‘tips and tricks’ for retaining and separating polar ... · ‘tips and tricks’ for retaining...

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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 Reversed Reversed-Phase and Hydrophilic Phase and Hydrophilic Interaction Chromatography (HILIC) Interaction Chromatography (HILIC)

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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]

THANK YOU

©2014 Waters Corporation 64

www.waters.com/hilic