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0.75 mL/min, 40°C5-95% ACN/0.1% H3PO410-min grads1 µL injections
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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
10
10
11
11
12
12
13
13
14
14
15
15
16
16
17
17
18
181
1
1
1
2
2
2
2
3
3
3
3
4
4,5
4
4
5
5
5
6
6
6
6
7
7,8
7
7
8
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
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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.
5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 6.0 min.
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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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
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