Part 2: Polar Compound Retention...Stationary phase is a POLAR material —Silica, hybrid, cyano,...

©2016 Waters Corporation 1

Part 2: Polar Compound Retention

Tuesday October 18, 2016 – 3 pm CET / 9 am EST

Considerations for Successful

HILIC Separations


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?


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.


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


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


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


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

http://www.waters.com/amide


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


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]


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


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


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.


Before You Start: Mobile Phase Preparation

Additives

– Replace 0.2% of mobile phase volume with additive [2 mL out of 1 L]

Buffers

– Prepare a stock buffer [typically 200 mM] and then dilute 20-fold into

the running mobile phase [10 mM concentration on column]

Example: Prepare stock of 200 mM ammonium formate, pH 3.

For a mobile phase containing 95% ACN and 5% water with 10 mM

ammonium formate, pH 3, add 50 mL of stock buffer to 950 mL of

ACN.

For the best gradient performance and reproducibility, it is

recommended that the additive or buffer be added to both

aqueous and organic mobile phase bottles


Screening Conditions: 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


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


Outline



– System setup

– Tips and tricks

o Injection solvent

o Buffers/additives

o Tuning the mobile phase for more retention


– Solid core HILIC columns- What is the advantage?


Influence of Needle Wash Solvent

1

3 4

2

1

3 4,2

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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


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


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Sample Diluent Considerations: Water as Polar Solvent

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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


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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


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


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.


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1.00

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1.00

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0.00

1.00

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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


N

NH

O

NH2

CytosinepKa = 12.2

NHCH3


O

N

OH



Effect of Buffer Concentration: pH 9.0

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1.00

0.00

1.00

0.00

1.00

0.00

1.00

0.00

1.00

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3

1 2

4

3

13

4

2

1

3 4

2

3

1

4

4

2

1,2

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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



2.5 mM ammonium

acetate

O

CH2OH

CH3


N

NH

O

NH2

CytosinepKa = 12.2

NHCH3


O

N

OH



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

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3

2

4

1

2 4

1

20 mM ammonium formate pH 3

0.2% formic acid pH 3 3



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


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


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


Outline



– System setup

– Tips and tricks


o Screening approach

o Optimization

o Rapid method development summary

– Solid-core HILIC columns - What is the advantage?


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


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


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


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


TIC

3.32e6


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


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


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


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


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



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


5.12e6

1 2

3

4 5


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


2.1 x 50 mm


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


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


Rapid HILIC Method Development

Screening approach Time c

Column conditioning* 30 minutes

3 Columns, 2 pH’s screening 30 minutes

Optimization

Column conditioning [temp. equilibration] 30 minutes

Gradient slope and temperature 30 minutes

Total method development time 2 Hours

*equilibration and 2 blank injections at each pH


Outline



– System setup

– Tips and tricks


– Solid-core HILIC columns

o What is the advantage?

o Application examples

– Summary


Outline



– 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?


– Solid-core HILIC columns - what is the advantage


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


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


Higher Efficiency: Sharper Peaks, Better Resolution

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0.15

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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


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

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5

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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


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.


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


Higher Resolution: Diquat/Paraquat in Drinking Water


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)


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]


HILIC Primer

HILIC primer [715001940]

– Comprehensive, 72-page Guide to

Hydrophilic Interaction

Chromatography [HILIC]

– Education is the key to success with

this technique


HILIC Method Development Wall Chart

HILIC method development

wall chart [720003484en]

– Efficient Hydrophilic Interaction

Chromatography [HILIC] Method

Development Strategy

– Reiterates key messages in the

HILIC method development

seminar

– www.waters.com/HILIC

http://www.waters.com/HILIC


Amide Column Selector: Sugar Analysis

www.waters.com/amide

• Carbohydrate method selection tool

• Link to BEH amide application notebook

• Link to ACQUITY UPLC column brand page

http://www.waters.com/amide


Other literature references

Peer reviewed publications – Monoamine neurotransmitters

o Danaceau JP, Chambers EE, Fountain KJ. Bioanalysis. 2012 Apr;4(7):783-94.


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


– 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


– Polar Basic Drugs in Environmental Samples

http://www.waters.com/waters/library.htm?cid=511436&lid=134817340







http://www.waters.com/waters/library.htm?cid=511436&lid=134817340


THANK YOU

Live Q&A Session

www.waters.com/hilic

Part 2: Polar Compound Retention...Stationary phase is a POLAR material —Silica, hybrid, cyano,...

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