2014PolarRetentionSeminar12 9 14FINAL

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‘Tips and Tricks’ for Retaining and

Separating Polar Compounds using
Reversed--Phase and Hydrophilic
Reversed
Interaction Chromatography (HILIC)
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About Today’s Presenter…
Kevin Jenkins, Product Manager, HPLC Columns,

Waters Corporation
Kevin has worked for Waters Corporation since 2000

and has been actively supporting chromatographers
with solutions for HPLC method development in the
pharmaceutical, chemical, food and environmental
industries. Prior to his current role, Kevin gained
expertise in LC method development strategies and
sample preparation techniques for bioanalytical assays
and other complex samples that require sensitivity and
resolution from sample constituents. His goal is to
promote these strategies and techniques to help other
scientists to attain better analytical results throughout
their method development process.

Outline
Retaining polar compounds in RPLC

– Elevated pH mobile phase
– Aqueous compatible C18 columns
– Pentafluorophenyl (PFP) columns for polar bases
Retaining polar compounds in HILIC

– Mobile phase preparation
– System setup
– Tips and Tricks
– Method Development
– Solid core HILIC columns- what is the advantage?

Outline


– System setup
– Tips and Tricks

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
– The unequal sharing of electrons within a bond results
in a separation of positive and negative electric charge.
Polarity is dependent on the electronegativity
difference between molecular atoms and compound
asymmetry

Examples of Polar Molecules
NH2 O
F CH3
S N HN
CH CH
O N O N
N N H
H
Thiourea (N) 5-Fluorocytosine (B) Thymine (N)
O
HN N
NH2
H2N N N N
O
HN
P O
HO O N
OH N
HO OH Adenine (N)
Guanosine-5’-monophosphate (A)
Reversed--Phase Retention Map:
Reversed
The Impact of pH on Ionizable Compounds
Maximum Maximum
acidic compound basic compound
retention range retention range
40
Note: Retention of neutral analytes not affected by pH
35
30 Acid Neutral
Retention Factor
25
(k)
20
15
10
5
Base
0
0 2 4 6 8 10 12
pH
pH Range for Silica Particles
pH Range for Hybrid Particles

Mobile Phase pH Selectivity:
Basic and Neutral Compounds
ACQUITY CSH C18,
0.1% formic acid
I Fl Acetonitrile
pH 3
A
Test Probes:
D
O I: Imipramine [B]
A: Amitriptyline [B]
Fl: Flavone [N]
P O: Octanophenone [N]
Fe
0.00 1.20 2.40 3.60 4.80 Observations:

Minutes
0.1% NH4OH At high pH, bases are
pH 10
in their neutral
Fl
(unionized) form,
P D I resulting in greater
A
O retention
Neutral compounds
Fe are unaffected by pH
0.00 1.20 2.40 3.60 4.80

Minutes
Improved Retention and Sensitivity:
High pH Mobile phase
HIGH pH
0.1% NH4OH
10-hydroxymorphine
5 3
%
6
1
4
Morphine-3ß–o-glucuronide
Morphine
0
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80
Morphine-6ß–o-glucuronide
LOW pH
0.1% HCOOH
Compounds
1. 10-hydroxymorphine
%
6 2. Morphine-3β-D-glucuronide Morphine N-oxide

4,5 3. Morphine-6β-D-glucuronide
2 4. Morphine
5. Morphine N-oxide
1 3 6. 6-acetylmorphine
0 6-acetylmorphine
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80
©2014 Waters Corporation
Minutes 10
Silica and Hybrid Particles:
Manufacturing Process
Anal. Chem. 2003, 75, 6781-6788

Reversed--Phase Chromatography:
Reversed
Challenging for Polar Compounds
To retain polar compounds on a 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?

Proper Wetting of Bonded
Chromatographic Surface
The particles are very porous, like the

pores of a sponge – 99% of
Mobile Phase chromatographic surface is inside the
pores
Mobile phase must

be allowed into the pore
Analyte in order for chromatographic
retention of the analyte
to take place.
If the pores are dry, the analyte

cannot get into the pores and it
will not be retained by the
chromatographic surface.
What influence does this have on chromatography?

Pore Dewetting:
Dewetting:
What Does It Look Like?
The observed reduction in V0 and sudden loss in retention after a

pressure release, indicates that material pores expel the aqueous
solvent*1
– 10% reduction in void volume is observed
– 23% retention loss of analyte
ACQUITY UPLC® BEH C18

L = 5 cm
F = 0.3 mL/min Vo Shift
P = 2,040
T = 90 oC
% Dewet = 23.10
0.20 0.40 0.60 0.80 1.00 1.20 1.25

Minutes
*1 T.H. Walter, P. Iraneta and M. Capparella J. Chromatogr. A 1075 (2005) 177-183

HSS T3 Versus HSS C18:
100% Aqueous Conditions
1 2 XSelect HSS T3
0.06
5
0.04 3 4
AU
0.02
Initial
0.00
0.06
0.04
AU
0.02
After dewetting
0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
5
1
0.06 2 4 XSelect HSS C18
0.04 3
AU
0.02
Initial
0.00
Pks 1 - 5
0.15
AU
0.10
0.05 After dewetting
0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
Minutes
Peak i.d.: 1) thiourea 2) 5-Fluorocytosine 3) adenine 4) thymidine-5’-monophosphate 5) thymine
Conditions: 10mM Ammonium Formate pH 3; 0.2mL/min; 30ºC; 2.1 x 50 mm
Polar Retention:
Why Does HSS T3 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
o Polar analytes can “fit” between C18 ligands and interact with
pores of material
o Optimized particle morphology (i.e. pore diameter/volume)
Secondary interactions due to residual silanols that are more
accessible due to reduced C18 coverage
– Cation-exchange interactions
– Hydrogen bonding interactions

Another Options for Polar Bases:
(Pentafluorophenyl)
PFP (Pentafluorophenyl
Multiple selectivity mechanisms including:

– hydrogen bonding
– dipole-dipole
– aromatic (pi-pi)
– hydrophobic (reversed-phase)
Target Analyses:
positional isomers, halogenated compounds and polar bases

Another Option for Polar Bases:
PFP versus C18
1 Compounds
ACQUITY HSS C18
1. Trimethoprim
2 2. Nordoxepin
3
3. Doxepin
5 4. Nortriptyline
4 5. Imipramine
6 6. Amitriptyline
7
7. Trimipramine
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
Minutes
1 ACQUITY HSS PFP
2
3
4 5 6
7
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes
Outline


– System setup
– Tips and Tricks

What is HILIC?
HILIC - Hydrophilic Interaction Chromatography

— Term coined in 1990 to distinguish from normal-phase*
HILIC is a variation of normal-phase chromatography without

the disadvantages of using solvents that are not miscible in
water
— “Reverse reversed-phase” or “aqueous normal-phase”
chromatography
Stationary phase is a POLAR material

— Silica, hybrid, cyano, amino, diol, amide
The mobile phase is highly organic (> 80% ACN) with a smaller
amount of aqueous mobile phase
— Water (or the polar solvent(s)) is the strong, eluting solvent
*Alpert, A. J. J.Chromatogr. 499 (1990) 177-196.

Multi--modal Retention Mechanisms:
Multi
HILIC
Combination of
partitioning, ion-exchange
and hydrogen bonding
• Polar analyte partitions

between bulk mobile phase
and partially immobilized
polar layer on material
surface
• Secondary interactions
between surface silanols
and/or functional groups
with the charged analyte
leading to ion-exchange
• Hydrogen bonding
between positively charged
analyte and negatively
charged surface silanols
D.V. McCalley, U. D. Neue, J. Chromatogr. A 1192 (2008) 225-229
E.S. Grumbach, D.M. Diehl, U.D. Neue, J. Sep. Sci. 31 (2008), 1511-1518
A. Méndez, E. Bosch, M. Rosés, U. D. Neue, J. Chromatogr. A 986 (2003), 33-44
Before You Start:
Mobile Phase Preparation
Additives
– Replace 0.2% of mobile phase volume with additive [2 mL out of 1 L]
Buffers
– Prepare a stock buffer [typically 200 mM] and then dilute 20-fold into
the running mobile phase [10 mM concentration on column]
– Example: Prepare stock of 200 mM ammonium formate, pH 3. For a
mobile phase containing 95% ACN and 5% water with 10 mM
ammonium formate, pH 3, add 50 mL of stock buffer to 950 mL of
ACN.
For the best gradient performance and reproducibility, it is

recommended that the additive or buffer be added to both
aqueous and organic mobile phase bottles

Screening Conditions:
Binary Pumping System
Low PH Conditions:
Mobile Phase A1: 50/50 ACN/H2O with 10 mM HCOONH4 and 0.125% HCOOH, pH 3.0
Mobile Phase B1: 95/5 ACN/H2O with 10 mM HCOONH4 and 0.125% HCOOH, pH 3.0
High pH Conditions:
Mobile Phase A2: 50/50 ACN/H2O with 10 mM CH3COONH4 and 0.04% NH4OH, pH 9.0
Mobile Phase B2: 95/5 ACN/H2O with 10 mM CH3COONH4 and 0.04% NH4OH, pH 9.0
Gradient: 99.9% B to 0.1% B in 5 min, reset (total run time = 6 min)

Flow rate: 0.5 mL/min
Injection volume: 5 µL
Sample Diluent: 75/25 Acetonitrile/Methanol
(0.2% HCOOH needed in some cases for solubility)
Column Temp.: 30oC
Strong and weak needle washes: 95/5 ACN/H2O (strong could also be 95/5 H2O/ACN)

Screening Conditions:
Quaternary Pumping System (allows for RP)
Mobile Phase A: H2O

Mobile Phase B: ACN
Mobile Phase C: 200 mM HCOONH4 and 0.125% HCOOH, pH 3.0
Mobile Phase D: 200 mM CH3COONH4 and 0.04% NH4OH, pH 9.0 (or pH 10.0)
Flow rate: 0.6 mL/min
Sample Diluent (HILIC): 75/25 ACN/MeOH (0.2% HCOOH may be needed for solubility)
Sample Diluent (RP): 95/5 H2O/ACN (or starting mobile phase composition)
Injection volume: 5 µL
Column Temperature: 30°C
Needle wash: 50/50 ACN/H2O
HILIC (low pH)

Time Flow % A % B % C % D Curve
(min) (mL/min)
Initial 0.60 5 90 5 0 *
5.00 0.60 45 50 5 0 6
6.00 0.60 45 50 5 0 6
6.01 0.60 5 90 5 0 6
Gradient conditions for 2.1 x 50 mm, 1.7 µm column 10.00 0.60 5 90 5 0 1

Before You Start:
Column Equilibration and Wash Solvents
Instrument Wash Solvents

– Strong needle wash: 9:1 acetonitrile:water
– Weak needle wash/purge solvent: initial mobile phase conditions [excluding salt,
additive or buffer]
Brand new column

– Run 50 empty column volumes of 50:50 acetonitrile:water with 10 mM buffer or
0.2% additive solution
Column equilibration
– Equilibrate with 20 empty column volumes of initial mobile phase conditions
Gradient separations
– Re-equilibrate with 5 to 8 empty column volumes
As with any column, insufficient equilibration can cause drifting retention times

Influence of Needle Wash Solvent
1. Methacrylic acid
2. Nicotinic acid
3. Nortriptyline
4. Cytosine
0.50
3 4
0.40
0.30
AU
0.20 1 Weak needle

wash: 95:5
0.10 2
MeCN/H2O
0.00
0.50 4,2
3
0.40
0.30
AU
0.20 1 Weak needle

0.10
wash: 95:5
H2O/MeCN
0.00
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
Minutes

Outline


– System setup
– Tips and Tricks
o Injection solvent
o Buffers/additives
o Tuning the mobile phase for more retention
o System Health- are you ready to inject samples? How do you know?

The Importance of Sample
Diluent/Injection
Diluent/Injection Solvent Selection
Sample diluent strongly influences solubility and peak shape

(just like reversed-phase)
Sample diluent should be at least 75% acetonitrile or as close to

initial mobile phase conditions as possible
– However, polar analytes often have low solubilities in organic
solvents
General purpose HILIC diluent

– 75:25 acetonitrile:methanol works for most polar analytes
– Offers a compromise between solubility and peak shape
– Adjust according to your analytes

Sample Diluent Considerations:
Water as Polar Solvent
0.60 100% H2O ACQUITY UPLC® BEH HILIC

0.50 S 2.1 x 100 mm, 1.7 µm
0.40
2
AU
0.30 3 Analytes
1 4 1. Methacrylic acid
0.20
0.10
2. Cytosine
0.00
3. Nortriptyline
0.00 0.50 1.00 1.50 2.00 2.50 3.00
4. Nicotinic acid
0.60 Minutes 50 ACN: 50 H2O
0.50
2
0.40
S 1
AU
0.30
4 Peak shape improves
0.20 3
as % ACN in the
0.10
diluent increases.
0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00

Minutes
What about alternative
0.60 75 ACN: 25 H2O
2 polar organic solvents?
0.50
1
0.40
3
AU
0.30
4
0.20
S
0.10
0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 Grumbach

Minutes

Sample Diluent Considerations:
Methanol as Polar Solvent
100% MeOH
0.60
S ACQUITY UPLC® BEH HILIC
2
0.50 2.1 x 100 mm, 1.7 µm
0.40 1
3
AU
0.30
4 Analytes
0.20 1. Methacrylic acid
0.10 2. Cytosine
0.00 3. Nortriptyline
0.00 0.50 1.00 1.50 2.00 2.50 3.00
4. Nicotinic acid
0.60
Minutes
50 ACN: 50 MeOH
0.50 S 2
0.40 1 3
AU
0.30 4 Peak shape and solubility improve by

0.20
replacing water with methanol
0.10
Peak shape improves
0.00 as % ACN in the
0.00 0.50 1.00 1.50 2.00 2.50 3.00 diluent increases.
0.60 Minutes 75 ACN: 25 MeOH
2
0.50
1
0.40 3
AU
0.30
4
0.20 S
0.10
0.00
Grumbach
0.00 0.50 1.00 1.50 2.00 2.50 3.00
Minutes
Retention and Selectivity:
Influence of Mobile Phase pH
ACQUITY UPLC BEH Amide, 2.1 x 50 mm , 1.7 µm N
2
pH 3 OH
Nicotinic Acid
1 pKa = 2.2, 4.8
Compounds
4 1. Methacrylic acid
2. Nortriptyline
3. Nicotinic acid
3 CH3
4. cytosine NH
Nortriptyline
pKa = 10
2 H
pH 9 N O
4
N
NH2
Cytosine
1 pKa = 12.2
3 O
H2C
OH
CH3
0.00 1.00 2.00 3.00
Minutes Methacrylic Acid
pKa 4.58

Before You Start:
Common HILIC mobile phases
Common buffers/additives*
– Ammonium formate, ammonium acetate
– Formic acid, ammonium hydroxide, acetic acid
– Phosphate salt buffers ARE NOT recommended due to precipitation
in the highly organic mobile phase (phosphoric acid is OK)
Recommended buffer concentration: 10 mM ON-COLUMN
Recommended additive concentration: 0.2% ON-COLUMN
*The actual pH of the mobile phase may be 1 pH unit closer to neutral due to the highly organic mobile phase
Canals, I.; Oumada, F. Z.; Roses, M.; Bosch, E. J. Chromatogr. A. 911 (2001) 191-202.
Espinosa, S.; Bosch, E.; Roses, M. Anal. Chem. 72 (2000) 5193-5200.

Effect of Buffer Concentration:
pH 3.0
N
All contain 90:10 MeCN:H2O
1.00 O
0 mM OH
AU
ammonium Nicotinic Acid

1,2 3 4 formate pKa = 2.2, 4.8
0.00
1.00
3 2.5 mM
AU
4
ammonium
1 2
CH3
formate NH
0.00 Nortriptyline
1.00
pKa = 10
3
5.0 mM
AU
4 ammonium
H
1 N O
2 formate
0.00 N
1.00 3
10 mM NH2
AU
1 4 ammonium Cytosine
2 formate pKa = 12.2
0.00
O
1.00
3
1. Methacrylic acid H2C
1 2. Nicotinic acid 20 mM OH
4 ammonium
AU
3. Nortriptyline CH3
2 4. Cytosine formate
0.00
Methacrylic Acid
pKa 4.58
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00
Minutes
Effect of Buffer Concentration:
pH 9.0
N
1.00 O
0 mM
AU
OH
1,2 ammonium Nicotinic Acid

3 4 acetate pKa = 2.2, 4.8
0.00
1.00
4 2.5 mM
AU
3 ammonium
1 2
CH3
acetate NH
0.00 Nortriptyline
1.00
pKa = 10
5.0 mM
4 H
AU
3 ammonium N O
1 acetate
2
0.00 N
1.00
10 mM NH2
ammonium
AU
3 4 Cytosine
acetate pKa = 12.2
1 2
O
0.00
1.00 1. Methacrylic acid H2C
2. Nicotinic acid 20 mM OH
3 3. Nortriptyline ammonium
CH3
AU
4 4. Cytosine acetate
1 2 Methacrylic Acid
0.00 pKa 4.58
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00
Minutes 34
Additives vs. Buffered Mobile Phases
4 3 0.2% ammonium hydroxide pH 9
1,2
pH 9 Observations
Acids are unretained without a buffered
mobile phase
3
4 20 mM ammonium acetate pH 9
Selectivity shifts for basic compounds
1
2
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00
Minutes
pH 3 Observations
1 3 0.2% formic acid pH 3 Poor peak shape and retention for bases
1. Methacrylic acid without a buffered mobile phase
2. Nicotinic acid
2 4 3. Nortriptyline Selectivity shifts for acidic compounds
4. Cytosine
3
20 mM ammonium formate pH 3
1
4
2
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00
Minutes
Solvent Selection for Elution Strength
Weakest
Acetone
Primary
[Weak]
Solvents
Acetonitrile
Isopropanol
Ethanol Elution
[Strong]
Solvents
Methanol
Use a less polar solvent to

Water Increase retention
Strongest of polar analytes
Influence of Polar Modifier
2 3 90:10 ACN:H2O
1
4
2 90:5:5 ACN:H2O:MeOH
1
3
4 Retention increases
with decreasing
2 90:5:5 ACN:H2O:EtOH solvent polarity
1
3
4
1 2 90:5:5 ACN:H2O:IPA 10 mM ammonium acetate with

0.02% acetic acid
3 Analytes:
4 1: methacrylic acid
2: cytosine
3: nortriptyline
4: nicotinic acid
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
Minutes

System Check Standard:
Triplicate Injection HILIC QCRM
8.0e-2
7.0e-2
1.31
0.49
6.0e-2
5.0e-2 0.67
1.86
AU
4.0e-2
3.0e-2
Acenaphthene
Adenine
2.0e-2
Thymine
Cytosine
1.0e-2
0.0
Time
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40
Isocratic separation of HILIC QCRM on a 4.6 x 100 mm CORTECS HILIC 2.7 µm

column demonstrating retention of polar analytes using HILIC mode
chromatography

Outline


– System setup
– Tips and Tricks

HILIC Screening Strategy
pH 3 pH 9
ACQUITY UPLC® BEH HILIC
2.1 x 50 mm, 1.7 µm
Optimization
ACQUITY UPLC® BEH Amide
2.1 x 50 mm, 1.7 µm
Atlantis HILIC Silica

2.1 x 50 mm, 3 µm OR
CORTECS HILIC, 2.1 x 50 mm, 1.6
µm
Where do I start?
• Initial scouting gradient from 95 to 50% acetonitrile over 5 minutes
• At least 5% should be a polar solvent (i.e., water or methanol)

Influence of Stationary Phase
3.0
2.0 r2 = 0.5250
BEH HILIC (ln k)
1.0
0.0
-1.0
Stationary phase has
larger influence on
-2.0 selectivity than
mobile phase
-3.0
-2.0 -1.0 0.0 1.0 2.0 3.0
BEH amide (ln k)

Implementing the Approach:
Organophosphonic Acids
O O
H3C P OH O
H3C
H3C HO P O
O H3C P O
CH3
CH3
OH CH3 H3C H3C
isobutyl hydrogen
Cyclohexyl methylphosphonic Pinacolyl methylphosphonic acid
methylphosphonate (IBMPA)
acid (CMPA) (PMPA)
O
O
H3C P O
H3C P O
CH3
OH CH3
H3C OH
Ethyl methylphosphonic acid (EMPA)

Isopropyl methylphosphonic acid (IMPA)

Stationary Phase Selectivity:
Organophosphonic Acids at Low pH
2,3 1: SIR of 5 Channels ES-

BEH Amide
TIC pH 3
1
3.32e6
4 Atlantis HILIC Silica yields greatest
5 retention
BEH Amide and Atlantis HILIC Silica

0.00 1.00 2.00 3.00 4.00 5.00 6.00
yield similar selectivity
BEH HILIC 1: SIR of 5 Channels ES-

TIC
2,3
1 3.32e6
4,5
0.00 1.00 2.00 3.00 4.00 5.00 6.00

Compounds
Atlantis HILIC Silica 1. PMPA
1: SIR of 5 Channels ES- 2. CMPA
1
TIC 3. MMPA
2,3
3.32e6 4. IMPA
4 5 5. EMPA
500 ng/mL each

0.00 1.00 2.00 3.00 4.00 5.00 6.00
©2014 Waters Corporation Minutes 43
Stationary Phase Selectivity:
Organophosphonic Acids at High pH
1: SIR of 5 Channels ES-
BEH HILIC TIC
3.32e6 pH 9
2,3
1
Greater Resolution for
BEH Amide
4 No resolution between
5
peaks 2 and 3
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Further optimization
BEH Amide 1: SIR of 5 Channels ES-
needed
1 2,3 TIC
3.32e6
Compounds
1. PMPA
2. CMPA
3. MMPA
4. IMPA
4 5. EMPA
5
500 ng/mL each
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes 44
Method Optimization Steps
Evaluate results after each step.

• Adjust gradient slope
1
Stop after criteria for success has been met
• Adjust column temperature
2
Consider injection solvent (sample diluent) if poor peak
shape/resolution
• Adjust column length and flow rate
3
• Isocratic mode instead of gradient

4 • 95:5 ACN:H2O with 10 mM buffer or 0.2% additive
• Replace a portion of the water in the mobile phase with a less polar
5 solvent [MeOH, EtOH or IPA]

Optimization Step 1:
Adjust Gradient Slope
99.9% to 0.1% B in 5 min
1
2,3 SIR of 5 Channels ES-
TIC
3.32e6 BEH Amide, pH 9
4
5 Shallower gradient
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 slope results in
99.9% to 50% B in 5 min improved resolution
1
SIR of 5 Channels ES-
2,3
TIC
4.18e6
Compounds
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 1. PMPA
99.9% to 90% B in 5 min 2. CMPA
1 3. MMPA
2 SIR of 5 Channels ES-
3 TIC 4. IMPA
5.12e6 5. EMPA
4
5 500 ng/mL each
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00

Minutes 46
Column Temperature
1
2 SIR of 5 Channels ES- BEH Amide, pH 9
30 °C TIC
3 Shallow gradient
5.12e6
4
5 Increased
temperature results in
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
improved resolution
1 2
50 °C 3
TIC
4 5.08e6
5
Compounds
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
1. PMPA
1 2 2. CMPA
SIR of 5 Channels ES- 3. MMPA
65 °C 3 TIC
5.01e6
4. IMPA
4 5. EMPA
5
500 ng/mL each
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes
Column Length
BEH Amide, pH 9
1 2
Shallow gradient, 65 oC
2.1 x 50 mm SIR of 5 Channels ES-
TIC
100 mm column results in
3 improved resolution
4
50 mm column results in
5 shorter run time
Select result that meets

method criteria
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
2.1 x 100 mm 1
TIC Compounds
3 1. PMPA
4 2. CMPA
3. MMPA
5
4. IMPA
5. EMPA
500 ng/mL each

0.00 2.00 4.00 6.00 8.00 10.00 12.00
Minutes
Rapid HILIC Method Development
Screening approach Time c

Column conditioning* 30 minutes
3 Columns, 2 pH’s screening 30 minutes
Optimization
Column conditioning [temp. equilibration] 30 minutes
Gradient slope and temperature 30 minutes
Total method development time 2 Hours

*equilibration and 2 blank injections at each pH
Outline


– System setup
– Tips and Tricks

CORTECS Solid-
Solid-Core Particle
Compared to Fully Porous Particles:

The center core is nonporous
dp = 1.6 µm
Only the outer chromatographic surface
CORTECS contains pores
Solid-core
The outer shell is typically “bumpy”
The particle size distribution is very
narrow
dcore = 1.1 µm
ρ = core diameter / particle diameter

Rho, ρ = 1.1/1.6 = 0.7
66% Porous Volume
ρ = 0 → fully porous particle

ρ = 1 → nonporous particle
G. Guiochon, F. Gritti, J. Chromatogr. A 1218 (2011) 1915–1938

©2014 Waters Corporation Omamogho et al., J. Chromatogr. A 1218 (2011) 1942-1953 51
Higher Efficiency with
Solid Core Columns
19,700 39% higher efficiency

20,000
16,000
14,150
Plates (4 sigma)
or up to 3x faster!
12,000
CORTECS UPLC 1.6 µm C18+

8,000
ACQUITY UPLC 1.7 µm BEH C18
4,000
0.00 0.25 0.50 0.75 1.00 1.25
Flow Rate (mL/min)
2.1 x 50 mm column. A standard ACQUITY UPLC I-Class using 70% Acetonitrile in H2O at 30 °C
with 0.5 µL injections from a 1 µL FL injector

Higher Efficiency:
Sharper Peaks, Better Resolution
1
0.20 5
1. Lidocaine
2. Butacaine
2 4
3. Tetracaine ACQUITY BEH HILIC
0.15 4. Procaine 2.1 x 50mm 1.7 µm
3
5. Procainamide
AU
0.10 USP Resolution2,3: 1.2
0.05
0.00
1
5
0.20 2
4 CORTECS UPLC HILIC
0.15 3 2.1 x 50 mm 1.6 µm
0.10 USP Resolution2,3: 2.2

AU
0.05
0.00
-0.05
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00
Minutes

Analysis of Neurotransmitters in
Artificial CSF using CORTECS HILIC
HA: Histamine
t-MHA: tele-methylhistamine ACh CORTECS UPLC HILIC
t-MIAA: tele-methylimidazoleacetic acid 2.1 x 100 mm 1.6 µm
ACh: Acetylcholine t-MIAA
Ch: Choline
iso-ACh: iso-Acetylcholine Ch ACQUITY UPLC with Xevo TQ-S MS
iso-ACh
HA
t-MHA
Minutes
These neurotransmitters are highly polar and poorly retained in RP-LC
Iso-ACh, an isobaric endogenous interference of ACh in CSF, is chromatographically

resolved using the CORTECS column.

Analysis of Basic Drugs in
Surface Water
80 ppb eq std (10 ng/mL) matrix atlantis15-May-2013
EPA_05152013_22a MRM of 10 Channels ES+
4.57 TIC
5.27e6
HPLC HILIC
%
6.28
5.30
7.83
7.69
0
2.00 4.00 6.00 8.00 10.00 12.00
EPA_05152013_14 MRM of 10 Channels ES+
1.45 TIC
5.27e6
CORTECS UPLC HILIC
Better resolution for metformin/ranitidine

%
1.92
2.61
Much faster analysis
0 Time
2.00 4.00 6.00 8.00 10.00 12.00

Different Selectivity for
HILIC Materials
3
0.06
12 CORTECS UPLC 1.6 µm HILIC
4
0.04
5
AU
0.02
0.00
3
0.06 1
2 4 BEH HILIC, 1.7 µm
0.04 5
AU
0.02
0.00
1
0.06
BEH Amide, 1.7 µm
0.04 2
3
AU
4
0.02
5
0.00
0.00 2.00 4.00 6.00 8.00 10.00
Minutes
1. Acenaphthene 2. Thymine 3. Adenine 4. Cytosine 5. 5-Fluoroorotic Acid

MeCN/ 100mM Ammonium Formate pH 3 (90/10 v/v), 0.5mL/min, 2µL injection, 30°C
Higher Resolution:
Diquat
iquat/
/Paraquat in Drinking Water

Summary
For RP
– Try elevated pH first- largest degree of selectivity change
– Low coverage C18 (HSS T3) next
– PFP columns last
For HILIC
– Don’t just reverse your RP mobile phases and go
– Remember the tips and tricks
– Use a screening protocol- changing the column gives highest degree
of selectivity change
– Use a check standard (QCRM) before you run samples
– Optimize with gradient slope, temperature, column length
(just like RP)

HILIC Primer
HILIC primer [715001940]
– Comprehensive, 72-page Guide to

Hydrophilic Interaction
Chromatography [HILIC]
– Education is the key to success with
this technique

HILIC Method Development Wall Chart
HILIC method development

wall chart [720003484en]
– Efficient Hydrophilic Interaction

Chromatography [HILIC] Method
Development Strategy
– Reiterates key messages in the
HILIC method development
seminar
– www.waters.com/HILIC

Amide Column Selector:
Sugar Analysis
www.waters.com/amide
• Carbohydrate method
selection tool
• Link to BEH amide

application notebook
• Link to ACQUITY UPLC

column brand page

Other literature references
Peer reviewed publications
– Monoamine neurotransmitters
o Danaceau JP, Chambers EE, Fountain KJ. Bioanalysis. 2012 Apr;4(7):783-94.
– Method development
o Fountain KJ, Xu J, Dieh, DM, Morrison D. J. Sep Sci. 2010, 33, 740-751.
– Use of hybrid particles for HILIC
o Grumbach ES, Diehl, DM, Neue UD. J. Sep. Sci. 2008, 31, 1511-1518
o Grumbach ES, Wagrowski-Diehl DM, Mazzeo JR, Alden B, Iraneta P. LCGC N. Am. 2004, 22, 1010-1023
Application notes
– Acetylcholine, Histamine, and their Metabolites in Human CSF
http://www.waters.com/waters/library.htm?lid=134744372&cid=511436
– Paraquat and Diquat: Drinking Water
– Paraquat and Diquat in Potato and Wheat
– Analysis of Metformin and Related Substances
– Metabolomic Assay for the Analysis of Polar Metabolites
– Amide-Bonded BEH HILIC Columns for High Resolution, HPLC-Compatible Separations of N-Glycans
– Polar Basic Drugs in Environmental Samples
http://www.waters.com/waters/library.htm?cid=511436&lid=134817340

Thank You for Attending!
Post-Event Home Pagehttp://www.waters.com/Dec9

30% Promotional Offer On ALL HILIC / T3 Columns
30% Off Analytical Standards and Reagents
30% Off CORTECS Solid Core LC Columns
30% Offer on LC Sample Vials
– Full Webinar Recording of Today’s Session w/PDF Slide
Deck
– Compilation of TODAY’S KEY Literature, Brochures etc…
For Questions and to Submit your Ideas for our Next Topic
– Please eMail - mychemrep@waters.com

THANK YOU
www.waters.com/hilic

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‘Tips and Tricks’ for Retaining and

©2014 Waters Corporation 1

Please use text chat functionality to submit your

©2014 Waters Corporation 2

Kevin Jenkins, Product Manager, HPLC Columns,

Kevin has worked for Waters Corporation since 2000

©2014 Waters Corporation 3

Retaining polar compounds in RPLC

Retaining polar compounds in HILIC

©2014 Waters Corporation 4

Retaining polar compounds in RPLC

Retaining polar compounds in HILIC

©2014 Waters Corporation 5

General chemistry definition:

©2014 Waters Corporation 6

pH Range for Hybrid Particles

0.00 1.20 2.40 3.60 4.80 Observations:

0.00 1.20 2.40 3.60 4.80

6 2. Morphine-3β-D-glucuronide Morphine N-oxide

Anal. Chem. 2003, 75, 6781-6788

©2014 Waters Corporation 11

To retain polar compounds on a non-polar surface we reduce

PROBLEM: Risk of dewetting (hydrophobic collapse) the particle

What is “dewetting” and

©2014 Waters Corporation 12

The particles are very porous, like the

Mobile phase must

If the pores are dry, the analyte

What influence does this have on chromatography?

The observed reduction in V0 and sudden loss in retention after a

ACQUITY UPLC® BEH C18

0.20 0.40 0.60 0.80 1.00 1.20 1.25

*1 T.H. Walter, P. Iraneta and M. Capparella J. Chromatogr. A 1075 (2005) 177-183

Dominant retention mechanism is reversed-phase (van der

©2014 Waters Corporation 16

Multiple selectivity mechanisms including:

©2014 Waters Corporation 17

1 ACQUITY HSS PFP

Retaining polar compounds in RPLC

Retaining polar compounds in HILIC

©2014 Waters Corporation 19

HILIC - Hydrophilic Interaction Chromatography

HILIC is a variation of normal-phase chromatography without

Stationary phase is a POLAR material

*Alpert, A. J. J.Chromatogr. 499 (1990) 177-196.

• Polar analyte partitions

For the best gradient performance and reproducibility, it is

©2014 Waters Corporation 22

Gradient: 99.9% B to 0.1% B in 5 min, reset (total run time = 6 min)

Column Temp.: 30oC

©2014 Waters Corporation 23

Mobile Phase A: H2O

HILIC (low pH)

©2014 Waters Corporation 24

Instrument Wash Solvents

Brand new column

©2014 Waters Corporation 25

0.20 1 Weak needle

0.20 1 Weak needle

©2014 Waters Corporation 26

Retaining polar compounds in RPLC