Study of Secondary Interaction
Based on Residual Silanol Groups
for Reversed-Phase Liquid
Chromatography
ChromaNik Technologies Inc.
Norikazu Nagae Ph.D
2008/03/03

Pittcon 20081

abstract

It 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 residual
silanol groups by an end-capping or embedding a polar groupin 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, onhydrophilic 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, apeak shape of a basic compound is good not tailing. In otherwords a silanol group on a silica stationary phase is not acause for tailing of a basic compound, at least on hilic mode.
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Pittcon 20082

We found that a silanol group controlled its activity by the heat
treatment didn’t make a basic compound tailing even if itexisted on the C18 reversed-phase. Secondary interaction
based on residual silanol groups for reversed-phase liquid
chromatography was evaluated using C18 stationary phasewith silanol groups controlled its activity. It was observed that
retention of basic compounds and polar compounds wasseveral times larger than that using a conventional end-capped
C18 silica stationary phase, furthermore it dropped as pH valueof a mobile phase decreased and salt concentration in a mobile
phase increased while retention of neutral compounds didn’tchange completely. It is considered that an ion exchangeinteraction occurred clearly by residual silanol groups on C18
silica stationary phase. As a result, a residual silanol groupcontrolled its activity could change selectivity of basic
compounds without peak tailing.
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Pittcon 20083

Form of silanol groups and peak shape ofpyridine H
Column AOOSiOSiOSi

Column BO

Isolated Silanol
OSiHOOH
Column CSiVicinal Silanol
HHOOSiPyridine peakGeminal Silanol
2008/03/03

60% CH3OH/H2O
Pittcon 2008

4

Our hypothesis
 Peak tailing of a basic compound is caused by

the state of silanol groups, not by existence ofsilanol groups on silica surface.
 Silanol groups near a strong hydrophobic site
don’t occur an ion-exchange interaction etc.
rapidly.

Consequence Decrease density of alkyl groups and makehydrophobicity low.
 Control activity of silanol groups near onlyhydrophobic site
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5

Variation of water on silica surface
H

Absorbed waterO
H

Bonded waterO
H
SiOOOOSiOO

H

HHOO

H

HOOSiOHOOOOSiOOSiO2008/03/03

OHO
SiSiH

Desorption ofabsorbed water(105 ∼ 150 °C)

H

Desorption ofbonded water(400 °C)

Desorption of silanol
OgroupH(800 °C)

Pittcon 2008

6

Change of silanol groups by heat treatmentOHOH

SiSiOO－H2O
Si+H2OOO
SiLow temp.

Silanol group

SiO

Si

High temp.

Warped siloxane

Siloxane• 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 byrehydration.
• Over 400 °C, rehydration do not occur.
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7

Control of silanol group
After heat treatment
Siloxane near hydrophobicsite (C18) is kept at thesame state and no silanol
goup produces byrehydration.

Active silanol
heatgroups for basic
compounds

Prevention of tailing for basic compound

H

H

Siloxane away fromhydrophobic site isrehydrated. Consequently
silanol group produces andit is unaffected byhydrophobic site.

O

Change toSiloxane
OO

H

SiOOOSiSiSiHOSiHSiHHOOOSiSiOSiHSiIncrease of retention of polar and basic compounds, inspite of no change of retention of a neutral compound
2008/03/03

Pittcon 20088Experiment

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

Bonded Octadecylsilane
 Carbon content: 14%
 As reference, prepareC18 phase with end-capping (TMS)Heat treatment in a vacuum Temperature: 200 °C，Treatment time: 48 hours
 As reference, prepare C18 phase without heat treatment

Evaluation
 Column: 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
2008/03/03

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9

Comparison of pyridine peak shape②

3
C18 with TMS

１Sunrise C18

Mobile phase: 30% CH3OH/H2O
Flow rate: 1.0 mL/min
Temperature: 40 ºC
3 C18 without heat Sample: 1=Uraciltreatment②=Pyridine
3=Phenol②a)
１b)

OH

3
１

Sunrise C18-SACc)
0

2008/03/03

C18 with heat treatment

HNO②
510Retention time / min

15

Pittcon 2008

NHON

2010

Separation of xanthine
C18 with TMS
Sunreise C18

32

1= Theobromine

43 times longer

N445CH34= Phenol

OOHO

3678NHN

3= Caffeine
H3C2HNN

3Sunrise C18-SAC

1H3CONHN
CH32C18 with heattreatment
1CH3HN
O0OO

12= Theophylline

91011CH3NNNN
H

CH3Time (min)Column dimension: 150 x 4.6 mm
Mobile phase: Methanol/20mM Phosphate buffer (pH4.5)＝(30:70)
Flow rate: 1.0 mL/min
Temperature: 40 ºC
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11Separation of antidepressants
1=Uracil

2=Propranolol

3=Nortriptyline

4=Amitriptyline

5=Toluene
CH3HNONHCH3OONHCH3OH4NHCH3N

2CH3CH33C18 with TMS Sunrise C181Retention increase by ionexchange interaction

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

51
5C18 with heat treatment Sunrise C18-SAC
Acetonitrile/20m phosphate buffer pH6.0 (80:20)
4205101520

32530

35404550556065

Time (min)2008/03/03

Pittcon 200812

Control of retention by mobile phase pH
42Acetonitrile/20mM phosphatebuffer pH3.0 (50:50)

3154123Acetonitrile/20mM
phosphate buffer pH4.5(50:50)

5Column: C18 with TMS, Sunrise C18,
C18 with heat treatment, Sunrise C18SAC
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Sample: 1=Uracil, 2=Propranolol,
3=Nortriptyline, 4=Amitriptyline,
5=Toluene

15

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

052008/03/03

1015204322530Time (min)35Pittcon 2008

40

45

5055

60

65

13

Separation of basic compounds withammonium acetate: Effect of pH12pH 2.7
1pH 3.5

1pH 5.0521 2

pH 4.143Only acetic acid

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

53

N=13,000453

4N=12,500235

4N=11,500

1 2pH 5.912008/03/03

434

5

N=11,500

2

pH 6.803

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

5101520Time (min)52530

Pittcon 2008

N=11,500

354014Separation of basic compounds withammonium acetate: Effect of salt concentration
1200 mM
1100 mM
150 mM23254

3

N=12,000

N=12,500

2531544

N=13,000

225 mM

3110 mM

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

452302008/03/03

2

N=13,500

4681012 14 16Time (min)54182022Pittcon 200824

N=12,00026151.4

14,0001.2

12,000

1.0

10,000

0.8

8,000

0.6

6,000

0.4

4,000

0.2

2,000

0.0

0
0.5

1.0

1.5

2.0

2.5

N [amitriptyline]

log kRelationship between bufferconcentration and retention

3.0

log [buffer concentration]
ToluenePropranolol

Nortriptyline

Amitriptyline

N (ami)zBzB
Vr
1log k = − log[ A] +log[ A] + log+log K AB
Vm z AzAzA
A：ion in eluent，eluent，B：ion of sample，sample， k：retention of B，K：selection coefficient
ZA, ZB：charge，
charge，Vr, Vm：resin volume and mobile phase volume in column

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Separation of metal chelating compound
1

Other pure ODS
1Column: Other pure ODS 5μm

C18 with heat treatment

C18 with heat treatment, Sunrise C18-SAC,
Mobile phase:2A)Acetonitrile/20mM phosphoric acid (10:90)

A: 20mM
phosphoric acid2B)acetonitrile/50mM formic acid (10:90)
C)acetonitrile/50mM acetic acid (10:90)
Flow rate: 1.0 mL/min

2

Temperature: 40 ºC

1

B: 50mM formicacid

1

Sample: 1=8-Quinolinol，2=Caffeine22
2

C: 50mM aceticacid

110246810 12Time (min)2008/03/03

14160246810 12 14Time min)1618

20Pittcon 20082217

Separation of nucleic acid bases
1HNO

N

2HN

NH2ON
HO

CH3

HNO
OH

OH
H

H
O

3O

H

H3C
HON
H

4Column:
C18 with heat treatment,
Sunrise C18-SAC
Mobile phase:
20mM Phosphate buffer pH4.5
Flow rate: 1.0 mL/min
Temperature: 25 ºC
Sample: 1=Cytosine，2=Uracil,
3=Cytidine, 4= Thymine,＊=impurity

NON

NH2

＊02008/03/03

510Retention time / min15Pittcon 200818

【Conclusions】
Residual silanol group in proposed C18 phase maderetention 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 ametal chelating compound. It is surmised thathydration or change of state of silanol groups
contributed to suppress influence of a metal impurity
on silica support.
2008/03/03

Pittcon 200819