Post on 07-Jun-2019
©2003 Waters Corporation
Understanding (and Creating) Polar Retention Using Reversed-Phase HPLC and Hydrophilic
Interaction Chromatography
Pittcon March 2003
©2003 Waters Corporation
Introduction
• Introduction• The Problem
•Background
•Hydrophilic Interaction Chromatography
•Reversed-Phase HPLC for Polar Molecules
•Applications
•Summary
©2003 Waters Corporation
Introduction - The Problem
•Conventional 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 compounds• Elute un-retained in the void volume• Co-elute at the beginning of the run – if retained at all
•Method Development• Isocratic - Many RP columns cannot be run under the
100% aqueous conditions necessary for polar compound retention– columns “dewet” (also termed -hydrophobic collapse)
©2003 Waters Corporation
Background
• Introduction•Background
• Polar molecules• Chromatographic methods for retaining polar
compounds
•Hydrophilic Interaction Chromatography•Reversed-Phase HPLC for Polar Molecules•Applications•Summary
©2003 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!
Electro-static attraction – opposite charge locations on different molecules attract
©2003 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
©2003 Waters Corporation
Comparison of Chromatographic Methods for Retention of Polar Compounds
• 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• Most ion pairing agents 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
• Sample and mobile phase solubility problems• Long re-equilibration times
• High percentage organic mobile phases give higher sensitivity in ESI-MS
HILIC
• Does not work well if the compound does not ionize• High salt buffers or pH gradients for elution – not compatible with MS
• Can retain any ionizable compoundIon
Exchange
DisadvantagesAdvantages
©2003 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 technique• Many stationary phase choices• Good reproducibility, stable equilibration• High 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 phase• Poor retention of polar analytes on a high coverage, non-polar C18
stationary phase
©2003 Waters Corporation
Hydrophilic Interaction Chromatography for Polar Compound Retention
• HILIC – Hydrophilic Interaction Chromatography• Polar stationary phase with >80% organic mobile phases• Strengths
• Good retention of very polar molecules• Volatile mobile phases
• Increased ESI-MS sensitivity• Lower backpressure
• Complementary selectivity vs reversed-phase• Weaknesses
• Not many stationary phases available• Poor retention of hydrophobic compounds
©2003 Waters Corporation
Hydrophilic Interaction Chromatography
• Introduction
• Background• Hydrophilic Interaction Chromatography (HILIC)
• What is it?• Why would someone use it?• AtlantisTM HILIC Silica
• Reversed-Phase HPLC for Polar Molecules
• Applications
• Summary
©2003 Waters Corporation
HILIC: What is it?
• HILIC – Hydrophilic Interaction Chromatography• “Reverse reversed-phase” (water is the eluting solvent)• Very polar analytes are retained and separated on a polar,
hydrophilic stationary phase• Analytes elute in order of increasing hydrophilicity• High organic/low aqueous conditions
• Mobile phases used• >70% ACN (with water/buffer) • Very volatile (very “ESI-MS-friendly”)
• Stationary phases• SiO2, diol, amino, amide, cyano, ion-exchange, SEC,
Zwitterion, etc.
©2003 Waters Corporation
HILIC: What is it?
Polar Phase
HILIC Retention• Polar analyte partitions into and out of adsorbed water layer
• The larger the water layer (greater aqueous concentration), the faster the compound(s) will elute
• Analytes elute in order of increasing hydrophilicity
• High Acetonitrile (>70%) with aqueous
• Polar Phases (Bonded and Unbonded)
HN
NH
O
O
HN
NH
O
O
OH
80% acetonitrile
20% aqueous,(e.g., pH 3.0)
uracil
©2003 Waters Corporation
HILIC: What is it?
Polar Phase
Weak Cation ExchangeRetention
• Charged polar analyte can undergo cation exchange with charged silanol groups
• High Acetonitrile (>70%) with aqueous
O-
80% acetonitrile
20% aqueous, (e.g., pH 5.5)
HILIC is defined as the high organic conditions
where polar compounds are retained longer
(reverse reversed-phase)
N
NH3+
NH
O
N
NH3+
NH
O
cytosine
©2003 Waters Corporation
HILIC Retention Characteristics
Cytosine
HILIC:Increased retention occurs when using
greater than 70% organic for polar bases
N
NH
O
NH2
-3.00
-2.00
-1.00
0.00
1.00
2.00
3.00
0 10 20 30 40 50 60 70 80 90% MeCN
LN (T
rmin
/cm
)
pH 5
pH 4
pH 3
Aqueous
Retention
©2003 Waters Corporation
-2.90-2.80-2.70-2.60-2.50-2.40-2.30-2.20-2.10-2.00
0 10 20 30 40 50 60 70 80 90% MeCN
LN (T
rmin
/cm
) pH 5
pH 4
pH 3
Reversed-Phase Retention Characteristics
Cytosine
Reversed-phase:Increased retention occurs when using less than 20% organic for polar bases
N
NH
O
NH2
Aqueous
Retention
©2003 Waters Corporation
HILIC:Why would someone use it?
• Why would someone use HILIC?• Retains very polar compounds not retainable by RP• Volatile mobile phase
• Increased ESI-MS sensitivity (lower LODs)• Increased flow rates• Increased sample throughput• Lower backpressures
• Facilitates sample prep (SPE, liquid/liquid, protein precipitation) as the final elution/extraction solvent does not have to be evaporated/reconstituted
©2003 Waters Corporation
Vo = 1.15 min
AU
0.00
0.05
0.10
0.15
Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
AtlantisTM HILIC Silica4.6 x 50 mm, 3 µm
95:5 ACN:Formate Buffer, pH 3.01.4 mL/min
AtlantisTM HILIC Silica: Retains very polar compounds not retainable by RP
AU
0.00
0.05
0.10
Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
AtlantisTM dC184.6 x 50 mm, 3 µm
100% Formate Buffer, pH 3.0 1.0 mL/min
Vo = 0.65 min
NH
NHNH
O
OO
NH2
Allantoin
HILIC offers retention when there is no retention by reversed-phase
k’= 1.0
k’= 0.0
©2003 Waters Corporation
AtlantisTM HILIC Silica : Enhanced ESI-MS Sensitivity
AtlantisTM dC18 Peak Area2.1 x 50 mm, 3 µm1. Albuterol 100 pg/µL 782. Bamethan 20 pg/µL 2
AtlantisTM HILIC Silica Peak Area2.1 x 50 mm, 3 µm2. Bamethan 20 pg/µL 93871. Albuterol 100 pg/µL 13131
HILIC requires high volatility solvents which increase ESI-MS sensitivity vs. high-aqueous mobile phases used in RP
7.63e3
0
100
%
1.802.13
ES+
239.8
1.07e5
209.90
100
%
1.80
2.13
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
0
100
%
1.311.63
ES+
239.8
1.07e5
209.9
1
2?
12
©2003 Waters Corporation
AtlantisTM HILIC Silica :Simplify Sample Preparation
Condition/Equilibrate*200 µL methanol/200 µL water
Load150 µL spiked plasma sample
150 µL internal standardwith 2% ammonium hydroxide
Wash200 µL 5% methanol in water
Elute300 µL 40% acetonitrile/60% isopropanol
with 2% formic acid
*Oasis® HLBµElution Plate
Inject eluent directly onto columnNo Evaporation and Reconstitution Step
©2003 Waters Corporation
AtlantisTM HILIC Silica :Simplify Sample Preparation
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00Time
0
100
%
SIR of 2 Channels ES+TIC239.8209.98.97e4
1.49 1.84
NHOH
OH
CH3
Bamethan
NH
OH
OH
OHCH3 CH3
CH3
Albuterol
AtlantisTM HILIC Silica2.1 x 50 mm, 3 µm1. Bamethan 10 pg/µL 2. Albuterol 50 pg/µL
1 2
SPE eluent injected directly onto AtlantisTM HILIC Silica column
©2003 Waters Corporation
Atlantis HILIC SIlica:Summary
• AtlantisTM HILIC Silica• Provides retention of some polar analytes not retained under
RP conditions• Enhanced sensitivity in ESI-MS is enjoyed due to the highly
volatile mobile phases (> 80% organic) used• Sample preparation procedures are shortened by eliminating
the evaporation and reconstitution steps and directly injecting the eluent
• AtlantisTM HILIC Silica columns are an alternative to RP chromatography for the retention of very polar basic analytes
New at Pittcon 2003
©2003 Waters Corporation
Reversed-Phase HPLC for Polar Molecules
• Introduction
• Background
• Hydrophilic Interaction Chromatography
• Reversed-Phase HPLC for Polar Molecules• The objective and challenge• Dewetting (not hydrophobic collapse)• What doesn’t work (and why)• AtlantisTM dC18 - an intelligent solution
• Applications
• Summary
©2003 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?
©2003 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
Chromatographically how does oneattempt to force polar Analyte X to retain on
the non-polar stationary phase?
Flow
PoorlyRetained
(like attracts like)
Well retained(like attracts like)
©2003 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?
©2003 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
©2003 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!
©2003 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=
4 θPc =4 θd
θcos
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
©2003 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
©2003 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?
©2003 Waters Corporation
Embedded Polar Groups –What Doesn’t Work
• 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?
©2003 Waters Corporation
Embedded Polar Groups –What Doesn’t Work
• 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!
©2003 Waters CorporationCarignan, 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: 7.0 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)
©2003 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
Minu tes1 .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
5Vo = 1.2min Column Z BR
(amide)
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 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
©2003 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
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
5Column S AZ
(amide)Vo = 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
12 3
45
Column M PCVo = 1.3min
1 234
5 Column W XR
Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00
Vo = 1.3min
©2003 Waters Corporation
Embedded Polar Groups –What Doesn’t Work & Why
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 for in order to obtain higher ligand
density
©2003 Waters Corporation
Embedded Polar Groups –What Doesn’t Work - Summary
•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.
©2003 Waters Corporation
The Ideal Column for Retention of Polar Compounds
•Retains and separates polar compounds•Compatible in 100% aqueous mobile phases –
i.e. does not dewet•Stable under acidic conditions•Compatible with all detectors•Enhanced polar compound retention without
extreme non-polar compound retention (a balance)
•Excellent peak shape and column-to-column reproducibility
©2003 Waters Corporation
AtlantisTM dC18 Intelligent Design
Intelligent Design of the Chromatographic Particle
We can: Optimize Silica-gel and Bonding Chemistry:* Pore size * Ligand type * Ligand density * Endcapping
Resulting in: Desired peak shape, dewetting, retention and stability characteristics
Liganddensity
Endcapping
Ligandtype
Poresize
DewettingPeak Shape
RetentionStability
©2003 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 lifetime• Enhanced low pH stability
• LC/MS compatible with ultra-low column bleed• Enhanced polar compound retention without infinite
non-polar compound retentionExamples of each attribute will be given
©2003 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
AtlantisTM 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
©2003 Waters Corporation
Operates and Thrives in 100% Aqueous Mobile Phase
• AtlantisTM was designed to resist the sudden loss of retention associated with pore dewetting.
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
©2003 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: H20Mobile 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 10
10.0 84 6 10Injection Volume: 10 µLTemperature: 30 oCDetection: UV @ 254 nmInstrument:: AllianceTM 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)
©2003 Waters Corporation
Benefit of a Fully Endcapped Column
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
1
2 4 5
3
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
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
Unendcapped “AQ-Type”column
AtlantisTM dC18 column
Longer base retention at the expense of peak shape
Excellent retention and peak for all compounds
©2003 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 difunctionalbond and why does it
provide longer column lifetimes at low pH?
©2003 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
©2003 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
©2003 Waters Corporation
Enhanced Polar Compound Retention without Infinite Non-polar Compound Retention
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
12
3 4 5
10
98
7
6
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
12 3
45 6
7
10
98V0 = 0.98 min
AtlantisTM dC18
Conventional C18with 2x ligand
density
More retention for polar compounds
Equal or less retention for non-polar compounds
Void Marker
Back Marker
V0 = 0.98 min
©2003 Waters Corporation
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
Atlantis dC18 - An Intelligent DesignWhy Does It Work?
Adapted from J.G. Dorsey and K.A. Dill, Chem. Rev., 1989, 89, 331-346
©2003 Waters Corporation
Low Coverage –Low Ligand
Density
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
Endcap
Atlantis dC18 - An Intelligent DesignWhy Does It Work?
N
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)
©2003 Waters Corporation
Atlantis dC18 - An Intelligent DesignWhy Does It 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 C18 coverage
• Cation-exchange interactions• Hydrogen bonding interactions
©2003 Waters Corporation
Atlantis dC18 - An Intelligent DesignExcellent Batch-to-Batch 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
©2003 Waters Corporation
Applications
• Introduction
•Background
•Hydrophilic Interaction Chromatography
•Reversed-Phase HPLC for Polar Molecules
•Applications
•Summary
©2003 Waters Corporation
Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
Minutes
1.00 2.00 3.00 4.00 5.00
Complementary Selectivity of HILIC as Compared to Reversed-Phase
Vo = 0.5 min
Compounds:1. Morphine 2. Morphine 3-β-D-glucuronide
AtlantisTM HILIC Silica4.6 x 50 mm, 3 µm
1 2
AtlantisTM dC184.6 x 50 mm, 3 µm
2
1Vo = 0.65 min
O
N
OH
OH
CH3
H
Morphine
Morphine 3-ß-D-Glucuronide
©2003 Waters Corporation
Gradient LC/MS Separation of Cytochrome C Tryptic Digest on AtlantisTM HILIC Silica versus AtlantisTM dC18
0
100
%
204 (T2)261 (T6,11)361 (T18)434 (T21)
678 (T14)
634 (T4)
585 (T8)
779 (T15)
729 (T10) 964 (T19) 1005 (T!2)
Peak AnnotationM/Z (Fragment ID)
779 (T15)
634 (T4)
678 (T14)434 (T21)
964 (T19)
261 (T6,11)204 (T2)
361 (T18)1005 (T!2)
585 (T8)
729 (T10)
0
100
%
0 45
HILIC
RP
AtlantisTM dC182.1 x 50 mm, 3 µm
AtlantisTM HILIC Silica2.1 x 50 mm, 3 µm
©2003 Waters Corporation
Isocratic RP Separation of Catecholamines and Metabolites on Atlantis dC18
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!!
©2003 Waters Corporation
Water Soluble Vitamins on Atlantis dC18
Compounds: USP Tailing1. L-ascorbic acid 1.122. Nicotinic acid 1.273. Thiamine 1.204. Pyridoxal 1.135. Pyridoxine 1.046. Folic acid 1.107. Caffeine 1.108. Riboflavin 1.14
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
12
3
45
87
6
V0 = 1.37 min
ConditionsColumn: AtlantisTM dC18 4.6 x 150 mm, 5 µm Mobile Phase A: 0.1% TFAMobile Phase B: ACNFlow Rate: 1.4 mL/minGradient: Time Profile
(min) %A %B0.0 100 04.0 97 36.0 85 15
15.0 80 20Injection Volume: 10 µLTemperature: 30 oCDetection: UV @ 260 nm
No ion-pairing agent necessary!!
©2003 Waters Corporation
Tryptic Cytochrome C Peptide Separation: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/min, 0.2 mL/min Split to MSGradient: 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
©2003 Waters Corporation
Summary
• Introduction
•Background
•Hydrophilic Interaction Chromatography
•Reversed-Phase HPLC for Polar Molecules
•Applications
•Summary
©2003 Waters Corporation
AtlantisTM Columns Summary
• AtlantisTM LC/MS columns are designed for retaining and separating polar compounds
• New AtlantisTM HILIC Silica columns• Retain very polar compounds for high throughput LC/MS• Simply sample preparation• Offer complementary selectivity as compared to RP
• AtlantisTM dC18 columns• Retain polar compounds without extreme retention of non-polar
compounds (universal low pH RP column)• Provide excellent peak shapes with LC/MS compatible mobile
phases • Thrive in aqueous mobile phases• Provide long column lifetimes at low pH
©2003 Waters Corporation
For More Information
•Visit Waters on-line at www.waters.com• Visit AtlantisTM columns microsite and download
brochure, applications notebook, LC/GC App Note,Care & Use, etc.
• C.talk for free on-line educational seminars• Applications database with over 17,000
applications
Thank you for attending today’s seminar!!Thank you for attending today’s seminar!!
©2003 Waters Corporation
For More Information
• Visit us at Booth 2953
• Presentations at Pittcon• Chromatographic Retention of Polar Compounds - What Are
the Options?• Tue AM - Session 800, Room 306AB
• Hydrophilic Interaction Chromatography (HILIC) for Small Molecules: Searching for the Right Stationary Phase
• Thu PM - Session 2370, Room 306AB
Thank you for attending today’s seminar!!Thank you for attending today’s seminar!!