.00

40.

006

0.00

80.

010

0.01

20.

014

Vol

ts

1.948

3.154

3.537

3.804 3.9104.012

4.215

4.581

4.732

5.102

5.635

6.350

6.6537.072

8.931

CH3CN/pH 3.8250% in 13.7 min.k* = 14.30.6 mL/min, 30 C for all 4

CH3CN/pH 8.2270% in 11.9 min.k* = 14.3

CH3CN/ pH 9.2270% in 11.9 min.k* = 14.3

3.0 4.0Time (min)

Vol

ts 3.154

3.537

3.8043.910 4.0124.215

4.581

4.732

2 4 6 8 10Time (min)

0.00

00.

002

0.00

40.

006

0.00

8V

olts

1.930

3.315

3.6383.926

4.155

4.719

4.7794.894

5.178

5.299

5.462

5.865

6.0586.1506.661

7.315 Gradient artifact

7.922

9.219

CH3CN/ pH 10.2270% in 11.9 min.k* = 14.3

4.0 5.0 6.0Time (min)

Volts 3.305

3.476

3.731

3.997 4.1474.274

4.505

4.9945.180 5.622

3/26/2015 16

0 2 4 6 8 10 12 14Time (min)

0.00

00.

002

0.00

40.

006

Vol

ts

3.824

5.303

5.764

6.564

6.941

7.454

8.226

8.849

9.230

9.545

9.887

10.370

11.273

12.040

0 2 4 6 8 10 12Time (min)

0.00

00.

002

Vol

ts

0.874

1.533

1.982

2.4964.132

4.354

4.536

4.938

5.225 6.727

10.829 12.065

0 2 4 6 8 10 12 14Time (min)

UltraCore SuperPhenylHexyl, 2.1 x 50 mm, 2.5 m: 4 pHs with CH3OH


CH3OH/pH 3.8250% in 13.7 min.k* = 14.30.6 mL/min, 30 C for all 4

CH3OH/pH 8.2270% in 11.9 min.k* = 14.3

CH3OH/pH 9.2270% in 11.9 min.k* = 14.3

0 2 4 6 8 10 12 14Time (min)

0.00

00.

002

0.00

4V

olts

CH3OH/pH 10.2270% in 11.9 min.k* = 14.3

0 10 20Time (min)

0.00

00.

002

0.00

40.

006

0.00

80.

010

0.01

2Vo

lts

2.7754.987

6.431

7.540

10.166

10.483

10.895

11.34011.527

11.705

13.944

18.774

19.514

20.190

0 2 4 6 8 10 12Time (min)

0.00

00.

002

0.00

40.

006

0.00

8Vo

lts

0.857

1.478

1.9602.466

4.091

4.287 4.455

4.878

5.194

6.018

6.712

10.67311.830

Scaling pH 3.8 and 10.2 Separations to 3 x 100 mm UltraCore SuperPhenylHexyl

18

2 4 6 8 10 12 14 16 18Time (min)

0.00

00.

002

0.00

40.

006

0.00

80.

010

Volts

3.4815.736

6.477 6.951

7.468

13.959

14.597

16.927

17.472

0 10 20Time (min)

0.00

00.

010

0.02

0Vo

lts 4.940

8.565

9.681

10.317

10.804

11.418

12.86713.354

14.060

14.287

14.526

15.25415.594

15.989

18.006 20.229

21.322

2.1 x 50 mm, 2.5 m250% in 13.7 min., 0.6 mL/min, 30 CCH3OH/pH 3.8 NH4OAc (10 mM), 2 Lk* = 14.35

3 x 100 mm, 2.5 m 1.0 mL/min270% in 23.8 min. k* = 14.35

2.1 x 50 mm, 2.5 m 295% in 16.3 min., 0.49 mL/min, 30 Ck* = 14.3CH3OH/pH 10.2 NH4OAc, 2 L

CH3OH

3 x 100 mm, 2.5 m1.0 mL/min, 30C295% in 32.5 min.CH3OH/pH 10.2 NH4OAc (10 mM), 8 L

pH 3.8

CH3OH

pH 10.2


14.0 15.0 16.0Time (min)

Volts 13.354

14.060

14.287 14.526

15.25415.594

15.766

11.0 12.0Time (min)

Volts

10.166 10.483

10.895 11.340

11.527

11.705

Separation on 3 x 100 mm, 2.5 m SuperPhenylHexyl: All 16 separated

0 2 4 6 8 10 12Time (min)

0.00

00.

010

0.02

0V

olts

1.711

2.5863.025

3.516 4.641

4.941

5.279

5.465

5.755

5.832

6.132 6.576

9.159

9.490

11.21811.886

5.0 6.0Time (min)

Volts

4.641

4.941

5.279

5.465

5.7555.832

6.132 6.576

0 2 4 6 8 10Time (min)

0.00

00.

010

0.02

0V

olts

2.022

2.653

2.931

3.212

3.805

3.971

4.169

4.3554.417

4.621

4.853

7.307 7.569

3.8 4.0 4.2 4.4 4.6 4.8Time (min)

Volts

3.805

3.971

4.169

4.3554.417

4.621

4.853


2–70% ACN/pH 3.8 in 30 min. slope2–70% ACN/pH 3.8 in 15 min. slope

Additional optimization with

DryLab

Summary

• ACE UltraCore SuperC18 and SuperPhenylHexyl columns were evaluated at several pHs for two mixtures of basic analytes.

• Peak shape and selectivity were best at pH 3.8 and pH 10.2 with ammonium formate and ammonium acetate/NH4OH mobile phases, respectively.

• Use of 2.1 x 50 mm, 2.5 m column geometries allows for rapid, yet thorough examination of various combinations of organic modifier, stationary phase and pH.

• Method transfer to larger column geometries went well, although inability to use injection delays did not permit completely accurate transfer.

• Useable conditions were found for both of the best conditions for each of the columns at low and high pH.

• For the drugs of abuse sample, pH 10.7 did not afford as good resolution as pH 10.2.


31015 waeghe 1045 am systematic method development with novel inert solid-core

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