2008/03/032008/03/03 Pittcon 2008Pittcon 2008 11

ChromaNik Technologies Inc.Norikazu Nagae Ph.D

Study of Study of SSecondary econdary IInteraction nteraction BBased on ased on RResidual esidual SSilanolilanol GGroups roups

for for RReversedeversed--PPhase hase LLiquid iquid CChromatographyhromatography

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abstractabstractIt has been said that residual silanol groups on C18 silica stationary phase yielded peak tailing for basic compounds. A lot of efforts have been exerted for reducing effect of residualsilanol groups by an end-capping or embedding a polar group in an alkyl chain. Consequently, a sharp peak of a basic compound has been obtained although retention of a basic compound has been getting little. On the other hand, on hydrophilic interaction chromatography (hilic) mode, silica column is recently used under a mixture of organic solvent and buffer solution, which is the same as a mobile phase used for reversed-phase liquid chromatography. In this mode, a peak shape of a basic compound is good not tailing. In other words a silanol group on a silica stationary phase is not a cause for tailing of a basic compound, at least on hilic mode.

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We found that a silanol group controlled its activity by the heat treatment didn’t make a basic compound tailing even if it existed on the C18 reversed-phase. Secondary interaction based on residual silanol groups for reversed-phase liquid chromatography was evaluated using C18 stationary phase with silanol groups controlled its activity. It was observed that retention of basic compounds and polar compounds was several times larger than that using a conventional end-capped C18 silica stationary phase, furthermore it dropped as pH value of a mobile phase decreased and salt concentration in a mobile phase increased while retention of neutral compounds didn’t change completely. It is considered that an ion exchange interaction occurred clearly by residual silanol groups on C18 silica stationary phase. As a result, a residual silanol group controlled its activity could change selectivity of basic compounds without peak tailing.

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Form of Form of silanolsilanol groups and peak shape of groups and peak shape of pyridinepyridine

OSi

O

OH

OSiSi

O

SiO

OH

Si

OH

OSi

O

HH

Pyridine peak60% CH3OH/H2O

Column C

Isolated Silanol

Vicinal Silanol

Geminal Silanol

Column AColumn B

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Our hypothesisOur hypothesis

Peak tailing of a basic compound is caused bythe state of silanol groups, not by existence of silanol groups on silica surface.Silanol groups near a strong hydrophobic site don’t occur an ion-exchange interaction etc. rapidly.

Decrease density of alkyl groups and make hydrophobicity low.Control activity of silanol groups near only hydrophobic site

Consequence

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Variation of waterVariation of water on son siilica surfacelica surface

Si

OSi

OSi

O

Si

O

SiO

Si

O

O

O

O

OO

OO

O

O

O

OO

Si

O

H H

H

H

H

O H

HO H

H

O H

H

H

H

O Desorption of bonded water

(400 °C)

Desorption of silanolgroup

(800 °C)

Desorption of absorbed water(105 ∼ 150 °C)

Bonded water

Absorbed water

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+H2O

Change of Change of silanolsilanol groups by heat treatmentgroups by heat treatment

OSi Si

OH OH

OSi Si

O

OSi Si

O－H2O

High temp.Low temp.Warped siloxane SiloxaneSilanol group

• Condensation of silanol group starts from near 100 °C.• Vacuum heat treatment makes an amount of silanolgroup low by water deprivation and siloxane produces.• Under 400 °C, siloxane reverts silanol groups by rehydration.• Over 400 °C, rehydration do not occur.

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Control of Control of silanolsilanol group group

Siloxane near hydrophobic site (C18) is kept at the same state and no silanolgoup produces by rehydration.

Siloxane away from hydrophobic site is rehydrated. Consequentlysilanol group produces and it is unaffected by hydrophobic site.

Prevention of tailing for basic compound

Increase of retention of polar and basic compounds, in spite of no change of retention of a neutral compound

After heat treatment

Active silanolgroups for basic

compounds Change to

Siloxane

O

Si

HO

Si

HO

SiO

O

SiSi

HO

HH

O

Si

HO

SiO

O

SiSi Si

OHH

heat

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ExperimentExperimentSilica gel

Pore diameter: 12 nm，Specific surface area: 340 m2/g，Particle size: 5 µm

Bonded OctadecylsilaneCarbon content: 14%As reference, prepareC18 phase with end-capping (TMS)

Heat treatment in a vacuumTemperature: 200 °C，Treatment time: 48 hours As reference, prepare C18 phase without heat treatment

EvaluationColumn: Sunrise C18-SAC (with heat treatment)

Sunrise C18 (with end-capping)Column dimension: 150 mm x 4.6 mm i.d. Mobile phase: acetonitrile or methanol/buffer solution

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Comparison of pyridine peak shape

Mobile phase: 30% CH3OH/H2OFlow rate: 1.0 mL/minTemperature: 40 ºCSample: 1=Uracil

②②=Pyridine=Pyridine3=Phenol

a)

b)

c)

１

１

１

3

②②

3

3

0 5 10 15 20Retention time / min

②②

②②

C18 with heat treatmentSunrise C18-SAC

C18 without heat treatment

C18 with TMSSunrise C18

OH

NNH

NH

O O

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Separation of Separation of xanthinexanthine

Column dimension: 150 x 4.6 mmMobile phase: Methanol/20mM Phosphate buffer (pH4.5)＝(30:70) Flow rate: 1.0 mL/minTemperature: 40 ºC

1= Theobromine 2=Theophylline

3=Caffeine 4=Phenol

N

N NH

NH

O

CH3

O

CH3

N

N N

NH

O

CH3

O

CH3

CH3 OH

N

NH N

NH

O

O

CH3

CH3

C18 with heat treatmentSunrise C18-SAC

0 1 2 3 4 5 6 7 8 9 10 11

Time (min)

1

1

2

2

3

3

4

4

C18 with TMSSunreise C18

3 times longer

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Separation of antidepressantsSeparation of antidepressants

C18 with TMS Sunrise C18

Acetonitrile/20mM phosphate buffer pH6.0 (80:20)

C18 with heat treatment Sunrise C18-SAC

Acetonitrile/20m phosphate buffer pH6.0 (80:20)

1

23

4

5

1

23

5

4

0 5 10 15 20 25 30 35 40 45 50 55 60 65

Time (min)

NH

NH

O OO

OH

NH CH3

CH3

NHCH3

NCH3

CH3

1=Uracil 2=Propranolol 3=Nortriptyline 4=Amitriptyline 5=TolueneCH3

Retention increase by ion-exchange interaction

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Control of retention by mobile phase pHControl of retention by mobile phase pH

Acetonitrile/20mM phosphate buffer pH3.0 (50:50)

1

1

2

2

3

3

4

5

5Acetonitrile/20mM phosphate buffer pH4.5(50:50)

Column: C18 with TMS, Sunrise C18,C18 with heat treatment, Sunrise C18-SACFlow rate: 1.0 mL/minTemperature: 40 ºCSample: 1=Uracil, 2=Propranolol, 3=Nortriptyline, 4=Amitriptyline, 5=Toluene

Acetonitrile/20mM phosphate buffer pH6.0 (80:20)

1

2 34

5

0 5 10 15 20 25 30 35 40 45 50 55 60 65

Time (min)

4

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Separation of basic compounds with Separation of basic compounds with ammonium acetate: Effect of pHammonium acetate: Effect of pH

Time (min)

0 5 10 15 20 25 30 35 40

Column: C18 with heat treatment,Sunrise C18-SACMobile phase:Acetonitrile/100mM ammonium acetate(70:30) Flow rate: 1.0 mL/minTemperature: 40 ºCSample: 1=Uracil, 2=Toluene, 3=Propranolol, 4=Nortriptyline, 5=Amitriptyline

pH 6.8

pH 4.1

pH 5.0

pH 5.9

pH 3.5

pH 2.7Only acetic acid

N=13,000

N=12,500

N=11,500

N=11,500

N=11,500

1

1

1

1

1

1

2

2

2

2

2

2

3

3

3

3

3

3 4

4

4

4

4

4

5

5

5

5

5

5

An ammonium ion makes a peak shape of a basic compound sharp.

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Separation of basic compounds with Separation of basic compounds with ammonium acetate: Effect of salt concentrationammonium acetate: Effect of salt concentration

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Column: C18 with heat treatment, Sunrise C18-SACMobile phase:Acetonitrile/ ammonium acetate (70:30) Flow rate: 1.0 mL/minTemperature: 40 ºCSample: 1=Uracil, 2=Toluene, 3=Propranolol, 4=Nortriptyline, 5=Amitriptyline

25 mM

100 mM

50 mM

200 mM

15

5

5

5

4

4

4

4

3

3

3

3

2

2

2

2

1

1

1

N=13,500

N=12,000

N=12,500

N=13,000

10 mM 543

21

N=12,000

25 mM

100 mM

50 mM

200 mM

15

5

5

5

4

4

4

4

3

3

3

3

2

2

2

2

1

1

1

N=13,500

N=12,000

N=12,500

N=13,000

0 2 4 6 8 10 12 14 16 18 20 22 24 26Time (min)

10 mM 543

21

N=12,000

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Relationship between buffer Relationship between buffer concentration and retentionconcentration and retention

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0.5 1.0 1.5 2.0 2.5 3.0log [buffer concentration]

log

k

0

2,000

4,000

6,000

8,000

10,000

12,000

14,000

N [a

mitr

ipty

line]

Toluene Propranolol Nortriptyline Amitriptyline N (ami)

log log[ ] log[ ] log logkzz

Azz

AVV z

KB

A

B

A

r

m AAB= − + + +

1

AA：：ion in ion in eluenteluent，，BB：：ion of sampleion of sample，， kk：：retention of retention of BB，，KK：：selectionselection coefficientcoefficientZZAA, , ZZBB：：chargecharge，，VVrr, , VVmm：：resin volume andresin volume and mobile phase volume in columnmobile phase volume in column

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Separation of metal chelating compoundSeparation of metal chelating compound

Column: Other pure ODS 5μmC18 with heat treatment, Sunrise C18-SAC,Mobile phase: A)Acetonitrile/20mM phosphoric acid (10:90)B)acetonitrile/50mM formic acid (10:90)C)acetonitrile/50mM acetic acid (10:90)Flow rate: 1.0 mL/minTemperature: 40 ºCSample: 1=8-Quinolinol，2=Caffeine

0 2 4 6 8 10 12 14 16Time (min)

0 2 4 6 8 10 12 14 16 18 20 22Time min)

1 2

1

1

1

1

1

2

2

2

2

2

A: 20mM phosphoric acid

C: 50mM acetic acid

B: 50mM formic acid

Other pure ODS C18 with heat treatment

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Separation of nucleicSeparation of nucleic acid basesacid basesColumn: C18 with heat treatment, Sunrise C18-SACMobile phase: 20mM Phosphate buffer pH4.5Flow rate: 1.0 mL/minTemperature: 25 ºCSample: 1=Cytosine，2=Uracil, 3=Cytidine, 4= Thymine, ＊=impurity

12

4

3

Retention time / min0 5 10 15

＊

N

NH

O NH2

NH

NH

O O

N

N

O NH2

O

H

OH

OH

H

CH3

HOH

NH

NH

O O

CH3

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【【ConclusionsConclusions】】Residual silanol group in proposed C18 phase made retention of polar compounds including basic compounds high.Silanol group controlled its activity by heat treatment did not make peak of basic compounds tailing.Furthermore, ion-exchange interaction was recognized and retention of basic compounds could be changed by pH or salt concentration in mobile phase.Proposed C18 phase was effective to separation of a metal chelating compound. It is surmised that hydration or change of state of silanol groups contributed to suppress influence of a metal impurity on silica support.