‘tips and tricks’ for retaining and separating polar ... · ‘tips and tricks’ for retaining...
TRANSCRIPT
Thank you for joining us!Our session will begin shortly…
‘Tips and Tricks’ for Retaining and ‘Tips and Tricks’ for Retaining and Separating Polar Compounds using Separating Polar Compounds using
©2014 Waters Corporation 1
While you are waiting, please feel free to browse our library of program content: www.waters.com/meettheexperts
Also, click below to learn more about CORTECS, our newest Solid-Core LC Column platform: www.waters.com/CORTECS
Separating Polar Compounds using Separating Polar Compounds using ReversedReversed--Phase and Hydrophilic Phase and Hydrophilic
Interaction Chromatography (HILIC)Interaction Chromatography (HILIC)
� Please use text chat functionality to submit your
questions today.
� Providing ‘Live’ Technical Support during today’s event
� Poll Questions – Audience participation
� Upon conclusion, follow up information will be available:
� http://www.waters.com/Dec9
Friendly Reminders…
©2014 Waters Corporation 2
� http://www.waters.com/Dec9
� Recorded version of today’s presentation
� PDF Copy of today’s slides
� Application specific discount offers
�HILIC / T3 Columns, Standards, Reagents, CORTECS
Columns and LC Sample Vials
� Product specific information and reference materials
About Today’s Presenter…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
©2014 Waters Corporation 3
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.
OutlineOutline
� Retaining polar compounds in RPLC
– Elevated pH mobile phase
– Aqueous compatible C18 columns
– Pentafluorophenyl (PFP) columns for polar bases
� Retaining polar compounds in HILIC
©2014 Waters Corporation 4
� Retaining polar compounds in HILIC
– Mobile phase preparation
– System setup
– Tips and Tricks
– Method Development
– Solid core HILIC columns- what is the advantage?
OutlineOutline
� Retaining polar compounds in RPLC
– Elevated pH mobile phase
– Aqueous compatible C18 columns
– Pentafluorophenyl (PFP) columns for polar bases
� Retaining polar compounds in HILIC
©2014 Waters Corporation 5
� Retaining polar compounds in HILIC
– Mobile phase preparation
– System setup
– Tips and Tricks
– Method Development
– Solid core HILIC columns- what is the advantage?
What is a Polar Molecule?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
©2014 Waters Corporation 6
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 MoleculesExamples of Polar Molecules
N N
S N
NCH
O
NH2
F
H
NH
O
CH
CH3
NH
O
©2014 Waters Corporation 7
Adenine (N)
Thiourea (N) 5-Fluorocytosine (B) Thymine (N)
Guanosine-5’-monophosphate (A)
NN
N
O
NH
O
NH2
OHOH
OP
OH
OH
O N
NN
NH
NH2
ReversedReversed--Phase Retention Map:Phase Retention Map:The Impact of pH on The Impact of pH on IonizableIonizable CompoundsCompounds
25
30
35
40
Retention Factor Acid Neutral
Note: Retention of neutral analytes not affected by pH
Maximum acidic compound retention range
Maximum basic compound retention range
©2014 Waters Corporation 8
pH Range for Silica Particles
pH
0
5
10
15
20
0 2 4 6 8 10 12
Retention Factor
(k)
Base
pH Range for Hybrid Particles
Mobile Phase pH Selectivity:Mobile Phase pH Selectivity:Basic and Neutral CompoundsBasic and Neutral Compounds
I
A
P
Fl
Fe
DO
0.1% formic acid pH 3
Test Probes:I: Imipramine [B]A: Amitriptyline [B]Fl: Flavone [N]O: Octanophenone [N]
ACQUITY CSH C18, Acetonitrile
©2014 Waters Corporation 9
IA
P
Fl
Fe
D
O
Minutes
0.00 1.20 2.40 3.60 4.80
Minutes
0.00 1.20 2.40 3.60 4.80
0.1% NH4OH pH 10
Observations:
At high pH, bases are in their neutral (unionized) form, resulting in greater retention
Neutral compounds are unaffected by pH
Improved Retention and Sensitivity:Improved Retention and Sensitivity:High pH Mobile phaseHigh pH Mobile phase
%
HIGH pH0.1% NH4OH
5
1
2
3
4
6
10-hydroxymorphine
Morphine-3ß–o-glucuronideMorphine
©2014 Waters Corporation 10
Compounds
1. 10-hydroxymorphine
2. Morphine-3β-D-glucuronide
3. Morphine-6β-D-glucuronide
4. Morphine
5. Morphine N-oxide
6. 6-acetylmorphine
Minutes0.20 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
%
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 3.800
LOW pH0.1% HCOOH
1
2
3
4,56
Morphine-6ß–o-glucuronide
Morphine N-oxide
6-acetylmorphine
Silica and Hybrid Particles: Silica and Hybrid Particles: Manufacturing ProcessManufacturing Process
©2014 Waters Corporation 11
Anal. Chem. 2003, 75, 6781-6788
ReversedReversed--Phase ChromatographyPhase Chromatography::Challenging for Polar CompoundsChallenging 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
©2014 Waters Corporation 12
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 Proper Wetting of Bonded Chromatographic SurfaceChromatographic Surface
The particles are very porous, like the
pores of a sponge – 99% of
chromatographic surface is inside the
pores
Mobile Phase
Mobile phase must
©2014 Waters Corporation 13
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.
Analyte
Mobile phase must
be allowed into the pore
in order for chromatographic
retention of the analyte
to take place.
Pore Pore DewettingDewetting::What Does It Look Like?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
©2014 Waters Corporation 14
Minutes
0.20 0.40 0.60 0.80 1.00 1.20
Vo Shift
ACQUITY UPLC® BEH C18
L = 5 cmF = 0.3 mL/minP = 2,040T = 90 oC% Dewet = 23.10
*1 T.H. Walter, P. Iraneta and M. Capparella J. Chromatogr. A 1075 (2005) 177-183
1.25
HSS T3 Versus HSS C18:HSS T3 Versus HSS C18:100% Aqueous Conditions100% Aqueous Conditions
15
2
3 4
AU
0.00
0.02
0.04
0.06
AU
0.00
0.02
0.04
0.06
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
XSelect HSS T3
Initial
After dewetting
©2014 Waters Corporation 15
Peak i.d.: 1) thiourea 2) 5-Fluorocytosine 3) adenine 4) thymidine-5’-monophosphate 5) thymineConditions: 10mM Ammonium Formate pH 3; 0.2mL/min; 30ºC; 2.1 x 50 mm
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
AU
0.00
0.02
0.04
0.06
AU
0.00
0.05
0.10
0.15
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
1
5
23 4
Pks 1 - 5
XSelect HSS C18
Initial
After dewetting
Polar Retention:Polar Retention:Why Does Why Does HSS HSS T3 Work?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
©2014 Waters Corporation 16
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:Another Options for Polar Bases:PFP (PFP (PentafluorophenylPentafluorophenyl))
�Multiple selectivity mechanisms including:
– hydrogen bonding
– dipole-dipole
– aromatic (pi-pi)
– hydrophobic (reversed-phase)
©2014 Waters Corporation 17
� Target Analyses:
� positional isomers, halogenated compounds and polar bases
Another Option for Polar Bases:Another Option for Polar Bases:PFP versus C18PFP versus C18
11
22
33 55
44
6677
Compounds 1. Trimethoprim 2. Nordoxepin 3. Doxepin4. Nortriptyline5. Imipramine6. Amitriptyline7. Trimipramine
ACQUITY HSS C18
©2014 Waters Corporation 18
Minutes
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
1
23
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 6.00Minutes
ACQUITY HSS PFP
OutlineOutline
� Retaining polar compounds in RPLC
– Elevated pH mobile phase
– Aqueous compatible C18 columns
– Pentafluorophenyl (PFP) columns for polar bases
� Retaining polar compounds in HILIC
©2014 Waters Corporation 19
� Retaining polar compounds in HILIC
– Mobile phase preparation
– System setup
– Tips and Tricks
– Method Development
– Solid core HILIC columns- what is the advantage?
What is HILIC?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
©2014 Waters Corporation 20
— “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.
MultiMulti--modal Retention Mechanisms: modal Retention Mechanisms: HILICHILIC
Combination of
partitioning, ion-exchange
and hydrogen bonding
• Polar analyte partitions
between bulk mobile phase
and partially immobilized
polar layer on material
surface
©2014 Waters Corporation 21
• 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:Before You Start:Mobile Phase PreparationMobile 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]
©2014 Waters Corporation 22
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: Screening Conditions: Binary Pumping SystemBinary 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)
©2014 Waters Corporation 23
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: Screening Conditions: Quaternary Pumping System (allows for RP)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)
©2014 Waters Corporation 24
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
Gradient conditions for 2.1 x 50 mm, 1.7 µm column
HILIC (low pH)
Time
(min)
Flow
(mL/min)
% A % B % C % D Curve
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
10.00 0.60 5 90 5 0 1
Before You Start: Before You Start: Column Equilibration and Wash SolventsColumn 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
©2014 Waters Corporation 25
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
AU
0.20
0.30
0.40
0.50
1. Methacrylic acid
2. Nicotinic acid
3. Nortriptyline
4. Cytosine
1
3 4
Weak needle
AU
0.20
0.30
0.40
0.50
AU
0.20
0.30
0.40
0.50
1. Methacrylic acid
2. Nicotinic acid
3. Nortriptyline
4. Cytosine
1
3 4
Weak needle
Influence of Needle Wash SolventInfluence of Needle Wash Solvent
©2014 Waters Corporation 26
0.00
0.10
0.20
AU
0.00
0.10
0.20
0.30
0.40
0.50
Minutes
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
1
2
1
34,2
Weak needle wash: 95:5 MeCN/H2O
Weak needle wash: 95:5 H2O/MeCN
0.00
0.10
0.20
AU
0.00
0.10
0.20
0.30
0.40
0.50
Minutes
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
0.00
0.10
0.20
AU
0.00
0.10
0.20
0.30
0.40
0.50
Minutes
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
1
2
1
34,2
Weak needle wash: 95:5 MeCN/H2O
Weak needle wash: 95:5 H2O/MeCN
OutlineOutline
� 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
©2014 Waters Corporation 27
– 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?
– Method Development
– Solid core HILIC columns- what is the advantage?
The Importance The Importance of Sample of Sample DiluentDiluent/Injection Solvent /Injection Solvent SelectionSelection
� 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
©2014 Waters Corporation 28
� 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
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00
Sample Sample DiluentDiluent Considerations:Considerations:Water as Polar SolventWater as Polar Solvent
0.50
0.60
100% H2O
50 ACN: 50 H2O
S
12
34
2
ACQUITY UPLC® BEH HILIC2.1 x 100 mm, 1.7 µm
Analytes1. Methacrylic acid2. Cytosine 3. Nortriptyline4. Nicotinic acid
©2014 Waters Corporation 29
AU
0.00
0.10
0.20
0.30
0.40
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00
75 ACN: 25 H2O
S 12
34
S
12
3
4
Peak shape improvesas % ACN in the diluent increases.
What about alternative polar organic solvents?
Grumbach
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00
0.50
0.60
Sample Sample DiluentDiluent Considerations:Considerations:Methanol as Polar SolventMethanol as Polar Solvent
100% MeOH
50 ACN: 50 MeOH
S
1
2
34
S 23
ACQUITY UPLC® BEH HILIC2.1 x 100 mm, 1.7 µm
Analytes1. Methacrylic acid2. Cytosine 3. Nortriptyline4. Nicotinic acid
©2014 Waters Corporation 30
AU
0.00
0.10
0.20
0.30
0.40
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00
75 ACN: 25 MeOH
S1 3
4
S
12
3
4
Peak shape and solubility improve by replacing water with methanol
Peak shape improvesas % ACN in the diluent increases.
Grumbach
Retention and Selectivity:Retention and Selectivity:Influence of Mobile Phase pHInfluence of Mobile Phase pH
1
2
3
4
pH 3
Compounds
1. Methacrylic acid
2. Nortriptyline
3. Nicotinic acid
4. cytosine
ACQUITY UPLC BEH Amide, 2.1 x 50 mm , 1.7 µm
NHCH3
O
N
OH
Nicotinic AcidpKa = 2.2, 4.8
©2014 Waters Corporation 31
Minutes0.00 1.00 2.00 3.00
pH 9
1
2
3
4
O
CH2OH
CH3
Methacrylic Acid pKa 4.58
N
NH
O
NH2
CytosinepKa = 12.2
NortriptylinepKa = 10
Before You Start:Before You Start:Common HILIC mobile phasesCommon 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)
©2014 Waters Corporation 32
� 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.
AU
0.00
1.00
AU
0.00
1.00
1,2 3 4
3
2
4
1
Effect of Buffer Concentration: Effect of Buffer Concentration: pH 3.0pH 3.0
0 mM
ammonium
formate
2.5 mM
ammonium
formate
All contain 90:10 MeCN:H2O
NHCH3
Nortriptyline
O
N
OH
Nicotinic AcidpKa = 2.2, 4.8
©2014 Waters Corporation 33
0.00
AU
0.00
1.00
AU
0.00
1.00
AU
0.00
1.00
Minutes
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
3
2
41
3
2
41
3
2
41
1. Methacrylic acid
2. Nicotinic acid
3. Nortriptyline
4. Cytosine
5.0 mM
ammonium
formate
10 mM
ammonium
formate
20 mM
ammonium
formate
O
CH2OH
CH3
Methacrylic Acid pKa 4.58
N
NH
O
NH2
CytosinepKa = 12.2
NortriptylinepKa = 10
Effect of Buffer Concentration: Effect of Buffer Concentration: pH 9.0pH 9.0
0.00
1.00
0.00
1.00
3
12
4
3
41,2
AU
AU
0 mM
ammonium
acetate
2.5 mM
ammonium
acetate
All contain 90:10 MeCN:H2O
NHCH3
Nortriptyline
O
N
OH
Nicotinic AcidpKa = 2.2, 4.8
©2014 Waters Corporation 34
0.00
0.00
1.00
0.00
1.00
0.00
1.00
Minutes
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
13
4
2
1
3 4
2
3
1
4
2
AU
AU
AU
5.0 mM
ammonium
acetate
10 mM
ammonium
acetate
20 mM
ammonium
acetate
O
CH2OH
CH3
Methacrylic Acid pKa 4.58
N
NH
O
NH2
CytosinepKa = 12.2
NortriptylinepKa = 10
1. Methacrylic acid
2. Nicotinic acid
3. Nortriptyline
4. Cytosine
Additives vs. Buffered Mobile PhasesAdditives vs. Buffered Mobile Phases
1,2
43
3
1
4
2
0.2% ammonium hydroxide pH 9
20 mM ammonium acetate pH 9
pH 9 Observations
Acids are unretained without a buffered
mobile phase
Selectivity shifts for basic compounds
©2014 Waters Corporation 35
Minutes
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
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
3
2
4
1
24
1
20 mM ammonium formate pH 3
0.2% formic acid pH 33
1. Methacrylic acid
2. Nicotinic acid
3. Nortriptyline
4. Cytosine
pH 3 Observations
Poor peak shape and retention for bases
without a buffered mobile phase
Selectivity shifts for acidic compounds
All contain 90:10 MeCN:H2O
Weakest
Primary
[Weak] Solvents
Acetone
Acetonitrile
Isopropanol
Solvent Selection for Elution StrengthSolvent Selection for Elution Strength
©2014 Waters Corporation 36
Strongest
Use a less polar solvent to
Increase retention
of polar analytes
Ethanol
Methanol
Water
Elution
[Strong] Solvents
Retention and Selectivity:Retention and Selectivity:Influence of Polar ModifierInfluence of Polar Modifier
1
2 3
4
12
3
4
90:10 ACN:H2O
90:5:5 ACN:H2O:MeOH
Retention increases
with decreasing
©2014 Waters Corporation 37
10 mM ammonium acetate with
0.02% acetic acid
Analytes:
1: methacrylic acid
2: cytosine
3: nortriptyline
4: nicotinic acid
12
3
4
1
34
90:5:5 ACN:H2O:EtOH
90:5:5 ACN:H2O:IPA
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
2
with decreasing
solvent polarity
System Check Standard:System Check Standard:Triplicate Injection HILIC QCRMTriplicate Injection HILIC QCRM
AU 4.0e-2
5.0e-2
6.0e-2
7.0e-2
8.0e-2
1.31
0.49
0.671.86
©2014 Waters Corporation 38
Time0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40
0.0
1.0e-2
2.0e-2
3.0e-2
Acenaphthene
Thymine
Adenine
Cytosine
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
OutlineOutline
� Retaining polar compounds in RPLC
– Elevated pH mobile phase
– Aqueous compatible C18 columns
– Pentafluorophenyl (PFP) columns for polar bases
� Retaining polar compounds in HILIC
©2014 Waters Corporation 39
� Retaining polar compounds in HILIC
– Mobile phase preparation
– System setup
– Tips and Tricks
– Method Development
– Solid core HILIC columns- what is the advantage?
pH 3
ACQUITY UPLC® BEH HILIC
2.1 x 50 mm, 1.7 µm
Op
tim
iza
tio
n
pH 9
ACQUITY UPLC® BEH Amide
2.1 x 50 mm, 1.7 µm
HILIC Screening StrategyHILIC Screening Strategy
©2014 Waters Corporation 40
Op
tim
iza
tio
n
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)
Retention and Selectivity:Retention and Selectivity:Influence of Stationary Phase Influence of Stationary Phase
BE
H H
ILIC
(ln
k)
1.0
2.0
3.0
r2 = 0.5250
©2014 Waters Corporation 41
BEH amide (ln k)
BE
H H
ILIC
(ln
k)
Stationary phase has
larger influence on
selectivity than
mobile phase
-3.0
-2.0
-1.0
0.0
-2.0 -1.0 0.0 1.0 2.0 3.0
OH
O
PCH3
O
Cyclohexyl methylphosphonic
acid (CMPA)
CH3
O
O
PCH3
OH CH3
isobutyl hydrogen
methylphosphonate (IBMPA)
O
O
POH
CH3
CH3
CH3
CH3CH3
Pinacolyl methylphosphonic acid
(PMPA)
Implementing the Approach:Implementing the Approach:OrganophosphonicOrganophosphonic AcidsAcids
©2014 Waters Corporation 42
acid (CMPA)
O
O
P
OHCH3
CH3
CH3
Isopropyl methylphosphonic acid (IMPA)
O
O
P
OHCH3
CH3
Ethyl methylphosphonic acid (EMPA)
Stationary Phase Selectivity:Stationary Phase Selectivity:OrganophosphonicOrganophosphonic Acids at Low pHAcids at Low pH
pH 3
Atlantis HILIC Silica yields greatest
retention
BEH Amide and Atlantis HILIC Silica
yield similar selectivity0.00 1.00 2.00 3.00 4.00 5.00 6.00
1: SIR of 5 Channels ES-
TIC
3.32e6
1: SIR of 5 Channels ES-
TIC
1
2,3
45
BEH Amide
BEH HILIC
©2014 Waters Corporation 43
Compounds
1. PMPA
2. CMPA
3. MMPA
4. IMPA
5. EMPA
500 ng/mL each
2,3
0.00 1.00 2.00 3.00 4.00 5.00 6.00
0.00 1.00 2.00 3.00 4.00 5.00 6.00
TIC
3.32e6
1: SIR of 5 Channels ES-
TIC
3.32e6
1
4,5
1
2,3
45
Atlantis HILIC Silica
Minutes
pH 9
Greater Resolution for
BEH Amide
No resolution between
peaks 2 and 3
1: SIR of 5 Channels ES-
TIC
3.32e6
1
2,3
4
5
BEH HILIC
Stationary Phase Selectivity:Stationary Phase Selectivity:OrganophosphonicOrganophosphonic Acids at High pHAcids at High pH
©2014 Waters Corporation 44
Compounds
1. PMPA
2. CMPA
3. MMPA
4. IMPA
5. EMPA
500 ng/mL each
peaks 2 and 3
Further optimization
needed
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: SIR of 5 Channels ES-
TIC
3.32e6
1 2,3
45
BEH Amide
Minutes
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• Adjust gradient slope
2• Adjust column temperature
• Adjust column length and flow rate
Method Optimization StepsMethod Optimization Steps
�Evaluate results after each step.
�Stop after criteria for success has been met
�Consider injection solvent (sample diluent) if poor peak
shape/resolution
©2014 Waters Corporation 45
3• Adjust column length and flow rate
4
• Isocratic mode instead of gradient
• 95:5 ACN:H2O with 10 mM buffer or 0.2% additive
5
• Replace a portion of the water in the mobile phase with a less polar solvent [MeOH, EtOH or IPA]
shape/resolution
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
12,3
45
99.9% to 0.1% B in 5 min
SIR of 5 Channels ES-
TIC
3.32e6
2,3
1SIR of 5 Channels ES-
99.9% to 50% B in 5 min
BEH Amide, pH 9
Shallower gradient
slope results in
improved resolution
Optimization Step 1:Optimization Step 1:Adjust Gradient SlopeAdjust Gradient Slope
©2014 Waters Corporation 46
2,3
4
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
SIR of 5 Channels ES-
TIC
4.18e6
Minutes
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
SIR of 5 Channels ES-
TIC
5.12e6
12
3
45
99.9% to 90% B in 5 min
Compounds
1. PMPA
2. CMPA
3. MMPA
4. IMPA
5. EMPA
500 ng/mL each
21
30 °C
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
SIR of 5 Channels ES-
TIC
5.12e6
12
3
45
BEH Amide, pH 9
Shallow gradient
Increased
temperature results in
improved resolution
Optimization Step 2:Optimization Step 2:Column TemperatureColumn Temperature
©2014 Waters Corporation 47
SIR of 5 Channels ES-
TIC
5.08e6
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
21
3
4
5
Minutes
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
21
3
4
5
SIR of 5 Channels ES-
TIC
5.01e6
50 °C
65 °C
Compounds
1. PMPA
2. CMPA
3. MMPA
4. IMPA
5. EMPA
500 ng/mL each
21
3
4
5
SIR of 5 Channels ES-
TIC
2.1 x 50 mm
BEH Amide, pH 9
Shallow gradient, 65 oC
100 mm column results in
improved resolution
50 mm column results in
shorter run time
Select result that meets
Optimization Step 3:Optimization Step 3:Column LengthColumn Length
©2014 Waters Corporation 48Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00
2
1
3
4
5
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
SIR of 5 Channels ES-
TIC
2.1 x 100 mmCompounds
1. PMPA
2. CMPA
3. MMPA
4. IMPA
5. EMPA
500 ng/mL each
Select result that meets
method criteria
Rapid HILIC Method DevelopmentRapid HILIC Method Development
Screening approach Time c
� Column conditioning* 30 minutes
� 3 Columns, 2 pH’s screening 30 minutes
Optimization
©2014 Waters Corporation 49
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
OutlineOutline
� Retaining polar compounds in RPLC
– Elevated pH mobile phase
– Aqueous compatible C18 columns
– Pentafluorophenyl (PFP) columns for polar bases
� Retaining polar compounds in HILIC
©2014 Waters Corporation 50
� Retaining polar compounds in HILIC
– Mobile phase preparation
– System setup
– Tips and Tricks
– Method Development
– Solid core HILIC columns- what is the advantage?
CORTECS SolidCORTECS Solid--Core ParticleCore Particle
Compared to Fully Porous Particles:
� The center core is nonporous
� Only the outer chromatographic surface
contains pores
� The outer shell is typically “bumpy”
� The particle size distribution is very
narrow
CORTECSSolid-core
dp= 1.6 µm
©2014 Waters Corporation 51
narrow
dcore = 1.1 µm
Rho, ρ = 1.1/1.6 = 0.7
66% Porous Volume
ρ = 0 → fully porous particle
ρ = 1 → nonporous particle
ρ = core diameter / particle diameter
G. Guiochon, F. Gritti, J. Chromatogr. A 1218 (2011) 1915–1938 Omamogho et al., J. Chromatogr. A 1218 (2011) 1942-1953
Higher Efficiency with Higher Efficiency with Solid Core ColumnsSolid Core Columns
19,700
14,15016,000
20,000
Plates (4 sigma)
39% higher efficiency
or up to 3x faster!
©2014 Waters Corporation 52
4,000
8,000
12,000
0.00 0.25 0.50 0.75 1.00 1.25
Plates (4 sigma)
Flow Rate (mL/min)
or up to 3x faster!
CORTECS UPLC 1.6 µm C18+
ACQUITY UPLC 1.7 µm BEH C18
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:Higher Efficiency:Sharper Peaks, Better ResolutionSharper Peaks, Better Resolution
AU
0.00
0.05
0.10
0.15
0.201
2
3
4
5
ACQUITY BEH HILIC2.1 x 50mm 1.7 µm
1. Lidocaine 2. Butacaine 3. Tetracaine4. Procaine5. Procainamide
USP Resolution2,3: 1.2
©2014 Waters Corporation 53
0.00
AU
-0.05
0.00
0.05
0.10
0.15
0.20
Minutes0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00
CORTECS UPLC HILIC2.1 x 50 mm 1.6 µm
1
2
3
4
5
USP Resolution2,3: 2.2
ACh
HA
t-MIAA
t-MHA
iso-AChCh
Analysis of Neurotransmitters in Analysis of Neurotransmitters in Artificial CSF using CORTECS HILICArtificial CSF using CORTECS HILIC
HA: Histamine t-MHA: tele-methylhistamine t-MIAA: tele-methylimidazoleacetic acidACh: Acetylcholine Ch: Choline iso-ACh: iso-Acetylcholine
CORTECS UPLC HILIC2.1 x 100 mm 1.6 µm
ACQUITY UPLC with Xevo TQ-S MS
©2014 Waters Corporation 54
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 Analysis of Basic Drugs in Surface WaterSurface Water
80 ppb eq std (10 ng/mL) matrix atlantis15-May-2013
%
EPA_05152013_22a MRM of 10 Channels ES+ TIC
5.27e64.57
6.28
5.307.83
7.69
HPLC HILIC
©2014 Waters Corporation 55
Time2.00 4.00 6.00 8.00 10.00 12.00
%
0
2.00 4.00 6.00 8.00 10.00 12.000
EPA_05152013_14 MRM of 10 Channels ES+ TIC
5.27e6
1.45
1.92
2.61
CORTECS UPLC HILIC
Better resolution for metformin/ranitidine
Much faster analysis
Different Selectivity for Different Selectivity for HILIC MaterialsHILIC Materials
0.04
0.06
AU
0.00
0.02
0.04
0.061 2
5
3
4
12
5
3
4
CORTECS UPLC 1.6 µm HILIC
BEH HILIC, 1.7 µm
©2014 Waters Corporation 56
1. Acenaphthene 2. Thymine 3. Adenine 4. Cytosine 5. 5-Fluoroorotic AcidMeCN/ 100mM Ammonium Formate pH 3 (90/10 v/v), 0.5mL/min, 2µL injection, 30°C
AU
0.00
0.02
0.04
0.06
Minutes0.00 2.00 4.00 6.00 8.00 10.00
AU
0.00
0.02
0.04 5
1
2
5
3
4
BEH Amide, 1.7 µm
Higher Resolution:Higher Resolution:DDiquatiquat//ParaquatParaquat in Drinking Waterin Drinking Water
©2014 Waters Corporation 57
SummarySummary
� For RP
– Try elevated pH first- largest degree of selectivity change
– Low coverage C18 (HSS T3) next
– PFP columns last
� For HILIC
©2014 Waters Corporation 58
� 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 PrimerHILIC Primer
�HILIC primer [715001940]
– Comprehensive, 72-page Guide to
Hydrophilic Interaction
Chromatography [HILIC]
©2014 Waters Corporation 59
Chromatography [HILIC]
– Education is the key to success with
this technique
HILIC Method Development Wall ChartHILIC Method Development Wall Chart
�HILIC method development
wall chart [720003484en]
– Efficient Hydrophilic Interaction
Chromatography [HILIC] Method
©2014 Waters Corporation 60
Chromatography [HILIC] Method
Development Strategy
– Reiterates key messages in the
HILIC method development
seminar
– www.waters.com/HILIC
Amide Column Selector:Amide Column Selector:Sugar AnalysisSugar Analysis
www.waters.com/amide
• Carbohydrate method
selection tool
• Link to BEH amide
©2014 Waters Corporation 61
• Link to BEH amide
application notebook
• Link to ACQUITY UPLC
column brand page
Other literature referencesOther 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
©2014 Waters Corporation 62
– Acetylcholine, Histamine, and their Metabolites in Human CSF http://www.waters.com/waters/library.htm?lid=134744372&cid=511436
– Paraquat and Diquat: Drinking Water
http://www.waters.com/waters/library.htm?lid=134744375&cid=511436
– Paraquat and Diquat in Potato and Wheat http://www.waters.com/waters/library.htm?lid=134776789&cid=511436
– Analysis of Metformin and Related Substances http://www.waters.com/waters/library.htm?lid=134735459&cid=511436
– Metabolomic Assay for the Analysis of Polar Metabolites http://www.waters.com/waters/library.htm?lid=134726984&cid=511436
– Amide-Bonded BEH HILIC Columns for High Resolution, HPLC-Compatible Separations of N-Glycans
http://www.waters.com/waters/library.htm?lid=134776151&cid=511436
– Polar Basic Drugs in Environmental Samples
http://www.waters.com/waters/library.htm?cid=511436&lid=134817340
Thank You for Attending!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
©2014 Waters Corporation 63
– 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 - [email protected]