©2004 Waters Corporation
Understanding (and Creating) Retention
of Polar Compounds in Reversed-Phase HPLC
Pittcon 2004
©2004 Waters Corporation
Overview
• Introduction– The problem
• Background
• Reversed-Phase HPLC for Polar Compounds
• An Intelligent Solution
• Applications
• Summary
©2004 Waters Corporation
Introduction - The Problem
• Conventional reversed-phase HPLC columns often do not provide adequate retention and separation of highly polar compounds
• Drug Discovery– Gradient - High-throughput screening (HTS) does not
work for polar compoundsElute un-retained in the void volumeCo-elute at the beginning of the run – if retained at all
• Method Development– Isocratic - Many columns cannot be run under the 100%
aqueous conditions necessary for polar compound retention– columns “dewet” (also termed -hydrophobic collapse)
©2004 Waters Corporation
Overview
• Introduction
• Background– Polar molecules– Chromatographic methods for retaining polar
compounds
• Reversed-Phase HPLC for Polar Molecules
• An Intelligent Solution
• Applications
• Summary
©2004 Waters Corporation
What is a Polar Molecule?
• General chemistry definition:– A molecule whose centers of positive and negative charges do not
coincide– The degree of polarity is measured by the dipole moment of the
molecule
• Dipole moment is the product of the charge at either end of the dipole times the distance between the charges
H
O
Hδ+
2δ-
δ+ Dipole moment = 1.85 D
This is a Polar Molecule!
“Like attract Like” orPolar compounds attract polar compounds
Electro-static attraction – opposite charge locations on different molecules attract
©2004 Waters Corporation
Examples of Polar Molecules
H N
NH
O
O
Uracil (N)
H N
NH
O
O
Thymine (N)
O
Acetone (N)
N
NH
NH2
O
Cytosine (B)
H N
H NF
O
O
OH O
5-Fluoroorotic acid (A)
OH
OH
NH
OH
Epinephrine (B) Ascorbic acid (A)
O
OH OH
OOHOH
©2004 Waters Corporation
Comparison of Chromatographic Methods for Retention of Polar Compounds
• Sample and mobile phase solubility problems• Often requires long re-equilibration times
• High % organic mobile phases give higher sensitivity in MS•Eliminate evaporation of SPE eluents
HILIC
• Dewetting under aqueous conditions• Poor retention of polar compounds with non-polar stationary phases
• Familiar technique• High efficiency• Rapid equilibration • Wide selection of columns
Reversed-Phase
• Long equilibration times• Difficult to run gradients• Not compatible with MS• Difficult method development
• Can retain any ionizable compound• Can perform ion pairing chromatography and reversed-phase chromatography on same column
Ion Pairing
• High salt buffers or pH gradients for elution – not compatible with mass spectrometry• Does not work well if the compound does not ionize
• Can retain any ionizable compoundIon
Exchange
DisadvantagesAdvantages
©2004 Waters Corporation
Reversed-Phase Chromatography for Polar Compound Retention
• Reversed-Phase Chromatography– Non-polar stationary phase with >80% aqueous mobile phases– Strengths
Familiar, well understood techniqueMany stationary phase choicesGood reproducibility, stable equilibrationHigh efficiency
– Weaknesses of modern C18 phases designed for improved peak shape for basic analytes: polar compound retention
Sudden loss of retention in 100% aqueous mobile phasePoor retention of polar analytes on a high coverage, non-polar C18stationary phase
©2004 Waters Corporation
Overview
• Introduction• Background• Reversed-Phase HPLC for Polar Molecules
– The objective and challenge– Dewetting– Embedded polar groups – dispelling the myth
• An Intelligent Solution• Applications• Summary
©2004 Waters Corporation
The Challenge for Stationary Phase Manufacturers
• The Objective–Create a stationary phase that retains all
analytes, gives good peak shape for bases, and is compatible with all LC detectors
• The Challenge–To retain a hydrophilic (polar) analyte on a
hydrophobic (non-polar) stationary phase
Why is this so difficult?
©2004 Waters Corporation
Reversed-Phase Chromatography
O-SiO-SiOHO-SiO-SiOHO-SiOHO-SiO-SiO-SiOH
O-SiO-Si
Non-polarstationary
phase
Polar mobilephase
Analyte XPolarAnalyte Y
Non-Polar
How does one develop a column that simultaneously retains
and separates analytes of different polarities?
Flow
PoorlyRetained
(like attracts like)
Well retained(like attracts like)
©2004 Waters Corporation
Challenge for Reversed-Phase Chromatography of Polar Compounds
• To retain polar compounds on this non-polar surface we reduce the amount of organic in the mobile phase (i.e., make mobile phase weaker, e.g., 100% aqueous)
• PROBLEM: Risk of dewetting (hydrophobic collapse) the particle surface – the chromatographic pores dry-out (non-polar pore surface expels the pure aqueous, polar mobile phase)
What is “dewetting” and why does it happen?
©2004 Waters Corporation
Overview
• Introduction
• Background
• Reversed-Phase HPLC for Polar Molecules– The objective and challenge– Dewetting (not hydrophobic collapse)– Embedded polar groups – dispelling the myth
• An Intelligent Solution
• Applications
• Summary
©2004 Waters Corporation
Mobile phase mustbe allowed into the pore
in order for chromatographic retention of the analyte
to take place.
Proper Wetting of Bonded Chromatographic Surface
What influence does this have on chromatography?
If the pores are dry, the analyte cannot get into the pores and it
will not be retained by the chromatographic surface.
The particles are very porous, like the pores of a
sponge – 99% of chromatographic surface is
inside the pores
Analyte
Mobile Phase
©2004 Waters Corporation
Minutes0 2 4 6 8 10
Initial(Column was wetted first
with organic)
After Flow Stoppage (Pores “dewet” 100%)
Mobile phase: 0.1% Acetic Acid
Amoxicillin
Vo: No retention of analyte
1,500psi
Note: Column is not broken --It just stopped working
Why?
1,500psi
Conventional C18 Column – Dewetting
©2004 Waters Corporation
Pore Dewetting Mechanism
Flow stoppage relieves the pressure that was forcing the aqueousmobile phase into the pores. When pressure is reduced (e.g., pump stopped), the hydrophobic pore surface can expel the polar mobile phase and “dewet” the pore.
At flow, with pressure on the mobile phase.
Stopped flow with no pressure on the mobile phase.
Pores de-wet – restart flow – pores still dewetted and analytes never enter pores – resulting in no retention.
Analytes
Analytes properly retained
Remember: Most of the surface area (> 95%) is inside the silica pores!
©2004 Waters Corporation
Water on C18:d = 100Åγ = 72.8 dynes/cmθ = 110.6°*
*B. Janczuk, T. Bialopiotrowicz and W. Wojcik, Colloids and Surfaces 36 (1989) 391-403
Methanol on C18:d = 100Åγ = 22 dynes/cmθ = 39.9°*
Pc < 0 psi
Pc = 1,500 psi
Note: Self-wetting – No pressure required
γEquation of Young and Laplace
γγ=
4θPc =
4θ
dθcos
Pressure on C18 pore requiredto force water back into the pore
Where:Pc = Capillary pressureγ = Surface tensiond = Capillary diameterθ = Contact angle
Stationary Phase Wetting
Solvent dependent
H2O
MeOH
θ
θ
Contact Angle
©2004 Waters Corporation
Re-wetting a Stationary Phase
• Use a mobile phase containing > 40% methanol or other polar organic solvent (other organic solvents may vary in % required for wetting)– This works by reducing the contact angle (θ)
• The use of pressure alone cannot force aqueous mobile phase back into silica pores – Not practical because column outlet is at atmospheric
pressure – not all pores see required pressure (1,500 psi)
1,500 psi 1 atm = 14.7 psi
Pressure gradient inside column
1,500 psi
Atmospheric pressure
©2004 Waters Corporation
Dewetting Summary
• Dewetting– Waters research suggests that dewetting - not
“hydrophobic collapse” is the reason for sudden loss of retention under highly aqueous conditions
– The observed reduction in V0 and sudden loss in retention after a pressure drop indicate that the pores expel the aqueous solvent (vs. ligand chains “folding” or “matting” as described by hydrophobic collapse)
– Rewetting with an organic solvent can rewet the pores by reducing the contact angle and surface tension
Ideally, it is best to avoid dewetting altogether.How have chromatographers
attempted to do this?
©2004 Waters Corporation
Overview
• Introduction
• Background
• Reversed-Phase HPLC for Polar Molecules– The objective and challenge– Dewetting (not hydrophobic collapse)– Embedded polar groups – dispelling the myth
• An Intelligent Solution
• Applications
• Summary
©2004 Waters Corporation
Embedded Polar Groups –Dispelling the Myth
• Embedded polar groups were designed to: – Improve peak shapes for basic compounds – Provide complementary selectivity as compared to
straight-chain alkyl stationary phases
• Examples of embedded polar groups include carbamate, ether, amide, urea, etc.
• Another feature of embedded polar groups is 100% aqueous mobile phase compatibility
This feature is confused with enhanced polar compound retention
Why?
©2004 Waters Corporation
Embedded Polar Groups –Dispelling the Myth
• Embedded polar groups resist pore dewetting by increasing the water layer at the surface of the pores
Embedded polar group behaves
like a “Polar Hook” –holding onto water.
Polar compound retention requires these very
weak polar mobile phasesi.e., highly aqueous mobile phases
with little-or-no organic modifier
This aqueous compatibility is confused with polar compound retention.
This is false!
©2004 Waters Corporation
Carignan, Iraneta
Embedded Polar Groups –What Doesn’t Work
Adenine
Minutes0.00 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
12
34
5
Compounds1. Thiourea2. 5-Fluorocytosine3. Adenine4. Guanosine-5’-monophosphate5. Thymine
ConditionsColumn: AtlantisTM dC18 4.6 x 150 mm, 5 µm Isocratic Mobile Phase: 10 mM NH4COOH, pH 3.0Flow Rate: 1.2 mL/minInjection Volume: 7 µLTemperature: AmbientDetection: UV @ 254 nm
Vo = 1.5minAtlantis dC18 100% Aqueous
QC Batch Test
Thiourea
N
NOH
FNH2
5-Fluorocytosine
NH
NH
O
O
Thymine
N
NH N
N
NH2
Guanosine-5’-monophosphateH2N NH2
SOP
NH
N
N
O
NH2N
O
OHOH
HHH
HO
H
O
OH
For highly polar compounds, embedded polar groups actually cause less retention (see next slide)
©2004 Waters Corporation
Embedded Polar Groups –What Doesn’t Work
Minutes0.00 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
12
3 4 5Vo = 1.5 min
AtlantisTM dC18
1 2
34
5Vo = 1.2min Column Z BR
(amide)
1 23
4 5Column I OEVo = 1.2min
Minutes1.00 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
1 2 34 5
Column P SPVo = 1.3min
Minutes1.00 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
Minutes1.00 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
©2004 Waters Corporation
Embedded Polar Groups –What Doesn’t Work
Minutes0.00 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
12
3 4 5Vo = 1.5 min
AtlantisTM dC18
1 2
34
5Column S AZ
(amide)Vo = 1.3min
12 3
45
Column M PCVo = 1.3min
1 234
5 Column W XRVo = 1.3min
Minutes1.00 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
Minutes1.00 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
Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00
©2004 Waters Corporation
Embedded Polar Groups –Dispelling the Myth
Why less retention of polar compounds with embedded polar group columns?
Reason 1Water layer“shields”
silanols and reduces cationic
interactions and H-bonding
This “water shield” results in less retention of polar compounds -
Both reasons given also describe the reasons for the
excellent peak shape observed for bases
+
N
N
N
NH
NH 3
Reason 2Shorter alkyl chain length in order to
obtain higher ligand density
©2004 Waters Corporation
Embedded Polar Groups –Dispelling the Myth
• Embedded polar groups – summary– Embedded polar groups were designed for
improved peak shape– Embedded polar groups provide aqueous
compatibility– For some compounds, embedded polar groups
can provide increased retention (e.g.,polyphenolics)
– For highly polar compounds, however, embedded polar groups cause less retention
Now that we’ve defined what is not an ideal column for polar retention, let’s define what an ideal column is for polar compound retention.
©2004 Waters Corporation
Overview
• Introduction• Background• Reversed-Phase HPLC for Polar Compounds• An Intelligent Solution – AtlantisTM dC18
– Designing the Ideal column for polar compounds– AtlantisTM dC18 attributes– Retention mechanisms (Why does AtlantisTM dC18
work?)
• Applications• Summary
©2004 Waters Corporation
Designing the Ideal Column
LigandDensity
Endcapping
LigandType
PoreSizeDewetting
Peak Shape
Retention
Stability
The result: Waters AtlantisTM dC18
Optimization of pore size,ligand type, ligand density and endcapping results in a good balance among peak shape,
stability, dewetting and polar retention.
©2004 Waters Corporation
Overview
• Introduction• Background• Reversed-Phase HPLC for Polar Compounds• An Intelligent Solution – AtlantisTM dC18
– Designing the Ideal column for polar compounds– AtlantisTM dC18 attributes– Retention mechanisms (Why does AtlantisTM dC18
work?)
• Applications• Summary
©2004 Waters Corporation
AtlantisTM dC18 Attributes
• What are the key attributes of AtlantisTM dC18?– Superior retention of polar compounds– Operates and thrives in 100% aqueous mobile phases– Excellent peak shape with Mass Spectrometry (MS)
compatible mobile phases– Difunctional bonding chemistry (dC18) results in:
Extended column lifetimeEnhanced low pH stability
– LC/MS compatible with ultra-low column bleed– Perfect balance between polar and non-polar retention– Excellent reproducibility
Examples of each attribute will be given
©2004 Waters Corporation
Superior Retention of Polar Compounds
Time (min)0.00 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
1
2
34
5
1 2 3
45
Time (min)
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
Conventional C18
Atlantis™ dC18ConditionsColumns: 4.6 x 150 mm, 5 µm Mobile Phase: 10mM NH4COOH, pH 3.0Flow rate: 1.2 mL/minInjection volume: 7 µLDetection: UV@254 nm
Compounds1. Thiourea2. 5- Fluorocytosine3. Adenine4. Guanosine-5’- monophosphate5. Thymine
Aqueous Separation of Polar Compounds for AtlantisTM vs. Conventional C18 Column
Vo = 1.5 min
Peak 1 elutesIn the void
35% - 60% more retention with
AtlantisTM dC18 ascompared to
high coverage C18
©2004 Waters Corporation
Operates and Thrives in 100% Aqueous Mobile Phase
AtlantisTM dC18 Columns Dewetting Test
Retention after stop flow test(Pore dewetting: ~100%)
Initial Retention
Initial Retention
Time (min)2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
Vo
Retention after stop flow test(Pore dewetting: ~ 3.5%)
Conventional C18
AtlantisTM dC18
Amoxicillin retention with 0.1% formic acid mobile phase
100% retention loss with conventional C18
<4% retention loss with AtlantisTM dC18
Note enhanced retention
Note change in V0in dewetted column
AtlantisTM dC18 columns resist the sudden loss of retention associated with pore dewetting.
©2004 Waters Corporation
Excellent Peak Shape Using AtlantisTM dC18with MS Compatible Mobile Phases
Note excellent peak shape for strong polar
base Adenine at pH 5.0
Compounds:1. Cytosine2. 5-Fluorocytosine3. Uracil4. 5-Fluorouracil5. Guanine6. Thymine7. Adenine
1
2 3
4
5 6
Minutes
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
7
V0 = 1.83 min
ConditionsColumn: AtlantisTM dC18 4.6 x 150 mm, 5 µm Mobile Phase A: H2OMobile Phase B: ACNMobile Phase C: 100 mM CH3COONH4, pH 5.0Flow Rate: 1.0 mL/minGradient: Time Profile
(min) %A %B %C0.0 90 0 1010.0 84 6 10
Injection Volume: 10 µLTemperature: 30 oCDetection: UV @ 254 nmInstrument:: Alliance® 2695, 2996 PDA
Grumbach, Diehl
The popular void marker Uracil is well retained and is actually peak 3
At pH 5.0, the strong polar base Adenine still exhibits excellent peak shape
(reason: fully endcapped)
©2004 Waters Corporation
Benefit of Fully Endcapped Column
1
2 3
4 5
Column Z SAVo = 1.5min
2 4 53
Minutes0.00 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
Column K AQVo = 1.3min
1Minutes
0.00 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
12
3 4 5AtlantisTM dC18
Minutes0.00 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
Longer base retention at the expense of peak shape
Excellent retention and peak for all compounds
ConditionsColumns: 4.6 x 150 mm, 5 µm Mobile Phase: 10 mM NH4COOH, pH 3.0Flow Rate: 1.2 mL/minInjection Volume: 7 µLDetection: UV @ 254 nm
Compounds1. Thiourea2. 5-Fluorocytosine3. Adenine4. Guanosine-5’-monophosphate5. Thymine
Vo = 1.5min
©2004 Waters Corporation
Extended Column Lifetime and Low pH Stability
• Difunctional C18 bonding chemistry results in: – Extended column lifetime– Enhanced low pH stability
Accelerated Low pH Degradation Test(30oC with 0.1% TFA)
1 20 50 100 200 300 700 800 900 950 990
Injection Number
% O
rigin
al
Ret
entio
n Ti
me
60
65
70
75
80
85
90
95
100
105
60
65
70
75
80
85
90
95
100
105
Thymine - AtlantisTM dC18
Thymine - Monofunctional C18
What is a difunctional
bond and why does it
provide longercolumn
lifetimes at low pH?
©2004 Waters Corporation
Hydrolysis of a Bonded Phase Material:Limitations of Monofunctional Ligands
+ HCl
+
+
Hydrolysis at low pH(monofunctional bond broken)
SiC
CC
CC
CC
CH3
CH3
H3C
Cl
OHSi
O
O
O
SiC
CC
CC
CC
CH3
CH3
H3C
OSi
O
O
O
SiC
CC
CC
CC
CH3
CH3
H3C
HOOH
SiO
O
O
Monofunctional = Attached to silica at one pointMore susceptible to low pH hydrolysis (column bleed)
Synthesis (monofunctional bond created)
Single siloxane bond
C8 Monochlorosilane Ligand
©2004 Waters Corporation
Making a Bonded Phase Material: Multifunctional Synthesis
+
+ HCl
C8 Dichlorosilane LigandOH
OH
OHSi
SiSi
O
OO
O
O OO
ClSi
Cl
CH3CH3
OS i
O
OH
Si
SiSi
O
OO
O
O OO
CH3
CH3
Difunctional = Attached to silica at two pointsLess susceptible to low pH hydrolysis (column bleed)
Synthesis
Two siloxane bonds
©2004 Waters Corporation
LC/MS Compatible with Ultra-low Column Bleed
Waters ZQ Detector Settings:Source: ESI (+)Capillary (kV): 3.5Cone (V): 30Temperature (°C)
Source: 150 Desolvation: 350
Gas Flow (L/Hr)Cone: 50Desolvation: 500
m/z: 300-1500
ConditionsColumn: AtlantisTM dC18 4.6 x 50 mm, 3 µmMobile Phase A: 0.1 % HCOOHMobile Phase B: 0.065 % HCOOH in ACNFlow Rate: 0.75 mL/min, 0.2 mL/min to MSGradient: Time Profile
(min) %A %B0.0 100 045.0 60 40
Injection Volume: 20 µLInjection Mass: 20.0 µgTemperature: Ambient
5.00 10.00 15.00 20.00 25.00 30.00Time0
%
Rainville, Russell
100 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.000
100
%
0
100
% 41.1432.9131.8929.0827.87
22.140.07
4.633.71 12.466.42 10.709.257.99 13.20 15.00 21.42 23.77 24.60 36.6633.35 38.49 42.09 44.28
0.95
2.26 2.964.15 43.1210.725.91
8.63 22.9813.86 21.4616.87 27.4824.25 28.84
42.7230.7332.18 39.08 43.52
No Column BlankScan ES+: TIC
8.00e6
AtlantisTM dC18 BlankScan ES+: TIC
8.00e6
Scan ESI+: TIC1.00e9
Cytochrome C Tryptic Digest
©2004 Waters Corporation
LC/MS Compatible with Ultra-low Column Bleed
No Column Blank Combined Spectrum over Entire Runtime
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z0
100
%
1: Scan ES+ 9.00e4490
355
327
429
384428
415
445
447
610
491536
503537
577539
653611
612
613
621
684
654
685
758687
744690832761 817
906835 909
No differences betweenthe combined spectra from
blank runs with column and without column.
AtlantisTM dC18 Column Blank Combined Spectrum over Entire Runtime
0
100
%
1: Scan ES+ 9.00e4
355
315 341
429367
371
384415
398
610445
536447503
538564
684611
613639
685 758687
744832761819 907835 861 909 982
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z
Combined Spectra of Blank TIC
©2004 Waters Corporation
ConditionsColumn: AtlantisTM dC18 4.6 x 150 mm, 5 µm Mobile Phase A: 0.1% TFA in H2OMobile Phase B: 0.1% TFA in ACNFlow Rate: 1.4 mL/minGradient: Time Profile
(min) %A %B0.0 100 05.0 97 36.0 85 1510.0 80 2012.0 0 10025.0 0 100
Injection Volume: 10 µLTemperature: 30 oCDetection: UV @ 268 nmInstrument: Alliance® 2695, 2996 PDA
Perfect Balance Between Polar and Non-Polar Compound Retention
Compounds: Concentration (µg/mL)1. L-ascorbic acid (vitamin C) 19.62. Nicotinic acid (niacin) 9.83. Thiamine (vitamin B1) 19.64. Pyridoxal 39.25. Pyridoxine 39.26. Folic acid (vitamin B11) 23.57. Caffeine 9.88. Riboflavin (vitamin B2) 3.99. Retinol (vitamin A) 19.610. Tocopherol (vitamin E) 39.211. Ergocalciferol (vitamin D2) 9.8
Grumbach, DiehlMinutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00
1 23
45
8
7
6
V0 = 1.37 min
109
11
Excellent retention for water-soluble vitamins
Not overly retentive for fat-soluble vitamins
©2004 Waters Corporation
Excellent Reproducibility
Minutes0.00 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
Eight Actual QC Batch Test Chromatograms Under 100% Aqueous Conditions
(not specially packed columns)
%RSD 0.16 1.18 1.39 1.92 1.96
©2004 Waters Corporation
Overview
• Introduction• Background• Reversed-Phase HPLC for Polar Compounds• An Intelligent Solution – AtlantisTM dC18
– Designing the Ideal column for polar compounds– AtlantisTM dC18 attributes– Retention mechanisms (Why does AtlantisTM dC18
work?)
• Applications• Summary
©2004 Waters Corporation
Surface of a Silica Gel Bonded-Phase Packing Material
High Coverage
– High Ligand Density
Low Coverage –Low Ligand
Density
H
SiO
O
OSi
O
O
OSi
O
O
OSi
O
O
OSi
O
O
OSi
O
O
OSi
O
O
OSi
O
O
OSi
O
O
OSiO
OO
SiO
O
O
SiCH2
H3C CH3
H2CCH2
H2CCH2
H2CCH2
H3C
SiCH3
H3C CH3 SiCH2
H3C CH3
H2CCH2
H2CCH2
H2CCH2
H3C
SiO
O
O
SiCH3
H3C CH3 SiCH3
H3C CH3H
HHHHH H
C8 alkyl chains
Residual silanolsEndcap
Polar analytes are not able to “energetically fit” between ligands –can’t interact with surface or ligands
Si
SiO
O
OSi
O
O
OSi
O
O
OSi
O
O
OSi
O
O
OSi
O
O
OSi
O
O
OSi
O
O
OSi
O
O
OSiO
OO
SiO
O
O
SiCH2H3C CH3
H2CCH2
H2CCH2
H2CCH2
H3C
SiCH2H3C CH3
H2CCH2
H2CCH2
H2CCH2
H3C
CH2H3C CH3
H2CCH2
H2CCH2
H2CCH2
H3C
SiCH2
H3C CH3
H2CCH2
H2CCH2
H2CCH2
H3C
SiO
O
O
SiCH3H3C CH3 Si
CH3H3C CH3
HHHHHH H
Residual silanolEndcap
Polar analytes easily interact with surface
and ligands
Adapted from J.G. Dorsey and K.A. Dill, Chem. Rev., 1989, 89, 331-346
©2004 Waters Corporation
Low Coverage –Low Ligand
Density
Surface of a Silica Gel Bonded-Phase Packing Material
H
SiO
O
O
SiO
O
O
SiO
O
O
SiO
O
O
SiO
O
O
SiO
O
O
SiO
O
O
SiO
O
O
SiO
O
O
Si
O
OO
SiO
O
O
Si
CH2H3C CH3
H2CCH2
H2C
CH2
H2C
CH2
H2C
Si
CH3H3C CH3 Si
CH2H3C CH3
H2CCH2
H2C
CH2
H2C
CH2
H2C
SiO
O
O
Si
CH3H3C CH3 Si
CH3H3C CH3
H
HHHHH H
Alkyl chains
Residual silanols
EndcapN
NH
NH2
O
Polar portion of analyte interacts
with silanols(hydrogen bonding
and/or ion exchange)
NH
NH
O
ONon-polarportion of
analyte interacts with bonded phase(hydrophobic)
©2004 Waters Corporation
Polar Retention:Why Does AtlantisTM dC18 Work?
• Dominant retention mechanism is reversed-phase (van der Waals forces – hydrophobic attraction)– Retention maximized using 100% aqueous mobile
phases– Retention maximized by using reduced C18 coverage
Polar analytes can “fit” between C18 ligands and interact with surface silanols and alkyl chains
• Secondary interactions due to residual silanols that are more accessible due to reduced C18coverage– Cation-exchange interactions– Hydrogen bonding interactions
©2004 Waters Corporation
Overview
• Introduction
• Background
• Reversed-Phase HPLC for Polar Compounds
• An Intelligent Solution – AtlantisTM dC18
• Applications
• Summary
©2004 Waters Corporation
Isocratic Separation of Catecholamines and Metabolites
Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
1
2
34
5
7
6V0 = 1.83 min
ConditionsColumn: AtlantisTM dC18 4.6 x 150 mm, 5 µm Mobile Phase A: H2OMobile Phase B: ACNMobile Phase C: 100 mM CH3COONH4, pH 5.0Flow Rate: 1.0 mL/minIsocratic Mobile Phase Composition: 88% A; 2% B; 10% C Injection Volume: 10 µLTemperature: 30 oCDetection: UV @ 280 nm
Compounds USP Tailing1. Norepinephrine (NE) 1.212. Epinephrine (E) 1.203. Dopamine (DA) 1.214. 3,4-Dihydroxyphenylacetic acid (DOPAC) 1.005. Serotonin (5-HT) 1.106. 5-Hydroxy-3-indoleacetic acid (5-HIAA) 0.977. 4-Hydroxy-3-methoxyphenylacetic acid (HVA) 0.97
No ion-pairing agent necessary!!
©2004 Waters Corporation
Low Level Acrylamide LC/MS/MS Analysis
LC Conditions:Column: AtlantisTM dC18 2.1 x 150 mm, 3 µmPart Number: 186001299Mobile Phase A: 0.1 % CH3COOHMobile Phase B: MeOHIsocratic Mobile Phase Composition: 99.5% A; 0.5% BFlow Rate: 0.2 mL/minInjection Volume: 20 µLTemperature: 26 oCInstrument:: Alliance® 2695, Quattro Premier API
CH
NH2
O
CH2
Acrylamide
2 pg on column(20 µL of 0.1 ppb)
1.00 2.00 3.00 4.00Minutes
71.7 > 43.9
71.7 > 54.84.2
x 10
3
Acrylamide
Smooth (Mn,1x2)
S/N = 10.82 pg on column
(20 µL of 0.1 ppb)
1.00 2.00 3.00 4.00Minutes
71.7 > 43.9
71.7 > 54.84.2
x 10
3
Acrylamide
Smooth (Mn,1x2)
S/N = 10.8
1.00 2.00 3.00 4.00Minutes
71.7 > 43.9
3.5
x 10
4
Smooth (Mn,1x2)
Acrylamide
71.7 > 54.8
2.50 3.00 3.50 4.00
2.4
x 10
350 pg on column(20 µL of 2.5 ppb)
S/N = 10.5
1.00 2.00 3.00 4.00Minutes
71.7 > 43.9
3.5
x 10
4
Smooth (Mn,1x2)
Acrylamide
71.7 > 54.8
2.50 3.00 3.50 4.00
2.4
x 10
350 pg on column(20 µL of 2.5 ppb)
S/N = 10.5
The mass detector was calibrated between m/z 19 to m/z2000 with unit resolution.
MS/MS Analysis Function (MRM of 4 mass pairs)
Transition Dwell Time Collision Energy Delay71.7 > 43.9 0.4 11.0 0.0171.7 > 54.8 0.4 10.0 0.0174.7 > 44.9 0.4 11.0 0.0174.7 > 57.8 0.4 10.0 0.01
The 74.7>57.8 and 71.7>44.9 for for the 13C3-Acrylamide Internal Standard at 50 ppb.
©2004 Waters Corporation
Peptides
Time (Min)
4 8 12 16 20 240
Compounds1. L22752. A66773. Bradykinin4. Angiotensin II5. Angiotensin I6. Substance P7. Renin Substrate8. Insulin B Chain9. Melittin
Atlantis™ dC18
Conventional C18
21
354 6
7
98
21
3 54 6 7
98V 0
ConditionsColumns: 4.6 x 50 mm, 5 µmMobile Phase A: 0.02% TFAMobile Phase B: 0.016% TFA in ACNFlow Rate: 0.75 mL/minGradient: Time Profile
(min) %A %B0.0 100 025.0 50 50
Injection Volume: 20 µLTemperature: 40 oCDetection: UV @ 220 nm
Gradient Peptide Separation Using TFA Mobile Phases
Note enhanced retention of peptides using AtlantisTM dC18 as compared to conventional C18 column
©2004 Waters Corporation
Cytochrome C Tryptic Digest:TFA Not Necessary
ConditionsColumn: AtlantisTM dC18 4.6 x 50 mm, 3 µm Mobile Phase A: 0.1% HCOOH or 0.02%TFAMobile Phase B: 0.065% HCOOH in ACN or
0.016% TFA in ACNFlow Rate: 0.75 mL/minGradient: Time Profile
(min) %A %B0.0 100 045.0 60 40
Injection Volume: 20 µLInjection Mass: 20 µgTemperature: Ambient
AtlantisTM dC18 columns permit the use of
MS-friendly mobile phase additives
©2004 Waters Corporation
Cytochrome C Tryptic Digest:Increased Mass Loading
No loss inresolution,
peak shape or peak capacity
ConditionsColumn: AtlantisTM dC18 4.6 x 50 mm, 3 µm Mobile Phase A: 0.1% HCOOH or 0.02%TFAMobile Phase B: 0.065% HCOOH in ACN or
0.016% TFA in ACNFlow Rate: 0.75 mL/minGradient: Time Profile
(min) %A %B0.0 100 045.0 60 40
Injection Volume: 20 µLInjection Mass: 20 µgTemperature: Ambient
©2004 Waters Corporation
ConditionsColumn: AtlantisTM dC18, 2.1x 20 mm IS™, 3 µmPart Number: 186002058Mobile Phase A: WaterMobile Phase B: MeOHMobile Phase C: 1% HCOOHFlow Rate: 0.4 mL/minGradient: Time Profile
(min) %A %B %C 0.0 50 40 101.0 30 60 10
Inj Volume: 2 µLConcentration: 10 µg/mLTemperature: 30 oCInstruments: Alliance® 2795 and Waters ZQTM
Compounds MWCinoxacin 262.2Oxolinic Acid 261.2Nalidixic Acid 232.2
Waters ZQTM
ES+
Capillary (kV) 3.5 Cone (V) 50 Extractor 3.0 RF Lens 0.1Source Temp (oC) 150 Desolvation Temp (oC) 400Cone Gas Flow (L/Hr) 50Desolvation Gas Flow (L/Hr) 500LM Resolution 15HM Resolution 15Ion Energy 0.5 Multiplier (V) 650
High-Throughput Separation: Nalidixic Acid Antibiotics
Cinoxacin
Nalidixic acid
Oxolinic acid
0.00 0.20 0.40 0.60 0.80 1.00Time0
100
%
0
100
%
0
100
%
0
100
%
1: Scan ES+ TIC
1.15e90.88
0.65
0.59
1: Scan ES+ 263
4.05e70.590.65
0.88
1: Scan ES+ 262
1.19e80.65
1: Scan ES+ 233
1.28e80.89
Separation in 1 minute!
©2004 Waters Corporation
Column: AtlantisTM dC18 4.6 x 100 mm, 5 µmPart Number:186001340Total Mass Load: 3,000 µg
Water Soluble Vitamins:Ease of Scale-up
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00
Time (min)
1
1
2
2
Conditions:Mobile Phase A: 0.1% HCOOH in H2OMobile Phase B: ACN/1% HCOOH (90/10)Sample Concentration: 5, 25 mg/mL in waterTemperature: AmbientInstrument:: AutoPurification™ SystemDetection: UV @ 290 nm
V0 = 1.73 min
V0 = 1.76min
Column: AtlantisTM dC18 30 x 100 mm, 5 µmPart Number:186001375Total Mass Load: 126,000 µg
Flow Rate: 1.0 mL/minGradient: Time Profile
(min) %A %B0.0 100 01.0 100 06.0 80 2016.0 60 40
Injection Volume: 100 µL
Flow Rate: 42.53 mL/minGradient: Time Profile
(min) %A %B0.0 100 03.25 100 08.25 80 2018.25 60 40
Injection Volume: 4200 µL
1. Pyridoxine 2. Caffeine
©2004 Waters Corporation
Time (min)
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00-0
100
%
11.7 mg total load
1
2
3
Preparative Separation –Nalidixic Acid Antibiotics
ConditionsColumn: AtlantisTM dC18 19 x 100 mm, 5 µmMobile Phase A: 0.1% HCOOH in H2OMobile Phase B: 0.1% HCOOH in ACNFlow Rate: 17.06 mL/minGradient: Time Profile
(min) %A %B0.0 80 204.02 80 2014.02 40 60
Injection Volume: 1800 µL (DMSO)Temperature: AmbientDetection: UV @ 300 nmInstrument: AutoPurification™ System
1. Cinoxacin (5 mg/mL)2. Oxolinic acid (0.5 mg/mL)3. Nalidixic acid (1 mg/mL)
©2004 Waters Corporation
Overview
• Introduction
• Background
• Reversed-Phase HPLC for Polar Compounds
• An Intelligent Solution – AtlantisTM dC18
• Applications
• Summary
©2004 Waters Corporation
AtlantisTM dC18 vs. Conventional C18 and Embedded Polar Group Columns
Column Resists Dewetting?
Increased Polar Retention?
Conventional alkyl C18 No No
Embedded polar group Yes No
AtlantisTM dC18 Yes Yes
Remember:1. Embedded polar group ≠ polar compound retention2. In order to create polar compound retention, choose
a column designed for polar compound retention
©2004 Waters Corporation
Polar Retention Summary
• Embedded polar groups were designed for peak shape, not polar compound retention
• The sudden loss in retention observed under highly aqueous conditions is due to pore dewetting nothydrophobic collapse
• Unendcapped C18 columns provide increased base retention at the expense of peak shape
• Increased RP retention can be realized with an optimized lower ligand density column
• A properly designed straight alkyl chain C18 column can provide the perfect balance of polar and non-polar compound retention
Thank you for attending today’s seminar!!Thank you for attending today’s seminar!!