understanding (and creating) retention of polar compounds ... · advantages disadvantages ©2004...

©2004 Waters Corporation

Understanding (and Creating) Retention

of Polar Compounds in Reversed-Phase HPLC

Pittcon 2004


Overview

• Introduction– The problem

• Background

• Reversed-Phase HPLC for Polar Compounds

• An Intelligent Solution

• Applications

• Summary


Introduction - The Problem

• Conventional reversed-phase 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 compoundsElute un-retained in the void volumeCo-elute at the beginning of the run – if retained at all

• Method Development– Isocratic - Many columns cannot be run under the 100%

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


Overview

• Introduction

• Background– Polar molecules– Chromatographic methods for retaining polar

compounds

• 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!

“Like attract Like” orPolar compounds attract polar compounds

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

• Sample and mobile phase solubility problems• Often requires long re-equilibration times

• High % organic mobile phases give higher sensitivity in MS•Eliminate evaporation of SPE eluents

HILIC

• 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• 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

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

• 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 techniqueMany stationary phase choicesGood reproducibility, stable equilibrationHigh 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 phasePoor retention of polar analytes on a high coverage, non-polar C18stationary phase


Overview

• Introduction• Background• Reversed-Phase HPLC for Polar Molecules

– The objective and challenge– Dewetting– Embedded polar groups – dispelling the myth

• 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

How does one develop a column that simultaneously retains

and separates analytes of different polarities?

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?


Overview

• Introduction

• Background

• Reversed-Phase HPLC for Polar Molecules– The objective and challenge– Dewetting (not hydrophobic collapse)– Embedded polar groups – dispelling the myth


• Applications

• Summary


Mobile phase mustbe allowed into the pore

in order for chromatographic retention of the analyte

to take place.

Proper Wetting of Bonded Chromatographic Surface

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.

The particles are very porous, like the pores of a

sponge – 99% of chromatographic surface is

inside the pores

Analyte

Mobile Phase


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=

4θPc =

4θ

dθcos

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?


Overview

• Introduction

• Background

• Reversed-Phase HPLC for Polar Molecules– The objective and challenge– Dewetting (not hydrophobic collapse)– Embedded polar groups – dispelling the myth


• Applications

• Summary


Embedded Polar Groups –Dispelling the Myth

• 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!


Carignan, Iraneta

Embedded Polar Groups –What Doesn’t Work

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 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

1 2

34

5Vo = 1.2min Column Z BR

(amide)

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

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

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



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

1 2

34

5Column S AZ

(amide)Vo = 1.3min

12 3

45

Column M PCVo = 1.3min

1 234

5 Column W XRVo = 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

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

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00



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 in order to

obtain higher ligand density



• 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.


Overview

• Introduction• Background• Reversed-Phase HPLC for Polar Compounds• An Intelligent Solution – AtlantisTM dC18

– Designing the Ideal column for polar compounds– AtlantisTM dC18 attributes– Retention mechanisms (Why does AtlantisTM dC18

work?)

• Applications• Summary


Designing the Ideal Column

LigandDensity

Endcapping

LigandType

PoreSizeDewetting

Peak Shape

Retention

Stability

The result: Waters AtlantisTM dC18

Optimization of pore size,ligand type, ligand density and endcapping results in a good balance among peak shape,

stability, dewetting and polar retention.


Overview



work?)



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 lifetimeEnhanced low pH stability

– LC/MS compatible with ultra-low column bleed– Perfect balance between polar and non-polar retention– Excellent reproducibility

Examples 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

Atlantis™ 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 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

AtlantisTM dC18 columns resist the sudden loss of retention associated with pore dewetting.


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: H2OMobile 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 1010.0 84 6 10

Injection Volume: 10 µLTemperature: 30 oCDetection: UV @ 254 nmInstrument:: Alliance® 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 Fully Endcapped Column

1

2 3

4 5

Column Z SAVo = 1.5min

2 4 53

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

Column K AQVo = 1.3min

1Minutes

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

12

3 4 5AtlantisTM dC18

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

Longer base retention at the expense of peak shape

Excellent retention and peak for all compounds

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

Vo = 1.5min


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 difunctional

bond and why does it

provide longercolumn

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


LC/MS Compatible with Ultra-low Column Bleed

Waters ZQ Detector Settings:Source: ESI (+)Capillary (kV): 3.5Cone (V): 30Temperature (°C)

Source: 150 Desolvation: 350

Gas Flow (L/Hr)Cone: 50Desolvation: 500

m/z: 300-1500

ConditionsColumn: AtlantisTM dC18 4.6 x 50 mm, 3 µmMobile Phase A: 0.1 % HCOOHMobile Phase B: 0.065 % HCOOH in ACNFlow Rate: 0.75 mL/min, 0.2 mL/min to MSGradient: Time Profile

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

Injection Volume: 20 µLInjection Mass: 20.0 µgTemperature: Ambient

5.00 10.00 15.00 20.00 25.00 30.00Time0

%

Rainville, Russell

100 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.000

100

%

0

100

% 41.1432.9131.8929.0827.87

22.140.07

4.633.71 12.466.42 10.709.257.99 13.20 15.00 21.42 23.77 24.60 36.6633.35 38.49 42.09 44.28

0.95

2.26 2.964.15 43.1210.725.91

8.63 22.9813.86 21.4616.87 27.4824.25 28.84

42.7230.7332.18 39.08 43.52

No Column BlankScan ES+: TIC

8.00e6

AtlantisTM dC18 BlankScan ES+: TIC

8.00e6

Scan ESI+: TIC1.00e9

Cytochrome C Tryptic Digest


LC/MS Compatible with Ultra-low Column Bleed

No Column Blank Combined Spectrum over Entire Runtime

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z0

100

%

1: Scan ES+ 9.00e4490

355

327

429

384428

415

445

447

610

491536

503537

577539

653611

612

613

621

684

654

685

758687

744690832761 817

906835 909

No differences betweenthe combined spectra from

blank runs with column and without column.

AtlantisTM dC18 Column Blank Combined Spectrum over Entire Runtime

0

100

%

1: Scan ES+ 9.00e4

355

315 341

429367

371

384415

398

610445

536447503

538564

684611

613639

685 758687

744832761819 907835 861 909 982

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z

Combined Spectra of Blank TIC


ConditionsColumn: AtlantisTM dC18 4.6 x 150 mm, 5 µm Mobile Phase A: 0.1% TFA in H2OMobile Phase B: 0.1% TFA in ACNFlow Rate: 1.4 mL/minGradient: Time Profile

(min) %A %B0.0 100 05.0 97 36.0 85 1510.0 80 2012.0 0 10025.0 0 100

Injection Volume: 10 µLTemperature: 30 oCDetection: UV @ 268 nmInstrument: Alliance® 2695, 2996 PDA

Perfect Balance Between Polar and Non-Polar Compound Retention

Compounds: Concentration (µg/mL)1. L-ascorbic acid (vitamin C) 19.62. Nicotinic acid (niacin) 9.83. Thiamine (vitamin B1) 19.64. Pyridoxal 39.25. Pyridoxine 39.26. Folic acid (vitamin B11) 23.57. Caffeine 9.88. Riboflavin (vitamin B2) 3.99. Retinol (vitamin A) 19.610. Tocopherol (vitamin E) 39.211. Ergocalciferol (vitamin D2) 9.8

Grumbach, DiehlMinutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

1 23

45

8

7

6

V0 = 1.37 min

109

11

Excellent retention for water-soluble vitamins

Not overly retentive for fat-soluble vitamins


Excellent 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


Overview



work?)



Surface of a Silica Gel Bonded-Phase Packing Material

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

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


Low Coverage –Low Ligand

Density

Surface of a Silica Gel Bonded-Phase Packing Material

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

EndcapN

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)


Polar Retention:Why Does AtlantisTM dC18 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

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 C18coverage– Cation-exchange interactions– Hydrogen bonding interactions


Overview

• Introduction

• Background


• An Intelligent Solution – AtlantisTM dC18

• Applications

• Summary


Isocratic Separation of Catecholamines and Metabolites

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!!


Low Level Acrylamide LC/MS/MS Analysis

LC Conditions:Column: AtlantisTM dC18 2.1 x 150 mm, 3 µmPart Number: 186001299Mobile Phase A: 0.1 % CH3COOHMobile Phase B: MeOHIsocratic Mobile Phase Composition: 99.5% A; 0.5% BFlow Rate: 0.2 mL/minInjection Volume: 20 µLTemperature: 26 oCInstrument:: Alliance® 2695, Quattro Premier API

CH

NH2

O

CH2

Acrylamide

2 pg on column(20 µL of 0.1 ppb)

1.00 2.00 3.00 4.00Minutes

71.7 > 43.9

71.7 > 54.84.2

x 10

3

Acrylamide

Smooth (Mn,1x2)

S/N = 10.82 pg on column

(20 µL of 0.1 ppb)

1.00 2.00 3.00 4.00Minutes

71.7 > 43.9

71.7 > 54.84.2

x 10

3

Acrylamide

Smooth (Mn,1x2)

S/N = 10.8

1.00 2.00 3.00 4.00Minutes

71.7 > 43.9

3.5

x 10

4

Smooth (Mn,1x2)

Acrylamide

71.7 > 54.8

2.50 3.00 3.50 4.00

2.4

x 10


S/N = 10.5

1.00 2.00 3.00 4.00Minutes

71.7 > 43.9

3.5

x 10

4

Smooth (Mn,1x2)

Acrylamide

71.7 > 54.8

2.50 3.00 3.50 4.00

2.4

x 10


S/N = 10.5

The mass detector was calibrated between m/z 19 to m/z2000 with unit resolution.

MS/MS Analysis Function (MRM of 4 mass pairs)

Transition Dwell Time Collision Energy Delay71.7 > 43.9 0.4 11.0 0.0171.7 > 54.8 0.4 10.0 0.0174.7 > 44.9 0.4 11.0 0.0174.7 > 57.8 0.4 10.0 0.01

The 74.7>57.8 and 71.7>44.9 for for the 13C3-Acrylamide Internal Standard at 50 ppb.


Peptides

Time (Min)

4 8 12 16 20 240

Compounds1. L22752. A66773. Bradykinin4. Angiotensin II5. Angiotensin I6. Substance P7. Renin Substrate8. Insulin B Chain9. Melittin

Atlantis™ dC18

Conventional C18

21

354 6

7

98

21

3 54 6 7

98V 0

ConditionsColumns: 4.6 x 50 mm, 5 µmMobile Phase A: 0.02% TFAMobile Phase B: 0.016% TFA in ACNFlow Rate: 0.75 mL/minGradient: Time Profile

(min) %A %B0.0 100 025.0 50 50

Injection Volume: 20 µLTemperature: 40 oCDetection: UV @ 220 nm

Gradient Peptide Separation Using TFA Mobile Phases

Note enhanced retention of peptides using AtlantisTM dC18 as compared to conventional C18 column


Cytochrome C Tryptic Digest: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/minGradient: 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


Cytochrome C Tryptic Digest:Increased Mass Loading

No loss inresolution,

peak shape or peak capacity

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/minGradient: Time Profile

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

Injection Volume: 20 µLInjection Mass: 20 µgTemperature: Ambient


ConditionsColumn: AtlantisTM dC18, 2.1x 20 mm IS™, 3 µmPart Number: 186002058Mobile Phase A: WaterMobile Phase B: MeOHMobile Phase C: 1% HCOOHFlow Rate: 0.4 mL/minGradient: Time Profile

(min) %A %B %C 0.0 50 40 101.0 30 60 10

Inj Volume: 2 µLConcentration: 10 µg/mLTemperature: 30 oCInstruments: Alliance® 2795 and Waters ZQTM

Compounds MWCinoxacin 262.2Oxolinic Acid 261.2Nalidixic Acid 232.2

Waters ZQTM

ES+

Capillary (kV) 3.5 Cone (V) 50 Extractor 3.0 RF Lens 0.1Source Temp (oC) 150 Desolvation Temp (oC) 400Cone Gas Flow (L/Hr) 50Desolvation Gas Flow (L/Hr) 500LM Resolution 15HM Resolution 15Ion Energy 0.5 Multiplier (V) 650

High-Throughput Separation: Nalidixic Acid Antibiotics

Cinoxacin

Nalidixic acid

Oxolinic acid

0.00 0.20 0.40 0.60 0.80 1.00Time0

100

%

0

100

%

0

100

%

0

100

%

1: Scan ES+ TIC

1.15e90.88

0.65

0.59

1: Scan ES+ 263

4.05e70.590.65

0.88

1: Scan ES+ 262

1.19e80.65

1: Scan ES+ 233

1.28e80.89

Separation in 1 minute!


Column: AtlantisTM dC18 4.6 x 100 mm, 5 µmPart Number:186001340Total Mass Load: 3,000 µg

Water Soluble Vitamins:Ease of Scale-up

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00

Time (min)

1

1

2

2

Conditions:Mobile Phase A: 0.1% HCOOH in H2OMobile Phase B: ACN/1% HCOOH (90/10)Sample Concentration: 5, 25 mg/mL in waterTemperature: AmbientInstrument:: AutoPurification™ SystemDetection: UV @ 290 nm

V0 = 1.73 min

V0 = 1.76min

Column: AtlantisTM dC18 30 x 100 mm, 5 µmPart Number:186001375Total Mass Load: 126,000 µg

Flow Rate: 1.0 mL/minGradient: Time Profile

(min) %A %B0.0 100 01.0 100 06.0 80 2016.0 60 40

Injection Volume: 100 µL

Flow Rate: 42.53 mL/minGradient: Time Profile

(min) %A %B0.0 100 03.25 100 08.25 80 2018.25 60 40

Injection Volume: 4200 µL

1. Pyridoxine 2. Caffeine


Time (min)

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00-0

100

%

11.7 mg total load

1

2

3

Preparative Separation –Nalidixic Acid Antibiotics

ConditionsColumn: AtlantisTM dC18 19 x 100 mm, 5 µmMobile Phase A: 0.1% HCOOH in H2OMobile Phase B: 0.1% HCOOH in ACNFlow Rate: 17.06 mL/minGradient: Time Profile

(min) %A %B0.0 80 204.02 80 2014.02 40 60

Injection Volume: 1800 µL (DMSO)Temperature: AmbientDetection: UV @ 300 nmInstrument: AutoPurification™ System

1. Cinoxacin (5 mg/mL)2. Oxolinic acid (0.5 mg/mL)3. Nalidixic acid (1 mg/mL)


Overview

• Introduction

• Background


• An Intelligent Solution – AtlantisTM dC18

• Applications

• Summary


AtlantisTM dC18 vs. Conventional C18 and Embedded Polar Group Columns

Column Resists Dewetting?

Increased Polar Retention?

Conventional alkyl C18 No No

Embedded polar group Yes No

AtlantisTM dC18 Yes Yes

Remember:1. Embedded polar group ≠ polar compound retention2. In order to create polar compound retention, choose

a column designed for polar compound retention


Polar Retention Summary

• Embedded polar groups were designed for peak shape, not polar compound retention

• The sudden loss in retention observed under highly aqueous conditions is due to pore dewetting nothydrophobic collapse

• Unendcapped C18 columns provide increased base retention at the expense of peak shape

• Increased RP retention can be realized with an optimized lower ligand density column

• A properly designed straight alkyl chain C18 column can provide the perfect balance of polar and non-polar compound retention

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

understanding (and creating) retention of polar compounds ... · advantages disadvantages ©2004...

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