solute attributes and molecular interactions contributing to “u … · 2016. 2. 23. · •...
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Solute Attributes and Molecular Interactions Contributing to “U-Shape” Retention on Fluorinated HPLC Stationary Phases
David S. Bell and A. Daniel JonesThe Pennsylvania State University
Department of Chemistry
T403154
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Introduction
• LC/MS rapidly becoming the cornerstone of analytical chemistry- Pharmaceutical- Environmental- Agricultural……
• Many efforts aimed at improving the technique- Improve sensitivity- Greater speed- More universal
• Much work centered on interfaces and instrumentation• Less attention paid to LC• Lack of retention/separation leads to
- Poor quantification- Problematic qualitative analysis
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Introduction
Reversed-Phase Normal-Phase
0
2
4
6
8
10
12
0 20 40 60 80 100% Acetonitrile
Ret
entio
n Ti
me
(min
)
verapamil amitriptyline cimetidine clonidinefluoxetine nifedipine trimethoprim
“U-Shape Retention”
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Introduction
• Retention at high organic modifier percentages may:- Increase sensitivity in LC/MS experiments through facilitated
desolvation.
0
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
0 10 20 30 40 50 60 70 80 90 100
% Acetonitrile
Area
Res
pons
e, c
ount
s
amitriptylinecimetidineclonidinefluoxetinenifedipinetrimethoprimverapamil
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Introduction
• Retention at high organic modifier percentages may also:- Speed up analyses.- Induce retention otherwise not obtained in RP.- Provide alternative mechanisms of interaction.
• Needham (J. Chromatogr. A, 869 (2000) 159)- Examined several stationary phase chemistries- Determined that pentafluorophenylpropyl (PFPP)-bonded silica
provided greatest retention with good peak shape and reproducibility
• Certain analytes under certain conditions using certain stationary phases……
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Introduction
• Paramount to understand- Solute attributes- Molecular interactions
• In this research we sought to better understand “U-Shape” retention by:- Investigating the relationship of retention and percent organic for
several pharmaceutical acids, bases and neutrals on PFPP compared to C18.
- Performing several studies aimed at elucidating the dominant molecular interactions responsible for the observed behavior.
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Introduction
• PFPP offers:- Dipole-dipole interactions- Pi-pi interactions- Charge-transfer interactions- Others?
F
F
F
F
F
SiOSi
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Acidic, Basic and Neutral Retention Profiles
• Experimental Design:- 6 analytes representing pharmaceutical acids, bases and
neutrals- Retention monitored from 40 to 90% acetonitrile at two pH
levels (4 and 6.7) on Discovery® HS F5 (PFPP) and Discovery HS C18
- Aqueous component-10 mM ammonium acetate
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Acidic Analyte Profiles on PFPPRetention Profiles (k’) of Acidic Probes on PFPP at pH 6.7
Retention (k’) of acidic probes x ibuprofen, � aspirin, + naproxen, � ketoprofen, �piroxicam and � diclofenac using PFPP from 40% to 90% acetonitrile under pH 6.7
conditions.
-0.5
0
0.5
1
1.5
2
2.5
3
30 40 50 60 70 80 90 100
% Acetonitrile
Rete
ntio
n (k
')
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Basic Analyte Profiles on PFPP
Retention Profiles (k’) of Basic Probes on PFPP at pH 6.7
Retention (k’) of basic probes � amitriptyline, � nortriptyline, � diphenhydramine, xverapamil, � alprenolol and � lidocaine using PFPP from 40% to 90% acetonitrile
under pH 6.7 conditions
0
10
20
30
40
50
60
30 40 50 60 70 80 90 100
% Acetonitrile
Rete
ntio
n (k
')
k’ values up to 30!
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Neutral Analyte Profiles on PFPP
Retention Profiles (k’) Neutral Probes on PFPP at pH 6.7
Retention (k’) of neutral probes + hydrocortisone, � hydrocortisone acetate, �progesterone, � corticosterone, � cortisone acetate and � prednisone using PFPP
from 40% to 90% acetonitrile under pH 6.7 conditions
0123456789
10
30 40 50 60 70 80 90 100
% Acetonitrile
Rete
ntio
n (k
')
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Basic Analyte Profiles on C18
Retention Profiles (k’) of Basic Probes on C18 at pH 6.7
Retention (k’) of basic probes � amitriptyline, � nortriptyline, � diphenhydramine, xverapamil, � alprenolol and � lidocaine using C18 from 40% to 90% acetonitrile under
pH 6.7 conditions
0
1
2
3
4
5
6
7
30 40 50 60 70 80 90 100
% Acetonitrile
Rete
ntio
n (k
')
k’ values up ~1.5
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Selectivity Analysis
• Secondary amines show relatively greater increase in the normal-phase region
• Except for verapamil, amines are located on structural “arms”
• Basic moiety in verapamil hindered• Lidocaine shows evidence of a pKa dependence
N
NO
OO
O
N
Amitriptyline Verapamil
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Retention Profiling Conclusions
• Only basic analytes exhibit appreciable “U-Shape” retention
• Not all bases• Not all to the same extent - selectivity• Also observed to a small degree on C18
- Commonality is the silica surface- Known to interact with bases via ion-exchange
• Pointed to ionic interactions with surface silanols
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“U-Shape” Retention Profile Dependence on MP Ionic Strength
• As % organic increased, ionic strength decreased• What if the ionic strength was held constant?
• Experimental:- Basic analytes nortriptyline and amitriptyline run from 50 to
90% acetonitrile on PFPP keeping the buffer concentration at 10 mM throughout
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“U-Shape” Retention Profile Dependence on MP Ionic Strength
Comparison of Amitriptyline and Nortriptyline Retention on PFPP Phase: Decreasing vs. Constant Buffer Concentration
•Retention at all % acetonitrile levels is attenuated•Little “U-Shape” character observed at constant ionic strength
0
10
20
30
40
50
60
30 40 50 60 70 80 90 100
% Acetonitrile
Rete
ntio
n (k
)
Amitriptyline (Constant) Amitriptyline (Decreasing)nortriptyline (Constant) nortriptyline (Decreasing)
Decreasing MP ionic strength
Constant MP ionic strength
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Contribution of Bonded Phase to Retention
• Compared basic analyte retention on bare silica to PFPP- 2 mM ammonium acetate (pH 6.7) at 90% acetonitrile
Compound k' Silica k' PFPP
amitriptyline 4.63 12.71
nortriptyline 6.92 18.98
diphenhydramine 4.69 10.98
verapamil 1.76 5.40
alprenolol 6.09 12.59
lidocaine 0.09 0.46
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Contribution of Bonded Phase to Retention
kappa-kappa plot: Retention of Basic Analytes on PFPP Versus Bare Silica
y = 0.8296x + 0.5189R2 = 0.9936
-0.6-0.4-0.2
00.20.40.60.8
11.21.4
-1.5 -1 -0.5 0 0.5 1
silica log k'
PFPP
log
k'
• good correlation indicative of similar dominant interactions
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Dependence of Retention on Ionic Strength at Constant % Acetonitrile
• Two-site model of retention (Yang, et. al., J Chromatogr. A 996 (2003) 13)
log k’ = log(k’RP + BIEX/[C+]m)
Reversed-Phase Contribution(independent of salt concentration)
Ion-Exchange Component(inversely proportional to salt concentration)
Plot of log k’ vs log[C+]mslope = -1, if exclusively ion-exchangeslope = ~0, if RP dominates
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Dependence of Retention on Ionic Strength at Constant % Acetonitrile
Amitriptyline Retention (logk’) vs. Ammonium Acetate Concentration (log[mM]) on PFPP at 85% Acetonitrile
y = -0.6659x + 1.2325R2 = 0.999
0
0.2
0.4
0.6
0.8
1
1.2
0 0.2 0.4 0.6 0.8 1 1.2 1.4log buffer concentration (mM)
log
k'• Slope of –0.6659 indicates more than just ion-exchange
•Note linear dependence of retention on buffer conc.
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Dependence of Silanol pKa on Bonding Chemistry
• For IEX silanols must be ionized• Neue (J. Chromatogr. A 925 (2001) 49) used
quaternary ammonium ion (bretylium) retention as a function of pH to estimate silanol pKa values.
• Bare silica, PFPP and C18 were subjected to similar experiment- Ammonium ion concentration held constant at 25 mM- pH varied from 2 to 8- Bretylium ion retention monitored
N+
Br
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Dependence of Silanol pKa on Bonding Chemistry
Retention (k’) of Bretylium Ion as a Function of pH
pH 2 to 8 on � bare silica, � PFPP and � C18
0
5
10
15
20
25
30
0 2 4 6 8 10pH
k'• Bonded-phase alters silanol acidity
• Bonded phase inhibits interaction
• Explains why PFPP and not C18
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Summary
• Only basic analytes exhibit appreciable “U-Shape” retention on PFPP
- Also observed on C18 to a lesser extent- Selectivity and good peak shape are obtained- Selectivity appears to be a function of pKa and silanol/base
accessibility• The “normal-phase” region of the profile was shown to be a
function of MP ionic strength• Based on the two-site model of retention, both RP and IEX
contribute to retention (hydrophobically-assisted ion-exchange)• Further supported by the preferential retention of bases on
PFPP compared to silica• Estimated pKa values for C18 and PFPP also support the
existence of IEX mechanisms
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Conclusions
• Ability to retain basic analytes at high organic modifier concentrations offers potential to increase LC/MS sensitivity
• Alternative mechanisms may provide unique options in method development
• Bonded-phase chemistry plays an important role in both RP and IEX
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Conclusions
• PFPP presents new opportunities to manipulate retention and selectivity- Buffer concentration- pH (pKa of analytes and surface silanol groups)
• Knowledge should lead to:- greater utilization- more robust methods- further advances in stationary phase chemistries- expansion to other forms of separation
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Acknowledgements
• A. Daniel Jones (Penn State)• Keith Duff (Supelco) and Shane Needham (Alturas
Analytics)• Supelco• Exygen Research• Organizers