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Examining the Selectivities of Several C18 and Modified C18 Phases: Advantages of Phenyl and Pentafluorophenyl (PFP) PhasesThomas J. Waeghe, Robert T. Moody, and Carl Zimmerman | MAC-MOD Analytical, Inc., 103 Commons Court, Chadds Ford, PA 19317

Abstract

Selectivity is, by far, the most powerful parameter for improving resolution in chromatographic separations. Column stationary phase in reversed-phase liquid chromatography is one of the most effective and easiest-to-change parameters for altering selectivity and achieving resolution. A variety of different stationary phases have been developed during the course of the history of HPLC, including alkyl chains of various lengths, phenyl and cyano phases, embedded polar phases having amide and carbamate groups, and many more. Recently, two new phases have been developed using phenylalkyl and pentafluorophenyl alkyl groups attached to ultrapure, low metal content silica. These phases not only afford selectivity differences versus each other and versus their predecessor C18 phase, but they’ve also been shown to provide selectivities different from many (most) other commercial phases. In this poster we will demonstrate the selectivity differences among the C18, C18-AR and C18-PFP phases, and will show their usefulness in developing new methods—especially for difficult-to-separate analytes. We will also attempt to show examples of the different separation mecha-nisms, which the various phases can exploit.

Resolution Equation Shows that Selectivity, α, is the Most Effective Parameter to Change Rs

ACE C18-AR and ACE C18-PFP Phases Complementary Selectivities Mean No Analyte Elutes at Same Time on All 3 Phases!

Changing Phases May Improve Resolution or just Change Selectivity

Large α Changes Often Occur Switching from ACN to MeOH Fast Column Screening Gradients for Method Development Using 3.0 x 50 mm, 3 µm ACE Columns

Large α Changes Also Occur Switching from MeOH to ACN

Additional Selectivity for C18-AR and C18-PFP from Other Analyte-Stationary Phase Interactions

HydropHobic interactions 80-85% of total retention

RPLC Method Selectivity Parameters

Rs = N(α - 1) 1 k

2

1 + k α4

k2

k1

α=

Resolution is directly proportional to selectivity (α), but is only proportional to the square root of efficiency, N.

The analysis condition parameters that most affect relative retention (selectivity, α) are1:

One of most powerful and easiest parameters to investigate

Chosen most often as part of comprehensive method development strategy

Column type (C18, phenyl, cyano, etc.) ++

B-solvent (acetonitrile, methanol, etc.) ++ Mobile phase pH ++ Ion-pair concentration ++ %B solvent/gradient steepness + Column temperature + Buffer concentration +

1 . 0 0 1 . 0 5 1 . 1 0 1 . 1 5 1 . 2 0 1 . 2 5

0 . 0

0 . 5

1 . 0

1 . 5

2 . 0

2 . 5

3 . 0

0 5 0 0 0 1 0 0 0 0 1 5 0 0 0 2 0 0 0 0 2 5 0 0 0

0 5 1 0 1 5 2 0 2 5

N

k

a

Resolution (R)

1 . 0 0 1 . 0 5 1 . 1 0 1 . 1 5 1 . 2 0 1 . 2 5

0 . 0

0 . 5

1 . 0

1 . 5

2 . 0

2 . 5

3 . 0

0 5 0 0 0 1 0 0 0 0 1 5 0 0 0 2 0 0 0 0 2 5 0 0 0

0 5 1 0 1 5 2 0 2 5

N

k

a

1 . 0 0 1 . 0 5 1 . 1 0 1 . 1 5 1 . 2 0 1 . 2 5

0 . 0

0 . 5

1 . 0

1 . 5

2 . 0

2 . 5

3 . 0

0 5 0 0 0 1 0 0 0 0 1 5 0 0 0 2 0 0 0 0 2 5 0 0 0

0 5 1 0 1 5 2 0 2 5

N

k

a

Resolution (R)

0 . 0

5 . 0

1 0 . 0

1 5 . 0

2 0 . 0

2 5 . 0

3 0 . 0

3 5 . 0

4 0 . 0

4 5 . 0

0 . 0 1 . 0 2 . 0 3 . 0 4 . 0 5 . 0 6 . 0 7 . 0 8 . 0

A l p h a

Resolution (Rs)

R s vs. alpha is nearly linear

between 1 < a < 2

Source: Jun Mao, PhD Thesis with Professor Peter Carr, U. of Minnesota, 2001

Res

olut

ion

(Rs)

Res

olut

ion

(Rs)

Dα of 1.00–1.25DR

s of 0–3

α

1 Adapted from “Introduction to Modern Liquid Chromatography”, 3rd Edition, L. R. Snyder, J. J. Kirkland, J. W. Dolan; p. 29, 2010, John Wiley & Sons, Inc.

F

F

F

F

F

features and benefits• C18 with integral phenyl group

• Leverages selectivity of both C18 and phenyl groups to separate mixtures not possible with C18 or phenyl alone

• Exceptional stability and low bleed of C18 phase with added aromatic (aryl) selectivity

• Compatible with highly aqueous mobile phases (no retention loss)

features and benefits• C18 with integral pentafluorophenyl

(PFP) group

• Augments hydrophobic selectivity of C18 with additional retention mechanisms of PFP group (π-π, hydrogen–bonding, dipole–dipole, shape selectivity)

• Provides stability, inertness, and low bleed comparable to other high perfor-mance C18-only phases

• Compatible with highly aqueous mobile phases (no retention loss)

π-π Interactions

R

F

F F

FF

Analyte

Stationary Phase

Dipole-dipole Interactions

Schematic of C18-AR Phase

Schematic of C18-PFP Phase

F

F

F

F

Fδ- +δR—RAnalyte

Stationary Phase

Hydrogen Bonding Interactions

F

F

F

F

Fδ- +δH–O–RAnalyte

Stationary Phase

Steric Interactions

F

F

F

F

F F

F

F

F

F F

F

F

F

F

More ordered stationary phase produces shape selectivity

Types and Strengths of Analyte Interactions

Strengths of Interactions ACE C18 ACE C18-AR ACE C18-PFP

Hydrophobic ++++ ++++ ++++

π-π – ++++ ++++ (electron-poor analytes) (electron-rich analytes)

Dipole–Dipole – ++ ++++

Hydrogen Bonding – ++ +++

Shape Selectivity + ++ +++

* C18-AR tends to have stronger retention for electron-poor analytes ** C18-PFP tends to have stronger retention for electron-rich analytes

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Conditions

2.1 x 50 mm, 3 µm columns, Flow rate: 0.6 mL/min, 40°C, 254 nm A: 0.1% v/v formic acid in water, B = 0.1% v/v formic acid in MeOH Gradient from 3 to 100%B in 6.5 min., hold for 1 min

Conditions

2.1 x 50 mm, 3 µm columns, Flow rate: 0.6 mL/min, 60°C, 254 nm A: 20 mM potassium phosphate, pH 2.7 B = 65:35 v/v MeOH/mobile phase A Gradient from 3 to 100%B in 5 min., hold for 1 min

A: water/0.1% formic acid

B: MeOH/0.1% formic acid

3 to 100% B in 5 min., hold 1 min Return to 3% at 6.5 min. Re-equil. 2.5 min.

A: water/0.1% formic acid

B: MeOH/0.1% formic acid

3 to 100% B in 5 min., hold 1 min Return to 3% at 6.5 min. Re-equil. 2.5 min.

A: water/0.1% formic acid

B: ACN/0.1% formic acid

3 to 100% B in 5 min., hold 1 min. Return to 3% at 6.5 min. Re-equil. 2.5 min.

A: water/0.1% formic acid

B: ACN/0.1% formic acid

3 to 100% B in 5 min., hold 1 min. Return to 3% at 6.5 min. Re-equil. 2.5 min.

Conditions

2.1 x 50 mm, 3 µm columns, Flow rate: 0.6 mL/min, 40°C, 254 nm A: 20 mM potassium phosphate, pH 2.7, B = 65:35 v/v MeOH/mobile phase A Gradient from 3 to 100%B in 5 min., hold for 1 min

1) sulindac, 2) chrysin, 3) piperine, 4) flurbiprofen, 5) ibuprofen

3) methylphenylsulfoxide, 4) methylphenylsulfone, 5) pindolol, 6) quinoxaline, 7) salicylaldehyde,8) 1,4-dinitrobenzene, 9) 1,3-dinitrobenzene, 10) 1,2-dinitrobenzene, 11) myrecetin, 12) juglone, 13) remacemide, 14) quercetin, 15) methdilazine, 16) plumbagin

1) 3-hydroxybenzoic acid, 2) methylphenylsulfoxide, 3) quinoxaline, 4) salicylic acid, 5) benzonitrile, 6) 1,2-dimethoxybenzene, 7) ethyl paraben, 8) 1,4-dimethoxy-benzene, 9) bendroflumethazide, 10) piroxicam, 11) benzyl chloride, 12) thioan-isole, 13) sulindac, 14) chrysin, 15) ibuprofen, 16) 1,2,3-trichlorobenzene, 17) meclofenamic acid

1) methylphenylsulfoxide, 2) eserine, 3) salicylamide, 4) phenacetin, 5) p-cresol, 6) acetophenone, 7) quercetin, 8) carvedilol, 9) 1,3-dinitrobenzene, 10) tolmetin, 11) 3,4-dichlorobenzoic acid, 12) ethyl benzoate, 13) toluene, 14) ibuprofen, 15) indomethacin, 16) 1,2-dichlorobenzene

Temperature: 40°C Injection vol.: 1 µL Flow: 0.6 mL/min Detection: 214 nm

Peak Order for ACE C18-PFP: ranitidine, pyrilamine, lomefloxacin, acebutolol, naringin, trazodone, prednisolone, prednisone, alprenolol, ketoprofen, tolmetin sodium, naproxen sodium

ACE Phase

C18

C18-AR C18-PFP

Retention Mechanism

Hydrophobic interactions

Hydrophobic and π–π interactions

Hydrophobic,π–π, hydrogen bonding, dipole-dipole, and steric interactions

Most Commonly Used For

Diverse analytes of differing polarities, acids, bases and neutrals at low to mid pH, strong acids or bases in ionized form using ion-pairing mode

Electron-poor compounds, analytes with electron-withdrawing groups (halogens, nitro groups, ketones)

Electron-rich compounds, analytes with electron-donating groups, (phenols, aromatic ethers, amines), analytes with π bonds or electron delocalization, proton donors, analytes with different dipole moments

Best Application

Analytes differing in hydrophobicity, homologous compounds differing by –CH2 groups

When selectivity different from C18 is needed; Basic analytes, heterocycles, highly aqueous conditions

When selectivity different from C18 is needed; Positional isomers, substituted aromatics, steroids, highly aqueous conditions, proton donors

6) salicylic acid, 7) quinoxaline, 8) benzoic acid, 9) quinine, 10) phenacetin, 11) 1,4-dinitrobenzene, 12) 1,3,5-trinitrobenzene, 13) furosemide, 14) 1,3,5-trimethoxybenzene, 15) piroxicam, 16) carvedilol, 17) ethyl benzoate, 18) nortriptyline

ACE C18 ACE C18

ACE C18

ACE C18-AR

ACE C18-AR

ACE C18-AR

ACE C18-PFP

ACE C18-PFP

ACE C18-AR, 2.1 x 50 mm, 3 µm ACE C18-AR, 2.1 x 50 mm, 3 µm

ACE C18-PFP, 2.1 x 50 mm, 3 µm

MeOH

MeOH

ACN

ACN

ACE C18-PFP

1 5

6

6

6,7

7 8 9

9

7,12

2

10

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10

11

11

12

12

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13

14

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15

16

16

17

17

18

181

1

1

1

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2

2

2

3

3

3

3

4

4,5

4

4

5

5

5

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6

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6

7

7,8

7

7

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8

8

9

9

9

9

10

10,12,13

10

10

11

11

11

11

12

12

12

13

13

13

14

14

14

14

15

15

15

15

16traz

alp

ran pyr

lom

ace

nar

traz

prednisolone

prednisone

ketotol

nap

napprednisolone + prednisone

16

16

16

C18

17

17

118

8

9

3

3,46

3

34

5

67 8 9

10

11 12

13

14

15 16

5

46

7

7

9

11

11 14 1213 15

16

12 1413

1516

8,9,10

8,10

4

15

14,16

5

17,18

1

13 2

4,5

2

3,4

5

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ACE C18

ACE C18-AR

ACE C18-PFP

Orthogonality of ACE C18-AR and ACE C18-PFP vs. ACE C18 analytes • 102 compounds screened on these and two other ACE phases (C8, Phenyl) – acidic and basic pharmaceuticals, aromatic acids and esters, substituted benzenes, flavonoidsanalysis conditions – Columns: 2.1 x 50 mm, 3 µm – Flow Rate, 0.6 mL/min – Temperature, 40° C – Gradients: 5.0 min from 3 to 100% MeOH (or ACN)/ water with 0.1% formic acid; hold 1 min, return in 0.5 min, post time, 2.5 min. • Produce X-Y plots of retention times on all three columns using ACN or MeOH as organic modifier • Regression analysis yields R2, coefficient of determination • S = 100 x (1 – R2)

Using ACE C18, C18-AR and C18-PFP in RPLC Method Development • ACE C18, C18-AR, and C18-PFP can be used both in simple and comprehensive, QBD method development strategies – Carry out column screening using multiple organic modifiers and blend(s) (ACN, MeOH, ACN/MeOH) at one or more pHs• ACE C18-AR and C18-PFP can be also exploited to: – Change selectivity or cause retention order reversals for a given separation on C18, C18-AR or C18-PFP

ACE C18-AR and C18-PFP Summary • Possess retention and stability advantages of ultrapure, ultra inert ACE C18 phase – Excellent peak shape – High efficiency – Excellent stability – Superior column-to-column and batch reproducibility• Possess complementary selectivities from additional types of molecular interactions – π–π (charge transfer), hydrogen bonding, dipole-dipole, and steric (shape-selective)• Provide a powerful toolset with ACE C18 for new method development or for method improvement and adjustment

Uwe Neue’s (Waters) selectivity descriptor

C18-ARC18-PFPS = 12

S = 11

S = 10

MeOH

S = 15

S = 10

MeCN

S = 12

S = 18C18-AR (MeOH)vs. C18 (ACN)

S = 19C18-PFP (MeOH)vs. C18 (ACN)

S = 100 x (1–R2)

Orthogonality Prism Comparing ACE C18, C18-AR, C18-PFP with ACN and MeOH Mobile Phases

ACE Bonded Phase Characteristics

α

α

5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 6.0 min.

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5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 6.0 min.

2 2.5 3 3.5 4 4.5 min

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0 2 4 6 8 10 min

1.5 2 2.5 3 3.5 4 .5 5 min

1.5 2 2.5 3 3.5 4 .5 5 min

0 2 4 6 8 10 min

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