Investigation of retention mechanisms in HILIC chromatography ...

Abstract
Investigation of retention mechanisms in
HILIC chromatography: Important
considerations for robust method
development
• Anders Fridström
• Sigma- Aldrich GMBH
Investigation of Retention Mechanisms in HILIC Chromatography: Important Considerations for Robust Method
Development
Anders Fridström 1 , David S. Bell 2
1 Sigma-Aldrich, Buchs SG/Switzerland, 2 Supelco/Sigma-Aldrich, Bellefonte/USA
Anders.fridstrom@sial.com
Hydrophilic interaction liquid chromatography (HILIC), especially in conjunction with mass spectrometry (MS), has
become a powerful tool for the analysis of a wide variety of challenging analytes. Applications of the technique have
increased dramatically over the past decade, especially for the analysis of polar analytes where reversed-phase
chromatography suffers. HILIC conditions employ a high percentage of acetonitrile which enables facilitated solvent
evaporation in LC/MS sources and thus often an increase in analyte response when compared to more aqueous based
systems. The increased retention of polar analytes afforded by HILIC provides improved selectivity and decreases the
impact of endogenous species, often leading to improved qualitative and quantitative analyses [1].
Although HILIC has proven useful, it has also been thwarted with complications including difficulties in method
development and method robustness.
In this presentation, studies investigating the underlying retention mechanisms dominant in HILIC chromatography are
presented and discussed. Along with reversed-partitioning HILIC is well known to exhibit, ion-exchange and the interplay
of the dominant mechanisms are unveiled and used to develop a model of overall retention and selectivity. Interactions
that operate using different stationary phase chemistries and conditions are presented. The impact of analyte polarity and
charge as well as the variations caused by high percentages of organic on these physiochemical parameters are
highlighted. Throughout the discussion, examples of use and misuse of HILIC are employed to illustrate these important
concepts to build a solid fundamental foundation for efficient and effective use of this powerful technique.
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Agenda
Introduction
Factors affecting the HILIC Separation
Modelling Ionic interactions on polar stationary phases in HILIC
Introduction
• HILIC Hydrophilic Interaction LIquid Chromatography
Retention
5-40 % Water
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Factors effecting the HILIC systems
• Column
• Mobile Phase
• pH
• Buffer Concentration
• Analytes
• pKa
• Log P OW or Log D OW
(Temperature)
Sample
Columns
• Ascentis Express Fused Core Particle Columns
• HILIC (Si)
• OH5 (Pentalol) Branched hydroxylated alkane New!!
• F5 (PFP)
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pH Effect of Acetonitrile on pH of Ammonium Acetate [4]
A Note on Buffer pH
s
pH w
pH Measured Following Addition of Organic
12
11
10
9
8
7
6
5
4
3
pKa
2
2 4 6 8 10 12
pH Measured Prior to Addition of Organic
w
pH
w
Measurements were taken at 25ºC.
Triangle: 90.0% ACN,
Square: 75% ACN,
Diamond: 50% ACN,
Circle: 32.5% ACN
• Analyte pK a values have also been shown to be impacted by the
presence of organic modifiers
• Figure 2 shows the results of an NMR experiment conducted that
explored the chemical shift of a proton near the ionizable group for
amitriptyline in 90% acetonitrile. From data such as this, effective pK a
values can be established for a variety of compounds.
• Table 1 shows the results for several basic pharmaceutical compounds.
The data indicates that the effective pK a value for a basic analyte in 90%
acetonitrile is approximately 1 pK a unit less than the aqueous-based
value
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Chemical Shift (ppm)
Determination of pK a Values using 1 H NMR [4]
Amitriptyline Chemical Shift as a Function of pH at 90% Acetonitrile
2.70
2.60
2.50
2.40
2.30
2.20
2.10
2.00
3 4 5 6 7 8 9 10 11 12 13
pH
Table 1: Determination of pK a Values using NMR
[4]
Amitriptyline
s
w
% Acetonitrile pK a Correlation
(R 2 )
25 9.32 0.9997
50 9.02 0.9996
75 8.88 0.9956
90 8.34 0.9923
• pK a values for bases decrease with
increasing acetonitrile
• At 90% each analyte exhibited a
pK a value about 1 full pH unit less
than the literature pK a value
Analyte Literature pKa pKa Correlation
(R2 s
w
)
Amitriptyline 9.4 8.34 0.9923
Nortriptyline 9.7 8.92 0.9920
Diphenhydramine 9.0 8.33 0.9978
Verapamil 8.9 7.98 0.9976
Alprenolol 9.7 8.73 0.9855
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Investigation of Retention Mechanisms on
Different HILIC Phases
• interaction differences for three different HILIC stationary phases: PFPP (F5), bare
silica (HILIC) and a new pentalol phase (OH5)
• Using ephedrine as a probe molecule, retention as a function of buffer
concentration was collected and interpreted.
• Related compounds and dominant interactions prevalent using each phase is
studied
Selected Probes
HO
C
H 3
C
H 3
C
H 3
C
H 3
NH
NH
OH
OH
HN
CH 3
OH
ACD/Labs, PhysChemProp, v. 12
Structure pKa(MB) LogD(8.0) LogP MW name
9.38 -0.37 1.08 165.23 pseudoephedrine
9.38 -0.37 1.08 165.23 ephedrine
9.37 -1.35 0.13 167.21 synephrine
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Experimental
• Conditions:
• Instrument: #9, Waters 2690/Micromass ZQ single quadrupole interfaced via ESI
operating in pos. ion mode
• Columns:
• Ascentis Express Pentalol (OH5), 10 cm x 3.0 mm
• Ascentis Express HILIC, 10 cm c 3.0 mm,
• Ascentis Express F5, 10 cm x 3.0 mm,
Mobile Phase A: 10 mM ammonium acetate (pH unadjusted) in 10:90
water:acetonitrile
• Mobile Phase B: 10:90 water:acetonitrile
• Mixtures of A and B were run at 0%B, 20%B, 40%B, 60%B and 80%B
corresponding to 10 mM, 8 mM, 2 mM, 4 mM and 2 mM buffer
concentrations, respectively
• Flow rate: 0.4 mL/min
• Temperature: ambient
• Detection: MS, ESI, pos ion mode, scan m/z 125 – 300
• Injection volume: 2 uL
Calculation for ion exchange impact in HILIC
• Samples were injected in triplicate at each buffer concentration using each of the
phases. A sample of ephedrine only was also injected under each condition to
discriminate from pseudoephedrine in the mix.
• Log k = -log[C + ] m + log IEX
• [C + ] m concentration of the competing ion in the mobile phase and
• IEXC constant for a given system
• phase ratio,
• ion-exchange capacity of the stationary phase ion-exchange equilibrium constant.
• Log k Retention
• A plot of log k vs log [C + ] m will thus yield a slope of -1 when ion-exchange is solely
responsible for retention,
• The plot would yield a slope of 0 where ion-exchange is not present.
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log k'
1.20
1.00
0.80
0.60
0.40
0.20
Response of Ephedrine Retention on Buffer
Concentration on Three HILIC Phases
y = -0.6525x + 1.2104
R 2 = 0.9984
y = -0.8187x + 1.1259
R 2 = 1
y = -0.2199x + 0.2455
R 2 = 0.9934
0.00
0.00 0.20 0.40 0.60 0.80 1.00 1.20
log buffer concentration (mM)
OH5 HILIC F5 Linear (HILIC) Linear (F5) Linear (OH5)
HILIC OH5 column at 10 and 2 mM ammonium
acetate
10 mM - 3
A1849_032_A005 Sm (Mn, 2x3) 1: Scan ES+
3.05
166
2.91e8
%
1
Time
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
2 mM - 3
A1849_032_A025 Sm (Mn, 2x3) 1: Scan ES+
6.49
168
7.84e7
%
1
10 mM
2 mM
1, 2
1, 2
3
Time
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
More polar synephrine elutes last
= partition
Little response of retention on
buffer conc.
3
1. Ephedrine
2. Pseudoephedrine
3. Synephrine
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4 mM - 1
A1849_032_A044 Sm (Mn, 2x3) 1: Scan ES+
11.49
166
1.59e8
%
2
Time
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
2 mM - 3
A1849_032_A051 Sm (Mn, 2x3) 1: Scan ES+
16.89
166
1.77e8
%
2
HILIC (bare silica) column at 4 and 2 mM
ammonium acetate
Synephrine still last
1
3
More Rt change
2
12.27
Both partition and IEX
Time
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
1
2
17.90
3
4 mM
2 mM
1. Ephedrine
2. Pseudoephedrine
3. Synephrine
F5 column at 8 and 2 mM ammonium acetate
8 mM - 1
A1849_032_A060 Sm (Mn, 2x3) 1: Scan ES+
5.16
166
1.91e8
%
2
Time
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
2 mM - 1
A1849_032_A075 Sm (Mn, 2x3) 1: Scan ES+
12.74
166
1.93e8
%
3
3
1
3
5.46
Time
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
2
1
13.33
2
8 mM
2 mM
Synephrine early!
Rt change with Buffer
Little or no partition,
IEX present
1. Ephedrine
2. Pseudoephedrine
3. Synephrine
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Proposed Model for Different HILIC Stationary Phases
OH5
Aqueous Layer
Aqueous Layer
- - - - - - -
-
Conclusions
Aqueous-Organic Mobile Phase
Silica
Polar Stationary Phase
• Columns described in this presentation
• Pentalol (OH5)– dominated by partition
• Bare silica (HILIC)– both partition and IEX
• Pentafluorophenylpropyl – mainly IEX
• In order to develop robust and reliable methods using HILIC
chromatography thefollowing factors should be considered
• pH changes in high % organics
• pKa of analytes in high % organics
• Mobile phase modifiers concentrations
F5
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Method Development and Optimization Chart
References
1. Hydrophilic Interaction Liquid Chromatography (HILIC) and Advanced
Applications, Wang Perry G., He Weixuan, CRC Press, Taylor & Francis Group.
2. Needham, S.R., Bell, D., J. Chromatogr., A. 2000, 869, 159-170.
3. McCalley, D. V., J. Chromatogr., A. 2010, 1217, 3408-3417.
4. Dinh, N. P., Jonsson T., Irgum K., J. Chromatogr., A. 2010, 1217, 3408-3417.
5. W. Naidong, Journal of Chromatography B 796 (2003) 209.
6. D.S. Bell, Jones, A. Daniel, Journal of Chromatography A 1073 (2005) 99.
7. D.S. Bell, Brandes, Hillel K., in 30th International Symposium and Exhibit on High
Performance Liquid Phase Separations and Related Techniques, San Francisco,
California USA, 2006.
8. D.S. Bell, Solute Attributes and Molecular Interactions Contributing to Retention
on a Fluorinated High-Performance Liquid Chromatography Stationary Phase,
Thesis, The Pennsylvania State University, 2005
9. ACD PhysChem, v. 12, Advanced Chemistry Development, Toronto, ON Canada
21
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Acknowledgements
• Dave Bell
• Hugh Cramer
• Craig Aurand
• Wayne Way
• Gaurang Parmar
Thanks!
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