©2016 Waters Corporation 1
Part 2: Polar Compound Retention
Tuesday October 18, 2016 – 3 pm CET / 9 am EST
Considerations for Successful
HILIC Separations
©2016 Waters Corporation 2
Outline
Retaining polar compounds in HILIC
– What is HILIC?
– Method selection
– Column choices
– Mobile phase preparation
– System setup
– Tips and tricks
– Method development
– Solid-core HILIC columns - What is the advantage?
©2016 Waters Corporation 3
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.
©2016 Waters Corporation 4
When To Use HILIC
When to Use HILIC:
Need improved retention of
hydrophilic or ionizable
compounds
Need improved MS response
for polar or ionizable
compounds
Need improved sample
throughput for assays using
organic extraction
Reversed-phase
polar non-polar
Compound Index
Normal-phase
ESI-
MS
Re
spo
nse
exce
llen
tp
oo
r
HILIC
©2016 Waters Corporation 5
Multi-modal Retention Mechanisms: 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
©2016 Waters Corporation 6
Typical HILIC Stationary Phases
Waters: XBridge BEH HILIC CORTECS HILIC Atlantis HILIC Competitors: Luna HILIC Kinetex HILIC Fortis HILIC Hypersil GOLD HILIC Hypersil GOLD Silica Syncronis HILIC AccuCore HILIC Ascentis Express HILIC
Waters: XBridge BEH AMIDE XSelect HSS CN Bonded Phases: TSK Amide Ascentis Express OH5 Ascentis Express ES-CN Charged Phases: NucleoShell HILIC SEQUENT ZIC HILIC
©2016 Waters Corporation 7
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
©2016 Waters Corporation 8
Method Selection Based on Analyte Classification
Is the polar compound an acid, neutral or
base?
Alternative Techniques: Ion-Pair Ion-Exchange Ion-Exclusion
Silica: CORTECS HILIC Atlantis HILIC Hybrid:
BEH HILIC
ACIDIC NEUTRAL BASIC
Insufficient retention?
yes
BEH Amide
Substitute water with polar organic solvent [MeOH or IPA]
Still insufficient retention?
yes
©2016 Waters Corporation 9
HILIC Retention and Selectivity Matrix
HILIC Retention and
Selectivity
Stationary Phase
Organic Modifier
Mobile Phase pH
•Decreasing solvent polarity increases retention •Screen multiple columns to maximize retention and selectivity differences •Analytes have greater retention when they are ionized [acids at high pH, bases at low pH]
©2016 Waters Corporation 10
HILIC Stationary Phases
Base particle and ligand influences HILIC separations
– Sorbent selection depends on analyte and sample conditions
CORTECS
HILIC
ACQUITY BEH HILIC
XBridge BEH HILIC
ACQUITY BEH Amide
XBridge BEH Amide
Atlantis HILIC
Intended
Use
Alkaline Polars
(Low pH)
Alkaline Polars
(Low-mid pH)
Acidic/Neutral Polars
(High pH)
Alkaline Polars
(Low pH)
pH Range 1-5 1-9 2-12 1-5
Particle
Type
Unbonded Solid-Core
Silica
Fully Porous Unbonded
Hybrid Silica
Fully Porous Amide
bonded Hybrid Silica
Fully Porous
Unbonded Silica
Maximum
Pressure
18,000 PSI (1240 bar) 18,000 PSI (1240 bar) 6,000 PSI (400 bar)
Particle Size 1.6 µm, 2.7 µm 1.7 µm, 2.5 µm, 3.5 µm, 5 µm, 10 µm 3 µm, 5 µm, 10 µm
Pore
Diameter
120 Å 100 Å 100 Å
Surface Area 100 m2/g 185 m2/g 330 m2/g
©2016 Waters Corporation 11
Influence of Stationary Phase on Retention
ACQUITY UPLC BEH HILIC
Unbonded hybrid with low silanol activity
4 5
Minutes
1 2
3
3
1 2 4
5
1 2 3
4
5
0 1 2 3
ACQUITY UPLC BEH Amide
Bonded hybrid
Atlantis HILIC Silica
Unbonded silica with high silanol activity
(1) acenaphthene (2) thymine (3) 5-fluoroorotic acid (4) adenine (5) cytosine; UV 254 nm
4 5
1 2
3 CORTECS HILIC Silica
Unbonded silica with moderate silanol activity
©2016 Waters Corporation 12
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.
©2016 Waters Corporation 13
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
©2016 Waters Corporation 14
Screening Conditions: Quaternary Pumping System
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: 30oC
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
©2016 Waters Corporation 15
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
©2016 Waters Corporation 16
Outline
Retaining polar compounds in HILIC
– Mobile phase preparation
– System setup
– Tips and tricks
o Injection solvent
o Buffers/additives
o Tuning the mobile phase for more retention
– Method development
– Solid core HILIC columns- What is the advantage?
©2016 Waters Corporation 17
Influence of Needle Wash Solvent
1
3 4
2
1
3 4,2
AU
0.00
0.10
0.20
0.30
0.40
0.50
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
AU
0.00
0.10
0.20
0.30
0.40
0.50
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
3 4
2
1
3 4,2
Poor Peak Shape
Weak Needle Wash: 95% Acetonitrile:5% Water
Weak Needle Wash: 95% Water:5% Acetonitrile
Needle Wash is too Strong!
1. Methacrylic acid 2. Nortryptyline 3. Nicotinic Acid 4. Cytosine
©2016 Waters Corporation 18
The Importance of Sample 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
©2016 Waters Corporation 19
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 Diluent Considerations: Water as Polar Solvent
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
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
100% H2O
50 ACN: 50 H2O
75 ACN: 25 H2O
S
1 2
3 4
S 1 2
3 4
S
1 2
3
4
Peak shape improves as % ACN in the diluent increases.
What about alternative
polar organic solvents?
ACQUITY UPLC® BEH HILIC 2.1 x 100 mm, 1.7 µm Analytes 1. Methacrylic acid 2. Cytosine 3. Nortriptyline 4. Nicotinic acid
©2016 Waters Corporation 20
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
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
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 Diluent Considerations: Methanol as Polar Solvent
100% MeOH
50 ACN: 50 MeOH
75 ACN: 25 MeOH
S
1
2
3 4
S 1
2 3
4
S
1 2
3
4
Peak shape and solubility improve by replacing water with
methanol
Peak shape improves as % ACN in the diluent increases.
ACQUITY UPLC® BEH HILIC 2.1 x 100 mm, 1.7 µm Analytes 1. Methacrylic acid 2. Cytosine 3. Nortriptyline 4. Nicotinic acid
©2016 Waters Corporation 21
Retention and Selectivity: Influence of Mobile Phase pH
Minutes 0.00 1.00 2.00 3.00
1
2
3
4
pH 3
pH 9
Compounds 1. Methacrylic acid 2. Nortriptyline 3. Nicotinic acid 4. cytosine
1
2
3
4
ACQUITY UPLC BEH Amide, 2.1 x 50 mm , 1.7 µm O
CH2OH
CH3
Methacrylic Acid pKa 4.58
N
NH
O
NH2
CytosinepKa = 12.2
NHCH3
NortriptylinepKa = 10
O
N
OH
Nicotinic AcidpKa = 2.2, 4.8
©2016 Waters Corporation 22
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.
©2016 Waters Corporation 23
AU
0.00
1.00
AU
0.00
1.00
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
1,2 3 4
3
2
41
3
2
41
3
2
41
3
2
41
Effect of Buffer Concentration: pH 3.0
0 mM ammonium
formate
2.5 mM ammonium
formate
1. Methacrylic acid 2. Nicotinic acid 3. Nortriptyline 4. Cytosine
All contain 90:10 MeCN:H2O
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
NHCH3
NortriptylinepKa = 10
O
N
OH
Nicotinic AcidpKa = 2.2, 4.8
©2016 Waters Corporation 24
Effect of Buffer Concentration: pH 9.0
0.00
1.00
0.00
1.00
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
3
1 2
4
3
13
4
2
1
3 4
2
3
1
4
4
2
1,2
AU
AU
AU
AU
AU
0 mM ammonium
acetate
2.5 mM ammonium
acetate
5.0 mM ammonium
acetate
10 mM ammonium
acetate
20 mM ammonium
acetate
All contain 90:10 MeCN:H2O
1. Methacrylic acid 2. Nicotinic acid 3. Nortriptyline 4. Cytosine
2.5 mM ammonium
acetate
O
CH2OH
CH3
Methacrylic Acid pKa 4.58
N
NH
O
NH2
CytosinepKa = 12.2
NHCH3
NortriptylinepKa = 10
O
N
OH
Nicotinic AcidpKa = 2.2, 4.8
©2016 Waters Corporation 25
Additives vs. Buffered Mobile Phases
1,2
4 3
3
1
4
2
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
0.2% ammonium hydroxide pH 9
20 mM ammonium acetate pH 9
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
2 4
1
20 mM ammonium formate pH 3
0.2% formic acid pH 3 3
1. Methacrylic acid 2. Nicotinic acid 3. Nortriptyline 4. Cytosine
All contain 90:10 MeCN:H2O
pH 3 Observations
Poor peak shape and retention for bases without a buffered mobile phase
Selectivity shifts for acidic compounds
pH 9 Observations
Acids are unretained without a buffered mobile phase
Selectivity shifts for basic compounds
©2016 Waters Corporation 26
Weakest
Strongest
Primary
[Weak] Solvents
Acetone
Acetonitrile
Isopropanol
Ethanol
Methanol
Water
Elution
[Strong] Solvents
Solvent Selection for Elution Strength
Low Polarity Solvents Increase Retention of polar analytes
©2016 Waters Corporation 27
Retention and Selectivity Influence of Polar Modifier
Retention increases with decreasing solvent polarity
90:10 ACN:H2O
90:5:5 ACN:H2O:MeOH
90:5:5 ACN:H2O:EtOH
90:5:5 ACN:H2O:IPA
10 mM ammonium acetate with 0.02% acetic acid
Analytes: 1. Methacrylic acid 2. Cytosine 3. Nortriptyline 4. Nicotinic acid
©2016 Waters Corporation 28
Outline
Retaining polar compounds in HILIC
– Mobile phase preparation
– System setup
– Tips and tricks
– Method development
o Screening approach
o Optimization
o Rapid method development summary
– Solid-core HILIC columns - What is the advantage?
©2016 Waters Corporation 29
pH 3
ACQUITY UPLC® BEH HILIC 2.1 x 50 mm, 1.7 µm
Op
tim
izat
ion
pH 9
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)
HILIC Screening Strategy
©2016 Waters Corporation 30
Retention and Selectivity: Influence of Stationary Phase
BEH amide (ln k)
BEH
HIL
IC (
ln k
)
-3.0
-2.0
-1.0
0.0
1.0
2.0
3.0
-2.0 -1.0 0.0 1.0 2.0 3.0
r2 = 0.5250
Stationary phase has larger influence on selectivity than
mobile phase
©2016 Waters Corporation 31
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)
O
O
P
OHCH3
CH3
CH3
Isopropyl methylphosphonic acid (IMPA)
O
O
P
OHCH3
CH3
Ethyl methylphosphonic acid (EMPA)
Implementing the Approach: Organophosphonic Acids
©2016 Waters Corporation 32
Stationary Phase Selectivity: Organophosphonic Acids at Low pH
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
0.00 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
3.32e6
1: SIR of 5 Channels ES-
TIC
3.32e6
1
2,3
45
1
4,5
1
2,3
4 5
BEH Amide
BEH HILIC
Atlantis HILIC Silica
Minutes
pH 3
Atlantis HILIC Silica yields greatest retention
BEH Amide and Atlantis HILIC Silica yield similar
selectivity
©2016 Waters Corporation 33
Compounds 1. PMPA 2. CMPA 3. MMPA 4. IMPA 5. EMPA
Stationary Phase Selectivity: Organophosphonic Acids at High pH
pH 9
Greater Resolution for BEH Amide
No resolution between
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 2,3
4 5
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
2,3
4
5
BEH HILIC
©2016 Waters Corporation 34
1 • Adjust gradient slope
2 • Adjust column temperature
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]
Method Optimization Steps
©2016 Waters Corporation 35
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 2,3
4 5
99.9% to 0.1% B in 5 min
SIR of 5 Channels ES- TIC
3.32e6
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
1 SIR of 5 Channels ES-
TIC 4.18e6
99.9% to 50% B in 5 min
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
1 2
3
4 5
99.9% to 90% B in 5 min
Compounds 1. PMPA 2. CMPA 3. MMPA 4. IMPA 5. EMPA
Optimization Step 1: Adjust Gradient Slope
BEH Amide, pH 9
Shallower gradient slope results in
improved resolution
©2016 Waters Corporation 36
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
2 1
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
2 1
3 4
5
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
1 2
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
Optimization Step 2: Column Temperature
BEH Amide, pH 9 Shallow Gradient
Increased
temperature results in improved resolution
©2016 Waters Corporation 37 Minutes
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
2 1
3
4
5
SIR of 5 Channels ES- TIC
2.1 x 50 mm
SIR of 5 Channels ES- TIC
2.1 x 100 mm Compounds 1. PMPA 2. CMPA 3. MMPA 4. IMPA 5. EMPA
Optimization Step 3: Column Length
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
method criteria
©2016 Waters Corporation 38
1 • Adjust gradient slope
2 • Adjust column temperature
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]
Method 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
©2016 Waters Corporation 39
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
©2016 Waters Corporation 40
Outline
Retaining polar compounds in HILIC
– Mobile phase preparation
– System setup
– Tips and tricks
– Method development
– Solid-core HILIC columns
o What is the advantage?
o Application examples
– Summary
©2016 Waters Corporation 41
Outline
Retaining polar compounds in HILIC
– Mobile phase preparation
– System setup
– Tips and Tricks
– Method Development
– Solid core HILIC columns- what is the advantage?
– 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
©2016 Waters Corporation 42
CORTECS Solid-Core 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
CORTECS Solid-core
dcore = 1.1 µm
dp =
1.6
µm
Rho, r = 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
©2016 Waters Corporation 43
Higher Efficiency with Solid Core Columns
19,700
14,150
4,000
8,000
12,000
16,000
20,000
0.00 0.25 0.50 0.75 1.00 1.25
Pla
tes (
4 s
igm
a)
Flow Rate (mL/min)
39% higher efficiency
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 30oC with 0.5 µL injections from a 1 µL FL injector
©2016 Waters Corporation 44
Higher Efficiency: Sharper Peaks, Better Resolution
AU
0.00
0.05
0.10
0.15
0.20
AU
-0.05
0.00
0.05
0.10
0.15
0.20
Minutes
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00
1
2
3
4
5 ACQUITY BEH HILIC 2.1 x 50mm 1.7 µm
CORTECS UPLC HILIC 2.1 x 50 mm 1.6 µm
1
2
3
4
5
1. Lidocaine 2. Butacaine 3. Tetracaine 4. Procaine 5. Procainamide
USP Resolution2,3: 2.2
USP Resolution2,3: 1.2
©2016 Waters Corporation 45
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
AU
0.00
0.02
0.04
0.06
Minutes 0.00 2.00 4.00 6.00 8.00 10.00
AU
0.00
0.02
0.04
0.06
AU
0.00
0.02
0.04
0.06 1 2
5
3
4
1 2
5
3
4
1
2
5
3
4
CORTECS UPLC 1.6 µm HILIC
BEH HILIC, 1.7 µm
BEH Amide, 1.7 µm
Different Selectivity for HILIC Materials
©2016 Waters Corporation 46
ACh
HA
t-MIAA
t-MHA
iso-ACh Ch
Analysis of Neurotransmitters in Artificial CSF using CORTECS HILIC
HA: Histamine t-MHA: tele-methylhistamine t-MIAA: tele-methylimidazoleacetic acid ACh: Acetylcholine Ch: Choline iso-ACh: iso-Acetylcholine
CORTECS UPLC HILIC 2.1 x 100 mm 1.6 µm ACQUITY UPLC with Xevo TQ-S MS
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.
©2016 Waters Corporation 47
Analysis of Basic Drugs in Surface Water
Time 2.00 4.00 6.00 8.00 10.00 12.00
0
2.00 4.00 6.00 8.00 10.00 12.00
Faster Analysis 1. Cimitidine 2. Clenbuterol 3. Albuterol 4. Metformin 5. Ranitidine
Better Resolution Metformin/ranitidine
1
2 3
4 5
CORTECS UPLC HILIC
Atlantis HILIC
©2016 Waters Corporation 48
Higher Resolution: Diquat/Paraquat in Drinking Water
©2016 Waters Corporation 49
Summary
For RP
– Try elevated pH first- largest degree of selectivity change
– Low coverage C18 (HSS T3) next
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)
©2016 Waters Corporation 50
Conclusions
For HILIC retention and selectivity:
– ACN is used the primary [weak] solvent in HILIC
– Water, methanol, ethanol or isopropanol are strong [elution] solvents
– Stationary phase charge and bonded phase can impact retention and
selectivity
– Analytes in their charged form exhibit greater retention [acids at
high pH, bases at low pH]
Practical considerations:
– At least 10 mM buffer or 0.2% additive is recommended in mobile
phase A and B
– Sample diluent should contain at least 75% acetonitrile for solubility
and peak shape
– Weak needle wash must be in a high organic solution [90 – 95%
ACN]
©2016 Waters Corporation 51
HILIC Primer
HILIC primer [715001940]
– Comprehensive, 72-page Guide to
Hydrophilic Interaction
Chromatography [HILIC]
– Education is the key to success with
this technique
©2016 Waters Corporation 52
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
©2016 Waters Corporation 53
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
©2016 Waters Corporation 54
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, Diehl, 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
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
©2016 Waters Corporation 55
THANK YOU
Live Q&A Session
www.waters.com/hilic