understanding (and creating) polar retention using ... · pdf fileusing reversed-phase hplc...

©2003 Waters Corporation

Understanding (and Creating) Polar Retention Using Reversed-Phase HPLC and Hydrophilic

Interaction Chromatography

Pittcon March 2003


Introduction

• Introduction• The Problem

•Background

•Hydrophilic Interaction Chromatography

•Reversed-Phase HPLC for Polar Molecules

•Applications

•Summary


Introduction - The Problem

•Conventional HPLC columns often do not provide adequate retention and separation of highly polar compounds

•Drug Discovery• Gradient - High-throughput screening (HTS) does not

work for polar compounds• Elute un-retained in the void volume• Co-elute at the beginning of the run – if retained at all

•Method Development• Isocratic - Many RP columns cannot be run under the

100% aqueous conditions necessary for polar compound retention– columns “dewet” (also termed -hydrophobic collapse)


Background

• Introduction•Background

• Polar molecules• Chromatographic methods for retaining polar

compounds

•Hydrophilic Interaction Chromatography•Reversed-Phase HPLC for Polar Molecules•Applications•Summary


What is a Polar Molecule?

• General chemistry definition:• A molecule whose centers of positive and negative charges do not

coincide• The degree of polarity is measured by the dipole moment of the

molecule

• Dipole moment is the product of the charge at either end of the dipole times the distance between the charges

H

O

Hδ+

2δ-

δ+ Dipole moment = 1.85 D

This is a Polar Molecule!

Electro-static attraction – opposite charge locations on different molecules attract


Examples of Polar Molecules

H N

NH

O

O

Uracil (N)

H N

NH

O

O

Thymine (N)

O

Acetone (N)

N

NH

NH2

O

Cytosine (B)

H N

H NF

O

O

OH O

5-Fluoroorotic acid (A)

OH

OH

NH

OH

Epinephrine (B) Ascorbic acid (A)

O

OH OH

OOHOH


Comparison of Chromatographic Methods for Retention of Polar Compounds

• Dewetting under aqueous conditions• Poor retention of polar compounds with non-polar stationary phases

• Familiar technique• High efficiency• Rapid equilibration • Wide selection of columns

Reversed-Phase

• Long equilibration times• Difficult to run gradients• Most ion pairing agents not compatible with MS• Difficult method development

• Can retain any ionizable compound• Can perform ion pairing chromatography and reversed-phase chromatography on same column

Ion Pairing

• Sample and mobile phase solubility problems• Long re-equilibration times

• High percentage organic mobile phases give higher sensitivity in ESI-MS

HILIC

• Does not work well if the compound does not ionize• High salt buffers or pH gradients for elution – not compatible with MS

• Can retain any ionizable compoundIon

Exchange

DisadvantagesAdvantages


Reversed-Phase Chromatography for Polar Compound Retention

• Reversed-Phase Chromatography• Non-polar stationary phase with >80% aqueous mobile phases• Strengths

• Familiar, well understood technique• Many stationary phase choices• Good reproducibility, stable equilibration• High efficiency

• Weaknesses of modern C18 phases designed for improved peak shape for basic analytes: polar compound retention

• Sudden loss of retention in 100% aqueous mobile phase• Poor retention of polar analytes on a high coverage, non-polar C18

stationary phase


Hydrophilic Interaction Chromatography for Polar Compound Retention

• HILIC – Hydrophilic Interaction Chromatography• Polar stationary phase with >80% organic mobile phases• Strengths

• Good retention of very polar molecules• Volatile mobile phases

• Increased ESI-MS sensitivity• Lower backpressure

• Complementary selectivity vs reversed-phase• Weaknesses

• Not many stationary phases available• Poor retention of hydrophobic compounds


Hydrophilic Interaction Chromatography

• Introduction

• Background• Hydrophilic Interaction Chromatography (HILIC)

• What is it?• Why would someone use it?• AtlantisTM HILIC Silica

• Reversed-Phase HPLC for Polar Molecules

• Applications

• Summary


HILIC: What is it?

• HILIC – Hydrophilic Interaction Chromatography• “Reverse reversed-phase” (water is the eluting solvent)• Very polar analytes are retained and separated on a polar,

hydrophilic stationary phase• Analytes elute in order of increasing hydrophilicity• High organic/low aqueous conditions

• Mobile phases used• >70% ACN (with water/buffer) • Very volatile (very “ESI-MS-friendly”)

• Stationary phases• SiO2, diol, amino, amide, cyano, ion-exchange, SEC,

Zwitterion, etc.


HILIC: What is it?

Polar Phase

HILIC Retention• Polar analyte partitions into and out of adsorbed water layer

• The larger the water layer (greater aqueous concentration), the faster the compound(s) will elute

• Analytes elute in order of increasing hydrophilicity

• High Acetonitrile (>70%) with aqueous

• Polar Phases (Bonded and Unbonded)

HN

NH

O

O

HN

NH

O

O

OH

80% acetonitrile

20% aqueous,(e.g., pH 3.0)

uracil


HILIC: What is it?

Polar Phase

Weak Cation ExchangeRetention

• Charged polar analyte can undergo cation exchange with charged silanol groups

• High Acetonitrile (>70%) with aqueous

O-

80% acetonitrile

20% aqueous, (e.g., pH 5.5)

HILIC is defined as the high organic conditions

where polar compounds are retained longer

(reverse reversed-phase)

N

NH3+

NH

O

N

NH3+

NH

O

cytosine


HILIC Retention Characteristics

Cytosine

HILIC:Increased retention occurs when using

greater than 70% organic for polar bases

N

NH

O

NH2

-3.00

-2.00

-1.00

0.00

1.00

2.00

3.00

0 10 20 30 40 50 60 70 80 90% MeCN

LN (T

rmin

/cm

)

pH 5

pH 4

pH 3

Aqueous

Retention


-2.90-2.80-2.70-2.60-2.50-2.40-2.30-2.20-2.10-2.00

0 10 20 30 40 50 60 70 80 90% MeCN

LN (T

rmin

/cm

) pH 5

pH 4

pH 3

Reversed-Phase Retention Characteristics

Cytosine

Reversed-phase:Increased retention occurs when using less than 20% organic for polar bases

N

NH

O

NH2

Aqueous

Retention


HILIC:Why would someone use it?

• Why would someone use HILIC?• Retains very polar compounds not retainable by RP• Volatile mobile phase

• Increased ESI-MS sensitivity (lower LODs)• Increased flow rates• Increased sample throughput• Lower backpressures

• Facilitates sample prep (SPE, liquid/liquid, protein precipitation) as the final elution/extraction solvent does not have to be evaporated/reconstituted


Vo = 1.15 min

AU

0.00

0.05

0.10

0.15

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

AtlantisTM HILIC Silica4.6 x 50 mm, 3 µm

95:5 ACN:Formate Buffer, pH 3.01.4 mL/min

AtlantisTM HILIC Silica: Retains very polar compounds not retainable by RP

AU

0.00

0.05

0.10

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

AtlantisTM dC184.6 x 50 mm, 3 µm

100% Formate Buffer, pH 3.0 1.0 mL/min

Vo = 0.65 min

NH

NHNH

O

OO

NH2

Allantoin

HILIC offers retention when there is no retention by reversed-phase

k’= 1.0

k’= 0.0


AtlantisTM HILIC Silica : Enhanced ESI-MS Sensitivity

AtlantisTM dC18 Peak Area2.1 x 50 mm, 3 µm1. Albuterol 100 pg/µL 782. Bamethan 20 pg/µL 2

AtlantisTM HILIC Silica Peak Area2.1 x 50 mm, 3 µm2. Bamethan 20 pg/µL 93871. Albuterol 100 pg/µL 13131

HILIC requires high volatility solvents which increase ESI-MS sensitivity vs. high-aqueous mobile phases used in RP

7.63e3

0

100

%

1.802.13

ES+

239.8

1.07e5

209.90

100

%

1.80

2.13

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

0

100

%

1.311.63

ES+

239.8

1.07e5

209.9

1

2?

12


AtlantisTM HILIC Silica :Simplify Sample Preparation

Condition/Equilibrate*200 µL methanol/200 µL water

Load150 µL spiked plasma sample

150 µL internal standardwith 2% ammonium hydroxide

Wash200 µL 5% methanol in water

Elute300 µL 40% acetonitrile/60% isopropanol

with 2% formic acid

*Oasis® HLBµElution Plate

Inject eluent directly onto columnNo Evaporation and Reconstitution Step


AtlantisTM HILIC Silica :Simplify Sample Preparation

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00Time

0

100

%

SIR of 2 Channels ES+TIC239.8209.98.97e4

1.49 1.84

NHOH

OH

CH3

Bamethan

NH

OH

OH

OHCH3 CH3

CH3

Albuterol

AtlantisTM HILIC Silica2.1 x 50 mm, 3 µm1. Bamethan 10 pg/µL 2. Albuterol 50 pg/µL

1 2

SPE eluent injected directly onto AtlantisTM HILIC Silica column


Atlantis HILIC SIlica:Summary

• AtlantisTM HILIC Silica• Provides retention of some polar analytes not retained under

RP conditions• Enhanced sensitivity in ESI-MS is enjoyed due to the highly

volatile mobile phases (> 80% organic) used• Sample preparation procedures are shortened by eliminating

the evaporation and reconstitution steps and directly injecting the eluent

• AtlantisTM HILIC Silica columns are an alternative to RP chromatography for the retention of very polar basic analytes

New at Pittcon 2003


Reversed-Phase HPLC for Polar Molecules

• Introduction

• Background

• Hydrophilic Interaction Chromatography

• Reversed-Phase HPLC for Polar Molecules• The objective and challenge• Dewetting (not hydrophobic collapse)• What doesn’t work (and why)• AtlantisTM dC18 - an intelligent solution

• Applications

• Summary


The Challenge for Stationary Phase Manufacturers

•The Objective• Create a stationary phase that retains all analytes,

gives good peak shape for bases, and is compatible with all LC detectors

•The Challenge• To retain a hydrophilic (polar) analyte on a

hydrophobic (non-polar) stationary phase

Why is this so difficult?


Reversed-Phase Chromatography

O-SiO-SiOHO-SiO-SiOHO-SiOHO-SiO-SiO-SiOH

O-SiO-Si

Non-polarstationary

phase

Polar mobilephase

Analyte XPolarAnalyte Y

Non-Polar

Chromatographically how does oneattempt to force polar Analyte X to retain on

the non-polar stationary phase?

Flow

PoorlyRetained

(like attracts like)

Well retained(like attracts like)


Challenge for Reversed-Phase Chromatography of Polar Compounds

• To retain polar compounds on this non-polar surface we reduce the amount of organic in the mobile phase(i.e., make mobile phase weaker, e.g., 100% aqueous)

• PROBLEM: Risk of dewetting (hydrophobic collapse) the particle surface – the chromatographic pores dry-out (non-polar pore surface expels the pure aqueous, polar mobile phase)

What is “dewetting” and why does it happen?


Minutes0 2 4 6 8 10

Initial(Column was wetted first

with organic)

After Flow Stoppage (Pores “dewet” 100%)

Mobile phase: 0.1% Acetic Acid

Amoxicillin

Vo: No retention of analyte

1,500psi

Note: Column is not broken --It just stopped working

Why?

1,500psi

Conventional C18 Column – Dewetting


Pore Dewetting Mechanism

Flow stoppage relieves the pressure that was forcing the aqueousmobile phase into the pores. When pressure is reduced (e.g., pump stopped), the hydrophobic pore surface can expel the polar mobile phase and “dewet” the pore.

At flow, with pressure on the mobile phase.

Stopped flow with no pressure on the mobile phase.

Pores de-wet – restart flow – pores still dewetted and analytes never enter pores – resulting in no retention.

Analytes

Analytes properly retained

Remember: Most of the surface area (> 95%) is inside the silica pores!


Water on C18:d = 100Åγ = 72.8 dynes/cmθ = 110.6°*

*B. Janczuk, T. Bialopiotrowicz and W. Wojcik, Colloids and Surfaces 36 (1989) 391-403

Methanol on C18:d = 100Åγ = 22 dynes/cmθ = 39.9°*

Pc < 0 psi

Pc = 1,500 psi

Note: Self-wetting – No pressure required

γEquation of Young and Laplace

γγ=

4 θPc =4 θd

θcos

Pressure on C18 pore requiredto force water back into the pore

Where:Pc = Capillary pressureγ = Surface tensiond = Capillary diameterθ = Contact angle

Stationary Phase Wetting

Solvent dependent

H2O

MeOH

θ

θ

Contact Angle


Re-wetting a Stationary Phase

• Use a mobile phase containing > 40% methanol or other polar organic solvent (other organic solvents may vary in % required for wetting)

• This works by reducing the contact angle (θ)

• The use of pressure alone cannot force aqueous mobile phase back into silica pores

• Not practical because column outlet is at atmospheric pressure – not all pores see required pressure (1,500 psi)

1,500 psi 1 atm = 14.7 psi

Pressure gradient inside column

1,500 psi

Atmospheric pressure


Dewetting Summary

•Dewetting• Waters research suggests that dewetting - not

“hydrophobic collapse” is the reason for sudden loss of retention under highly aqueous conditions

• The observed reduction in V0 and sudden loss in retention after a pressure drop indicate that the pores expel the aqueous solvent (vs. ligand chains “folding” or “matting” as described by hydrophobic collapse)

• Rewetting with an organic solvent can rewet the pores by reducing the contact angle and surface tension

Ideally, it is best to avoid dewetting altogether.How have chromatographers

attempted to do this?


Embedded Polar Groups –What Doesn’t Work

• Embedded polar groups were designed to: • Improve peak shapes for basic compounds • Provide complementary selectivity as compared to straight-

chain alkyl stationary phases

• Examples of embedded polar groups include carbamate, ether, amide, urea, etc.

• Another feature of embedded polar groups is 100% aqueous mobile phase compatibility

This feature is confused with enhanced polar compound retention

Why?



• Embedded polar groups resist pore dewetting by increasing the water layer at the surface of the pores

Embedded polar group behaves

like a “Polar Hook” –holding onto water.

Polar compound retention requires these very

weak polar mobile phasesi.e., highly aqueous mobile phases

with little-or-no organic modifier

This aqueous compatibility is confused with polar compound retention.

This is false!

©2003 Waters CorporationCarignan, Iraneta


Adenine

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

12

34

5

Compounds1. Thiourea2. 5-Fluorocytosine3. Adenine4. Guanosine-5’-monophosphate5. Thymine

ConditionsColumn: AtlantisTM dC18 4.6 x 150 mm, 5 µm Isocratic Mobile Phase: 10 mM NH4COOH, pH 3.0Flow Rate: 1.2 mL/minInjection Volume: 7.0 µLTemperature: AmbientDetection: UV @ 254 nm

Vo = 1.5minAtlantis dC18 100% Aqueous

QC Batch Test

Thiourea

N

NOH

FNH2

5-Fluorocytosine

NH

NH

O

O

Thymine

N

NH N

N

NH2

Guanosine-5’-monophosphateH2N NH2

SOP

NH

N

N

O

NH2N

O

OHOH

HHH

HO

H

O

OH

For highly polar compounds, embedded polar groups actually cause less retention (see next slide)



Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

12

3 4 5Vo = 1.5 min

AtlantisTM dC18

Minu tes1 .00 2 .00 3 .00 4 .00 5 .00 6 .00 7 .00 8 .00 9 .00 10 .00 11.00 12 .00 13 .00 14. 00 15 .00

1 2

34

5Vo = 1.2min Column Z BR

(amide)

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

1 23

4 5Column I OEVo = 1.2min

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

1 2 34 5

Column P SPVo = 1.3min



Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

12

3 4 5Vo = 1.5 min

AtlantisTM dC18

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

1 2

34

5Column S AZ

(amide)Vo = 1.3min

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

12 3

45

Column M PCVo = 1.3min

1 234

5 Column W XR

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00

Vo = 1.3min


Embedded Polar Groups –What Doesn’t Work & Why

Why less retention of polar compounds with embedded polar group columns?

Reason 1Water layer“shields”

silanols and reduces cationic

interactions and H-

bonding

This “water shield” results in less retention of polar compounds -

Both reasons given also describe the reasons for the

excellent peak shape observed for bases

+

N

N

N

NH

NH 3

Reason 2Shorter alkyl chain

length for in order to obtain higher ligand

density


Embedded Polar Groups –What Doesn’t Work - Summary

•Embedded polar groups – summary• Embedded polar groups were designed for

improved peak shape• Embedded polar groups provide aqueous

compatibility• For some compounds, embedded polar groups

can provide increased retention (e.g.,polyphenolics)

• For highly polar compounds, however, embedded polar groups cause less retention

Now that we’ve defined what is not an ideal column for polar retention, let’s define what an ideal column is for polar compound retention.


The Ideal Column for Retention of Polar Compounds

•Retains and separates polar compounds•Compatible in 100% aqueous mobile phases –

i.e. does not dewet•Stable under acidic conditions•Compatible with all detectors•Enhanced polar compound retention without

extreme non-polar compound retention (a balance)

•Excellent peak shape and column-to-column reproducibility


AtlantisTM dC18 Intelligent Design

Intelligent Design of the Chromatographic Particle

We can: Optimize Silica-gel and Bonding Chemistry:* Pore size * Ligand type * Ligand density * Endcapping

Resulting in: Desired peak shape, dewetting, retention and stability characteristics

Liganddensity

Endcapping

Ligandtype

Poresize

DewettingPeak Shape

RetentionStability


AtlantisTM dC18 Attributes

•What are the key attributes of AtlantisTM dC18?• Superior retention of polar compounds• Operates and thrives in 100% aqueous mobile phases• Excellent peak shape with Mass Spectrometry (MS)

compatible mobile phases• Difunctional bonding chemistry (dC18) results in:

• Extended column lifetime• Enhanced low pH stability

• LC/MS compatible with ultra-low column bleed• Enhanced polar compound retention without infinite

non-polar compound retentionExamples of each attribute will be given


Superior Retention of Polar Compounds

Time (min)0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

1

2

34

5

1 2 3

45

Time (min)

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

Conventional C18

AtlantisTM dC18ConditionsColumns: 4.6 x 150 mm, 5 µm Mobile Phase: 10mM NH4COOH, pH 3.0Flow rate: 1.2 mL/minInjection volume: 7 µLDetection: UV@254 nm

Compounds1. Thiourea2. 5- Fluorocytosine3. Adenine4. Guanosine-5’- monophosphate5. Thymine

Aqueous Separation of Polar Compounds for AtlantisTM vs. Conventional C18 Column

Vo = 1.5 min

Peak 1 elutesIn the void

35% - 60% more retention with

AtlantisTM dC18 ascompared to

high coverage C18


Operates and Thrives in 100% Aqueous Mobile Phase

• AtlantisTM was designed to resist the sudden loss of retention associated with pore dewetting.

AtlantisTM dC18 Columns Dewetting Test

Retention after stop flow test(Pore dewetting: ~100%)

Initial Retention

Initial Retention

Time (min)2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

Vo

Retention after stop flow test(Pore dewetting: ~ 3.5%)

Conventional C18

AtlantisTM dC18

Amoxicillin retention with 0.1% formic acid mobile phase

100% retention loss with conventional C18

<4% retention loss with AtlantisTM dC18

Note enhanced retention

Note change in V0in dewetted column


Excellent Peak Shape Using AtlantisTM dC18with MS Compatible Mobile Phases

Note excellent peak shape for strong polar

base Adenine at pH 5.0

Compounds:1. Cytosine2. 5-Fluorocytosine3. Uracil4. 5-Fluorouracil5. Guanine6. Thymine7. Adenine

1

2 3

4

5 6

Minutes

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

7

V0 = 1.83 min

ConditionsColumn: AtlantisTM dC18 4.6 x 150 mm, 5 µm Mobile Phase A: H20Mobile Phase B: ACNMobile Phase C: 100 mM CH3COONH4, pH 5.0Flow Rate: 1.0 mL/minGradient: Time Profile

(min) %A %B %C0.0 90 0 10

10.0 84 6 10Injection Volume: 10 µLTemperature: 30 oCDetection: UV @ 254 nmInstrument:: AllianceTM 2695, 2996 PDA

Grumbach, Diehl

The popular void marker Uracil is well retained and is actually peak 3

At pH 5.0, the strong polar base Adenine still exhibits excellent peak shape

(reason: fully endcapped)


Benefit of a Fully Endcapped Column

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

12

34

5

1

2 4 5

3

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

ConditionsColumns: 4.6 x 150 mm, 5 µm Mobile Phase: 10 mM NH4COOH, pH 3.0Flow Rate: 1.2 mL/minInjection Volume: 7 µLDetection: UV @ 254 nm

Compounds1. Thiourea2. 5-Fluorocytosine3. Adenine4. Guanosine-5’-monophosphate5. Thymine

Unendcapped “AQ-Type”column

AtlantisTM dC18 column

Longer base retention at the expense of peak shape

Excellent retention and peak for all compounds


Extended Column Lifetime and Low pH Stability

• Difunctional C18 bonding chemistry results in: • Extended column lifetime• Enhanced low pH stability

Accelerated Low pH Degradation Test(30oC with 0.1% TFA)

1 20 50 100 200 300 700 800 900 950 990

Injection Number

% O

rigin

al

Ret

entio

n Ti

me

60

65

70

75

80

85

90

95

100

105

60

65

70

75

80

85

90

95

100

105

Thymine - AtlantisTM dC18

Thymine - Monofunctional C18

What is a difunctionalbond and why does it

provide longer column lifetimes at low pH?


Hydrolysis of a Bonded Phase Material:Limitations of Monofunctional Ligands

+ HCl

+

+

Hydrolysis at low pH(monofunctional bond broken)

SiC

CC

CC

CC

CH3

CH3

H3C

Cl

OHSi

O

O

O

SiC

CC

CC

CC

CH3

CH3

H3C

OSi

O

O

O

SiC

CC

CC

CC

CH3

CH3

H3C

HOOH

SiO

O

O

Monofunctional = Attached to silica at one pointMore susceptible to low pH hydrolysis (column bleed)

Synthesis (monofunctional bond created)

Single siloxane bond

C8 Monochlorosilane Ligand


Making a Bonded Phase Material: Multifunctional Synthesis

+

+ HCl

C8 Dichlorosilane LigandOH

OH

OHSi

SiSi

O

OO

O

O OO

ClSi

Cl

CH3CH3

OS i

O

OH

Si

SiSi

O

OO

O

O OO

CH3

CH3

Difunctional = Attached to silica at two pointsLess susceptible to low pH hydrolysis (column bleed)

Synthesis

Two siloxane bonds


Enhanced Polar Compound Retention without Infinite Non-polar Compound Retention

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

12

3 4 5

10

98

7

6

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

12 3

45 6

7

10

98V0 = 0.98 min

AtlantisTM dC18

Conventional C18with 2x ligand

density

More retention for polar compounds

Equal or less retention for non-polar compounds

Void Marker

Back Marker

V0 = 0.98 min


High Coverage

– High Ligand Density

Low Coverage –Low Ligand

Density

H

SiO

O

OSi

O

O

OSi

O

O

OSi

O

O

OSi

O

O

OSi

O

O

OSi

O

O

OSi

O

O

OSi

O

O

OSiO

OO

SiO

O

O

SiCH2

H3C CH3

H2CCH2

H2CCH2

H2CCH2

H3C

SiCH3

H3C CH3 SiCH2

H3C CH3

H2CCH2

H2CCH2

H2CCH2

H3C

SiO

O

O

SiCH3

H3C CH3 SiCH3

H3C CH3H

HHHHH H

C8 alkyl chains

Residual silanolsEndcap

Polar analytes are not able to “energetically fit” between ligands –can’t interact with surface or ligands

Si

SiO

O

OSi

O

O

OSi

O

O

OSi

O

O

OSi

O

O

OSi

O

O

OSi

O

O

OSi

O

O

OSi

O

O

OSiO

OO

SiO

O

O

SiCH2H3C CH3

H2CCH2

H2CCH2

H2CCH2

H3C

SiCH2H3C CH3

H2CCH2

H2CCH2

H2CCH2

H3C

CH2H3C CH3

H2CCH2

H2CCH2

H2CCH2

H3C

SiCH2

H3C CH3

H2CCH2

H2CCH2

H2CCH2

H3C

SiO

O

O

SiCH3H3C CH3 Si

CH3H3C CH3

HHHHHH H

Residual silanolEndcap

Polar analytes easily interact with surface

and ligands

Atlantis dC18 - An Intelligent DesignWhy Does It Work?

Adapted from J.G. Dorsey and K.A. Dill, Chem. Rev., 1989, 89, 331-346


Low Coverage –Low Ligand

Density

H

SiO

O

O

SiO

O

O

SiO

O

O

SiO

O

O

SiO

O

O

SiO

O

O

SiO

O

O

SiO

O

O

SiO

O

O

Si

O

OO

SiO

O

O

Si

CH2H3C CH3

H2CCH2

H2C

CH2

H2C

CH2

H2C

Si

CH3H3C CH3 Si

CH2H3C CH3

H2CCH2

H2C

CH2

H2C

CH2

H2C

SiO

O

O

Si

CH3H3C CH3 Si

CH3H3C CH3

H

HHHHH H

Alkyl chains

Residual silanols

Endcap


N

NH

NH2

O

Polar portion of analyte interacts

with silanols(hydrogen bonding

and/or ion exchange)

NH

NH

O

ONon-polarportion of

analyte interacts with bonded phase(hydrophobic)



• 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

• Polar analytes can “fit” between C18 ligands and interact with surface silanols and alkyl chains

• Secondary interactions due to residual silanols that are more accessible due to reduced C18 coverage

• Cation-exchange interactions• Hydrogen bonding interactions


Atlantis dC18 - An Intelligent DesignExcellent Batch-to-Batch Reproducibility

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

Eight Actual QC Batch Test Chromatograms Under 100% Aqueous Conditions

(not specially packed columns)

%RSD 0.16 1.18 1.39 1.92 1.96


Applications

• Introduction

•Background



•Applications

•Summary


Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Minutes

1.00 2.00 3.00 4.00 5.00

Complementary Selectivity of HILIC as Compared to Reversed-Phase

Vo = 0.5 min

Compounds:1. Morphine 2. Morphine 3-β-D-glucuronide


1 2


2

1Vo = 0.65 min

O

N

OH

OH

CH3

H

Morphine

Morphine 3-ß-D-Glucuronide


Gradient LC/MS Separation of Cytochrome C Tryptic Digest on AtlantisTM HILIC Silica versus AtlantisTM dC18

0

100

%

204 (T2)261 (T6,11)361 (T18)434 (T21)

678 (T14)

634 (T4)

585 (T8)

779 (T15)

729 (T10) 964 (T19) 1005 (T!2)

Peak AnnotationM/Z (Fragment ID)

779 (T15)

634 (T4)

678 (T14)434 (T21)

964 (T19)

261 (T6,11)204 (T2)

361 (T18)1005 (T!2)

585 (T8)

729 (T10)

0

100

%

0 45

HILIC

RP




Isocratic RP Separation of Catecholamines and Metabolites on Atlantis dC18

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

1

2

34

5

7

6V0 = 1.83 min

ConditionsColumn: AtlantisTM dC18 4.6 x 150 mm, 5 µm Mobile Phase A: H2OMobile Phase B: ACNMobile Phase C: 100 mM CH3COONH4, pH 5.0Flow Rate: 1.0 mL/minIsocratic Mobile Phase Composition: 88% A; 2% B; 10% C Injection Volume: 10 µLTemperature: 30 oCDetection: UV @ 280 nm

Compounds USP Tailing1. Norepinephrine (NE) 1.212. Epinephrine (E) 1.203. Dopamine (DA) 1.214. 3,4-Dihydroxyphenylacetic acid (DOPAC) 1.005. Serotonin (5-HT) 1.106. 5-Hydroxy-3-indoleacetic acid (5-HIAA) 0.977. 4-Hydroxy-3-methoxyphenylacetic acid (HVA) 0.97

No ion-pairing agent necessary!!


Water Soluble Vitamins on Atlantis dC18

Compounds: USP Tailing1. L-ascorbic acid 1.122. Nicotinic acid 1.273. Thiamine 1.204. Pyridoxal 1.135. Pyridoxine 1.046. Folic acid 1.107. Caffeine 1.108. Riboflavin 1.14

Time (Min)1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

12

3

45

87

6

V0 = 1.37 min

ConditionsColumn: AtlantisTM dC18 4.6 x 150 mm, 5 µm Mobile Phase A: 0.1% TFAMobile Phase B: ACNFlow Rate: 1.4 mL/minGradient: Time Profile

(min) %A %B0.0 100 04.0 97 36.0 85 15

15.0 80 20Injection Volume: 10 µLTemperature: 30 oCDetection: UV @ 260 nm

No ion-pairing agent necessary!!


Tryptic Cytochrome C Peptide Separation:TFA Not Necessary

ConditionsColumn: AtlantisTM dC18 4.6 x 50 mm, 3 µm Mobile Phase A: 0.1% HCOOH or 0.02%TFAMobile Phase B: 0.065% HCOOH in ACN or

0.016% TFA in ACNFlow Rate: 0.75 mL/min, 0.2 mL/min Split to MSGradient: Time Profile

(min) %A %B0.0 100 045.0 60 40

Injection Volume: 20 µLInjection Mass: 20 µgTemperature: Ambient

AtlantisTM dC18 columns permit the use of

MS-friendly mobile phase additives


Summary

• Introduction

•Background



•Applications

•Summary


AtlantisTM Columns Summary

• AtlantisTM LC/MS columns are designed for retaining and separating polar compounds

• New AtlantisTM HILIC Silica columns• Retain very polar compounds for high throughput LC/MS• Simply sample preparation• Offer complementary selectivity as compared to RP

• AtlantisTM dC18 columns• Retain polar compounds without extreme retention of non-polar

compounds (universal low pH RP column)• Provide excellent peak shapes with LC/MS compatible mobile

phases • Thrive in aqueous mobile phases• Provide long column lifetimes at low pH


For More Information

•Visit Waters on-line at www.waters.com• Visit AtlantisTM columns microsite and download

brochure, applications notebook, LC/GC App Note,Care & Use, etc.

• C.talk for free on-line educational seminars• Applications database with over 17,000

applications

Thank you for attending today’s seminar!!Thank you for attending today’s seminar!!


For More Information

• Visit us at Booth 2953

• Presentations at Pittcon• Chromatographic Retention of Polar Compounds - What Are

the Options?• Tue AM - Session 800, Room 306AB

• Hydrophilic Interaction Chromatography (HILIC) for Small Molecules: Searching for the Right Stationary Phase

• Thu PM - Session 2370, Room 306AB

Thank you for attending today’s seminar!!Thank you for attending today’s seminar!!

understanding (and creating) polar retention using ... · pdf fileusing reversed-phase hplc...

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