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
δ+
H
δ+H
O2δ
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
O
O
HN
HN
O
NH2
HN
O
N
F
HN
O
N
H
Uracil (N)
O
N
H
O
O
Acetone (N)
HO
O
Cytosine (B)
Thymine (N)
OH
N
H
HO
5-Fluoroorotic acid (A)
HO
H
N
O
HO
HO
HO
Epinephrine (B)
O
OH
Ascorbic acid (A)
©2003 Waters Corporation
Comparison of Chromatographic Methods
for Retention of Polar Compounds
Advantages
Ion
Exchange
HILIC
Ion
Pairing
• Can retain any ionizable
Disadvantages
• Does not work well if the
compound
compound does not ionize
• High salt buffers or pH gradients
for elution – not compatible with MS
• High percentage organic mobile
• Sample and mobile phase
• Can retain any ionizable
• Long equilibration times
• Difficult to run gradients
• Most ion pairing agents not
phases give higher sensitivity in
ESI-MS
compound
• Can perform ion pairing
chromatography and reversedphase chromatography on same
column
• Familiar technique
• High efficiency
Reversed- • Rapid equilibration
Phase
• Wide selection of columns
solubility problems
• Long re-equilibration times
compatible with MS
• Difficult method development
• Dewetting under aqueous
conditions
• Poor retention of polar
compounds with non-polar
stationary phases
©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?
HILIC Retention
O
OH
HN
O
O
HN
N
H
Polar
Phase
80% acetonitrile
20% aqueous,
(e.g., pH 3.0)
O
N
H
uracil
• 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)
©2003 Waters Corporation
HILIC: What is it?
Weak Cation Exchange
Retention
H
N
O-
O
N
+
NH3
Polar
Phase
80% acetonitrile
20% aqueous,
(e.g., pH 5.5)
H
N
O
N
+
NH3
cytosine
• Charged polar analyte can
undergo cation exchange with
charged silanol groups
• High Acetonitrile (>70%) with
aqueous
HILIC is defined as the
high organic conditions
where polar compounds are
retained longer
(reverse reversed-phase)
©2003 Waters Corporation
HILIC
Retention Characteristics
3.00
H
N
Aqueous
pH 5
2.00
LN (Tr min/cm)
O
Retention
N
1.00
NH2
0.00
pH 4
Cytosine
-1.00
-2.00
pH 3
-3.000
10
20
30
40
50
60
70
80
90
% MeCN
HILIC:
Increased retention occurs when using
greater than 70% organic for polar bases
©2003 Waters Corporation
Reversed-Phase
Retention Characteristics
-2.00
Aqueous
-2.10
LN (Tr min/cm)
H
N
pH 5
-2.20
N
Retention
-2.30
-2.40
pH 4
-2.50
NH2
Cytosine
-2.60
-2.70
pH 3
-2.80
-2.90
O
0
10
20
30
40
50
% MeCN
60
70
80
90
Reversed-phase:
Increased retention occurs when using
less than 20% organic for polar bases
©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
AtlantisTM HILIC Silica: Retains very polar
compounds not retainable by RP
k’= 0.0
AtlantisTM dC18
4.6 x 50 mm, 3 µm
100% Formate Buffer, pH 3.0
1.0 mL/min
0.10
AU
Vo = 0.65 min
0.05
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
k’= 1.0
AU
Vo = 1.15 min
0.05
O
O
HILIC Silica
4.6 x 50 mm, 3 µm
95:5 ACN:Formate Buffer, pH 3.0
1.4 mL/min
0.15
H
N
5.00
Minutes
0.10
NH
H2N
0.00
AtlantisTM
O
N
H
Allantoin
0.00
0.50
1.00
1.50
2.00
2.50
3.00
Minutes
3.50
4.00
4.50
5.00
HILIC offers retention when there
is no retention by reversed-phase
©2003 Waters Corporation
AtlantisTM HILIC Silica :
Enhanced ESI-MS Sensitivity
100
1
100
7.63e3
ES+
%
%
1.80
2?
2.13
239.8
209.9
0
1.07e5
1.80
0
100
2
AtlantisTM dC18
2.1 x 50 mm, 3 µm
1. Albuterol 100 pg/µL
2. Bamethan 20 pg/µL
Peak Area
78
2
2.13
1.31
1.63
ES+
1
239.8
%
209.9
1.07e5
AtlantisTM HILIC Silica Peak Area
2.1 x 50 mm, 3 µm
2. Bamethan 20 pg/µL
9387
1. Albuterol 100 pg/µL
13131
0
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
HILIC requires high volatility solvents which increase ESI-MS
sensitivity vs. high-aqueous mobile phases used in RP
©2003 Waters Corporation
AtlantisTM HILIC Silica :
Simplify Sample Preparation
Condition/Equilibrate*
200 µL methanol/200 µL water
Load
150 µL spiked plasma sample
150 µL internal standard
with 2% ammonium hydroxide
Wash
200 µL 5% methanol in water
Elute
300 µL 40% acetonitrile/60% isopropanol
with 2% formic acid
*Oasis® HLB
µElution Plate
Inject eluent directly onto column
No Evaporation and Reconstitution Step
©2003 Waters Corporation
AtlantisTM HILIC Silica :
Simplify Sample Preparation
HO
H3C
H3C
HO
NH
OH
OH
NH
CH3
CH3
Albuterol
Bamethan
1.49
100
1
OH
AtlantisTM HILIC Silica
2.1 x 50 mm, 3 µm
1. Bamethan 10 pg/µL
2. Albuterol 50 pg/µL
1.84
SIR of 2 Channels ES+
TIC
239.8
209.9
8.97e4
2
%
0
0.00
0.50
1.00
1.50
2.00
2.50
Time
3.00
3.50
4.00
4.50
5.00
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
Polar mobile
phase
Non-polar
stationary
phase
Analyte Y
Non-Polar
Well retained
(like attracts like)
Analyte X
Polar
Flow
O-S
i Si
OOH
O-Si
O-Si
OH
O-Si
OH
O-Si
O-Si
O-Si
OH
O-Si
O-Si
Poorly
Retained
(like attracts like)
Chromatographically how does one
attempt to force polar Analyte X to retain on
the non-polar stationary phase?
©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 dryout (non-polar pore surface expels the pure aqueous,
polar mobile phase)
What is “dewetting” and
why does it happen?
©2003 Waters Corporation
Conventional C18 Column – Dewetting
Mobile phase: 0.1% Acetic Acid
Initial
Amoxicillin
(Column was wetted first
with organic)
1,500psi
1,500psi
0
2
4
Vo: No retention of analyte
After Flow Stoppage
(Pores “dewet” 100%)
6
8
10
Minutes
Note: Column is not broken -It just stopped working
Why?
©2003 Waters Corporation
Pore Dewetting Mechanism
Flow stoppage relieves the pressure that was forcing the aqueous
mobile 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.
Analytes
Analytes
properly retained
At flow, with pressure on
the mobile phase.
Stopped flow with no pressure on the
mobile phase.
Remember: Most of the
surface area (> 95%) is
inside the silica pores!
Pores de-wet – restart flow – pores
still dewetted and analytes never
enter pores – resulting in no retention.
©2003 Waters Corporation
Stationary Phase Wetting
Pressure on C18 pore required
to force water back into the pore
Water on C18:
d = 100Å
γ = 72.8 dynes/cm
θ = 110.6°*
Pc = 1,500 psi
Methanol on C18:
H 2O
θ
Contact
Angle
Solvent dependent
Pc =
4γ
d
cos θ
Where:
Pc = Capillary pressure
γ = Surface tension
d = Capillary diameter
θ = Contact angle
θ
d = 100Å
γ = 22 dynes/cm
θ = 39.9°*
Pc < 0 psi
Equation of Young and Laplace
MeOH
Note: Self-wetting – No pressure required
*B. Janczuk, T. Bialopiotrowicz and W. Wojcik,
Colloids and Surfaces 36 (1989) 391-403
©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
1,500 psi
Atmospheric pressure
Pressure gradient
inside column
©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 phases
i.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 Corporation
Embedded Polar Groups –
What Doesn’t Work
For highly polar compounds, embedded polar groups
actually cause less retention (see next slide)
Compounds
1. Thiourea
2. 5-Fluorocytosine
3. Adenine
4. Guanosine-5’-monophosphate
O
5. Thymine
Conditions
Column: AtlantisTM dC18 4.6 x 150 mm, 5 µm
Isocratic Mobile Phase: 10 mM NH4COOH, pH 3.0
Flow Rate: 1.2 mL/min
Injection Volume: 7.0 µL
Temperature: Ambient
Detection: UV @ 254 nm
NH2
S
F
N
1
H2N
N
NH2
Thiourea
3
HO
P
O
H
OH
H
5-Fluorocytosine
2
O
N
HO
N
O
OH
1.00
N
4
NH2
O
NH2
N
HN
H
O
H
OH
Guanosine-5’-monophosphate
N
H
N
H
Thymine
N
N
Adenine
5
Atlantis dC18 100% Aqueous
QC Batch Test
Vo = 1.5min
0.00
NH
2.00
3.00
4.00
5.00
6.00
7.00
8.00
Minutes
9.00
10.00
11.00
12.00
13.00
14.00
15.00
Carignan, Iraneta
©2003 Waters Corporation
Embedded Polar Groups –
What Doesn’t Work
1
2
4
3
5
AtlantisTM dC18
Vo = 1.5 min
0.00
1.00
2.00
3.00
4.00
1 2
Vo = 1.2min
1 .0 0
Vo
5.00
6.00
5
3
2 .0 0
3 .0 0
Vo = 1.3min
1.00
2.00
4
3.00
9.00
10.00
11.00
12.00
4 .0 0
5 .0 0
6 .0 0
7 .0 0
8 .0 0
Min u te s
9 .0 0
1 0 .0 0
1 1 .0 0
1 2 .0 0
2.00
3.00
14.00
15.00
1 3 .0 0
1 4 . 00
1 5 .0 0
Column I OE
4.00
5
5.00
6.00
7.00
8.00
Minutes
9.00
10.00
11.00
12.00
2 3
1
13.00
Column Z BR
(amide)
4
4 5
1 2
3
= 1.2min
1.00
7.00
8.00
Minutes
13.00
14.00
15.00
Column P SP
4.00
5.00
6.00
7.00
8.00
Minutes
9.00
10.00
11.00
12.00
13.00
14.00 ©2003
15.00Waters Corporation
Embedded Polar Groups –
What Doesn’t Work
1
2
4
3
5
AtlantisTM dC18
Vo = 1.5 min
0.00
1.00
2.00
3.00
4.00
5.00
1 2
Vo = 1.3min
5
2.00
9.00
10.00
11.00
12.00
4.00
2 3
5.00
6.00
7.00
8.00
Minutes
9.00
10.00
11.00
12.00
5
1
3.00
2
Vo = 1.3min
2.00
4.00
3
4
5.00
6.00
7.00
8.00
Minutes
9.00
10.00
11.00
12.00
15.00
13.00
14.00
15.00
6.00
13.00
14.00
15.00
Column W XR
5
4.00
14.00
Column M PC
4
2.00
13.00
Column S AZ
(amide)
4
3.00
Vo = 1.3min
1.00
7.00
8.00
Minutes
3
1.00
1
6.00
8.00
Minutes
10.00
12.00
14.00
©2003 Waters Corporation
Embedded Polar Groups –
What Doesn’t Work & Why
Reason 1
Water layer
“shields”
silanols and
reduces
cationic
interactions
and Hbonding
Why less retention of polar compounds
with embedded polar group columns?
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
+
NH 3
N
N
N
N
H
Reason 2
Shorter 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
Endcapping
Ligand
type
Peak Shape
Dewetting
Retention
Stability
Ligand
density
Pore
size
©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 retention
Examples of each attribute will be given
©2003 Waters Corporation
Superior Retention of
Polar Compounds
Aqueous Separation of Polar Compounds
for AtlantisTM vs. Conventional C18 Column
Conditions
Columns: 4.6 x 150 mm, 5 µm
Mobile Phase: 10mM NH 4COOH, pH 3.0
Flow rate: 1.2 mL/min
Injection volume: 7 µL
5 Detection: UV@254 nm
AtlantisTM dC18
1
2
4
3
Vo = 1.5 min
0.00
1.00
1
2.00
2
3
3.00
5.00
6.00
7.00
8.00
9.00
Time (min)
5
4
2.00
3.00
4.00
5.00
6.00
7.00
8.00
10.00
11.00
12.00
13.00
14.00
15.00
Compounds
1. Thiourea
2. 5- Fluorocytosine
3. Adenine
4. Guanosine-5’- monophosphate
5. Thymine
Conventional C18
Peak 1 elutes
In the void
1.00
4.00
9.00
10.00
11.00
12.00
13.00
14.00
35% - 60% more
retention with
AtlantisTM dC18 as
compared to
high coverage C18
15.00
Time (min)
©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
Note change in V0
in dewetted column
Amoxicillin retention with
0.1% formic acid mobile phase
Initial
Retention
100% retention loss with
conventional C18
Retention after stop flow test
(Pore dewetting: ~100%)
Conventional C18
Initial
Retention
Vo
AtlantisTM
<4% retention loss with
AtlantisTM dC18
Retention after stop flow test
(Pore dewetting: ~ 3.5%)
dC18
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
Time (min)
Note enhanced retention
©2003 Waters Corporation
Excellent Peak Shape Using AtlantisTM dC18
with MS Compatible Mobile Phases
The popular void marker Uracil is
well retained and is actually peak 3
Conditions
Column: AtlantisTM dC18 4.6 x 150 mm, 5 µm
Mobile Phase A: H20
Mobile Phase B: ACN
Mobile Phase C: 100 mM CH3COONH4, pH 5.0
Flow Rate: 1.0 mL/min
Gradient:
Time
Profile
(min)
%A %B
%C
0.0
90
0
10
10.0
84
6
10
Injection Volume: 10 µL
Temperature: 30 oC
Detection: UV @ 254 nm
Instrument:: AllianceTM 2695, 2996 PDA 1
2
2.00
3.00
Note excellent peak
shape for strong polar
base Adenine
at pH 5.0
7
4
5
V0 = 1.83 min
1.00
Compounds:
1. Cytosine
2. 5-Fluorocytosine
3. Uracil
4. 5-Fluorouracil
5. Guanine
6. Thymine
7. Adenine
6
3
4.00
5.00
Minutes
6.00
7.00
8.00
At pH 5.0, the strong polar base
Adenine still exhibits excellent peak shape
(reason: fully endcapped)
9.00
10.00
Grumbach, Diehl
©2003 Waters Corporation
Benefit of a Fully Endcapped Column
1
Conditions
Columns: 4.6 x 150 mm, 5 µm
Mobile Phase: 10 mM NH4COOH, pH 3.0
Flow Rate: 1.2 mL/min
Injection Volume: 7 µL
Detection: UV @ 254 nm
Compounds
1. Thiourea
2. 5-Fluorocytosine
3. Adenine
4. Guanosine-5’-monophosphate
5. Thymine
Unendcapped “AQ-Type”column
2
4
Longer base retention at
the expense of peak shape
5
3
0.00
1.00
2.00
3.00
4.00
5.00
6.00
1
7.00
8.00
Minutes
9.00
10.00
11.00
12.00
13.00
14.00
15.00
AtlantisTM dC18 column
2
4
3
5
Excellent retention and
peak for all compounds
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
Minutes
9.00
10.00
11.00
12.00
13.00
14.00
15.00
©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
% Original
Retention Time
(30oC with 0.1% TFA)
105
105
100
100
95
95
90
90
85
85
80
80
75
75
70
Thymine - AtlantisTM dC18
70
65
Thymine - Monofunctional C18
65
60
1
20
50
100
200
300
700
800
900
950
What is a difunctional
bond and why does it
provide longer column
lifetimes at low pH?
60
990
Injection Number
©2003 Waters Corporation
Hydrolysis of a Bonded Phase Material:
Limitations of Monofunctional Ligands
O
O
Si
O
O
O
OH
+
Cl
Si
O
Si
C
C
CH3
C
C
C
C
C
CH3
Synthesis
(monofunctional bond created)
H 3C
Single siloxane bond
C8 Monochlorosilane Ligand
H 3C
Si
C
C
C
C
C
C
C
+ HCl
CH3
CH3
O
Hydrolysis at low pH
(monofunctional bond broken)
O
O
Si
O
OH
H3C
+
HO
Si
C
C
C
C
C
C
C
CH3
CH3
Monofunctional = Attached to silica at one point
More susceptible to low pH hydrolysis (column bleed)
©2003 Waters Corporation
Making a Bonded Phase Material:
Multifunctional Synthesis
O
OH
O Si
O
OH
Si
OH
O
Si
O
O O
C8 Dichlorosilane Ligand
+
CH3
Si
CH3
Cl
Cl
Synthesis
OH CH3
O
O Si
Si
O
O
Si
O
Si
O
O
O O
CH3
Two siloxane bonds
+ HCl
Difunctional = Attached to silica at two points
Less susceptible to low pH hydrolysis (column bleed)
©2003 Waters Corporation
Enhanced Polar Compound Retention without
Infinite Non-polar Compound Retention
More retention for
polar compounds
V0 = 0.98 min
2
1
1.00
Equal or less retention for
non-polar compounds
7
2.00
Conventional C18
with 2x ligand
density
8
3
3.00
4.00
10
5
6.00
7.00
8.00
Minutes
Void
Marker
9.00
10.00
2
2.00
3.00
4.00
12.00
5.00
5
6.00
14.00
15.00
9
6
7.00
8.00
Minutes
13.00
Back
Marker
8
3
4
1.00
11.00
7
V0 = 0.98 min
1
AtlantisTM dC18
6
4
5.00
9
10
9.00
10.00
11.00
12.00
13.00
14.00
15.00
©2003 Waters Corporation
Atlantis dC18 - An Intelligent Design
Why Does It Work?
Polar analytes are not able to “energetically fit” between ligands –
can’t interact with surface or ligands
H3C
CH2
CH2
H2C
H2C
CH2
CH2
H2C
H2C
H2C
CH2
Residual silanol
H2C
CH2
CH3
H3C
H3C
CH
3
Si
Si CH3H
H
O
O
O
O
O
Si
Si
Si
Si
Si
O
O
O
O
O O
O
O
O
O
H3C
H
CH2
H2C
CH2
H2C
O
Si
O
H2C
CH2
H2C
H2C
CH2
H2C
CH2
H2C
H2C
CH2
Endcap
H3C
CH2
H2C
CH2
Residual silanols
CH2
CH2
CH2
CH2
CH2
H3C
H3C
Si CH3 H3C
Si CH3 H
Si CH3 H
H
O
H
O O
O
O
O
O
Si
Si
Si
Si
Si
O
O
O O O
O Si O
O
O
O
O
Polar analytes easily
interact with surface
and ligands
H2C
H2C
CH2
CH3
H3C
CH
CH3H3C
3
Si
Si
H
H
O
O
O
O
Si
Si
Si
Si
O
O
O
O
O O
O
O
O
H3C
H
CH2
CH2
C8 alkyl chains
H3C
H2C
H3C
H3C
H3C
CH3
H
Si CH3 H
O
O
O
Si
Si
Si
O
O O
O
O
CH2
CH3
Si CH3
O
Si
O
O
Low
Coverage –
Low Ligand
Density
Endcap
CH2
H3C
CH3 H3C
Si
H
H
H
O O
O
O
Si
Si
O
O
O Si O
O
O
Adapted from J.G. Dorsey and K.A. Dill, Chem. Rev., 1989, 89, 331-346
High
Coverage
– High
Ligand
Density
CH3
Si CH3
O
Si
O
O
©2003 Waters Corporation
Atlantis dC18 - An Intelligent Design
Why Does It Work?
Low
Coverage –
Low Ligand
Density
Alkyl chains
O
H2C
Non-polar
portion of
analyte
HC
interacts with 2
bonded phase
(hydrophobic)
CH2
N
H
O
H2C
H2C
CH2
H2C
Si
CH3
CH3
CH3
H3C
H
H3C
Si
CH3
H
Si
CH3
O
H
H
O
O
O
O
O
O
Si
Si
Si
Si
Si
Si
Si
Si
O
O
O
O
O
O
O
O
O
O
O
O
H3C
N
H
O
O
Endcap
CH2
O
O
CH2
N
CH2
H
H2C
NH2
Residual silanols
CH2
H3C
CH2
Polar portion of
analyte interacts
with silanols
(hydrogen bonding
and/or ion exchange)
NH
CH2
H2C
H2C
O
O
Si
H
O
O
Si
O
O
Si
H
O
O
CH3 H3C
Si
H
CH3
O
Si
O
CH3
O
Si
O O
O
©2003 Waters Corporation
Atlantis dC18 - An Intelligent Design
Why 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 Design
Excellent Batch-to-Batch Reproducibility
Eight Actual QC Batch Test Chromatograms
Under 100% Aqueous Conditions
(not specially packed columns)
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
Minutes
%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
Complementary Selectivity of HILIC
as Compared to Reversed-Phase
Compounds:
1. Morphine
2. Morphine 3-β-D-glucuronide
1
HO
2
AtlantisTM HILIC Silica
4.6 x 50 mm, 3 µm
O
H
N
Vo = 0.5 min
CH3
HO
Morphine
1.00
2.00
2
3.00
4.00
5.00
Minutes
AtlantisTM dC18
4.6 x 50 mm, 3 µm
1
Vo = 0.65 min
0.50
1.00
1.50
2.00
2.50
Minutes
3.00
3.50
4.00
4.50
5.00
Morphine 3-ß-D-Glucuronide
©2003 Waters Corporation
Gradient LC/MS Separation of Cytochrome C Tryptic
Digest on AtlantisTM HILIC Silica versus AtlantisTM dC18
100
204 (T2)
261 (T6,11)
361 (T18)
434 (T21)
Peak Annotation
M/Z (Fragment ID)
RP
AtlantisTM dC18
2.1 x 50 mm, 3 µm
779 (T15)
%
678 (T14)
634 (T4)
729 (T10)
1005 (T!2)
964 (T19)
585 (T8)
0
634 (T4)
100
AtlantisTM HILIC Silica
HILIC
779 (T15)
%
678 (T14)
434 (T21)
2.1 x 50 mm, 3 µm
261 (T6,11)
204 (T2)
585 (T8)
964 (T19) 729 (T10)
1005 (T!2)
361 (T18)
0
0
45
©2003 Waters Corporation
Isocratic RP Separation of Catecholamines
and Metabolites on Atlantis dC18
Conditions
Column: AtlantisTM dC18 4.6 x 150 mm, 5 µm
Mobile Phase A: H2O
Mobile Phase B: ACN
Mobile Phase C: 100 mM CH3COONH4, pH 5.0
Flow Rate: 1.0 mL/min
Isocratic Mobile
Phase Composition: 88% A; 2% B; 10% C
Injection Volume: 10 µL
Temperature: 30 oC
Detection: UV @ 280 nm
Compounds
USP Tailing
1. Norepinephrine (NE)
1.21
2. Epinephrine (E)
1.20
3. Dopamine (DA)
1.21
4. 3,4-Dihydroxyphenylacetic acid (DOPAC)
1.00
5. Serotonin (5-HT)
1.10
6. 5-Hydroxy-3-indoleacetic acid (5-HIAA)
0.97
7. 4-Hydroxy-3-methoxyphenylacetic acid (HVA)
0.97
No ion-pairing agent necessary!!
2
1
3
4
V0 = 1.83 min
6
5
7
2.00
4.00
6.00
8.00
10.00
12.00
14.00 16.00
Minutes
18.00
20.00
22.00
24.00
26.00
28.00
30.00
©2003 Waters Corporation
Water Soluble Vitamins
on Atlantis dC18
Conditions
Column: AtlantisTM dC18 4.6 x 150 mm, 5 µm
Mobile Phase A: 0.1% TFA
Mobile Phase B: ACN
Flow Rate: 1.4 mL/min
Gradient:
Time
Profile
(min)
%A %B
0.0
100
0
4.0
97
3
6.0
85 15
15.0
80 20
Injection Volume: 10 µL
Temperature: 30 oC
3
Detection: UV @ 260 nm
Compounds:
1. L-ascorbic acid
2. Nicotinic acid
3. Thiamine
4. Pyridoxal
5. Pyridoxine
6. Folic acid
7. Caffeine
8. Riboflavin
No ion-pairing agent necessary!!
2
7
1
V0 = 1.37 min
8
4
6
5
1.00
USP Tailing
1.12
1.27
1.20
1.13
1.04
1.10
1.10
1.14
2.00
3.00
4.00
5.00
6.00
7.00
8.00
Time (Min)
9.00
10.00
11.00
12.00
13.00
14.00
15.00
©2003 Waters Corporation
Tryptic Cytochrome C Peptide Separation:
TFA Not Necessary
Conditions
Column: AtlantisTM dC18 4.6 x 50 mm, 3 µm
Mobile Phase A: 0.1% HCOOH or 0.02%TFA
Mobile Phase B: 0.065% HCOOH in ACN or
0.016% TFA in ACN
Flow Rate: 0.75 mL/min, 0.2 mL/min Split to MS
Gradient:
Time
Profile
(min)
%A %B
0.0
100
0
45.0
60
40
Injection Volume: 20 µL
Injection Mass: 20 µg
Temperature: 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
Thank you for attending today’s seminar!!
• 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
•
©2003 Waters Corporation
For More Information
Thank you for attending today’s seminar!!
• 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
©2003 Waters Corporation