Abstract Number: 250 - 38P

Study of Secondary Interaction
Based on Residual Silanol Groups
for Reversed-Phase Liquid
Chromatography II
Norikazu Nagae Ph.D, Kouji Yamamoto, Chiaki Kadota
ChromaNik Technologies Inc.
Email: info@chromanik.co.jp
Website: http://chromanik.co.jp
Pittcon 20091

abstract

It has been said that residual silanol groups on a 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 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, a silica
column is recently used under a mixture of an organic solvent
and a buffer solution, which is the same as a mobile phaseused for reversed-phase liquid chromatography. In this mode,a peak 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.
Pittcon 20092

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 a 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 a C18
silica stationary phase. As a result, a residual silanol groupcontrolled its activity could change selectivity of basic
compounds without peak tailing.
Pittcon 20093

Form of silanol groups and peak shape ofpyridine H
Column AOOSiOSiOSiColumn BO

Isolated SilanolOSiHOOHColumn CSiVicinal SilanolHHOOSiPyridine peakGeminal Silanol

60% CH3OH/H2O
Pittcon 20094

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
Pittcon 20095

Variation of water on silica surface
HAbsorbed waterO
H

Bonded waterO
HSiOOOOSiOOHHHOOHHOOSiOHOOOO

SiOOOHOSiSiH

Desorption ofabsorbed water(105 ~ 150 °C)

SiO

HDesorption ofbonded water(~ 400 °C)

Desorption of silanol
OgroupH(~ 800 °C)

Pittcon 20096

Change of silanol groups by heat treatmentOHOH

SiSiOO－H2O
Si+H2OOOSi

Low temp.

Silanol group

SiO

SiHigh 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.
Pittcon 20097

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

Active silanol
heatgroups for basic
compounds

Prevention of tailing for basic compound

HH

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

OChange toSiloxaneOOHSiOOOSiSiSiHOHSiHHSiOOOSiSiOSiHSi

Increase of retention of polar and basic compounds, inspite of no change of retention of a neutral compound
Pittcon 20098

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
Pittcon 2009

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=Phenol
②a)
１b)

OH3１

Sunrise C18-SACc)
0C18 with heat treatment

HNO②510Retention time / min

15

Pittcon 2009

NHON2010Separation of xanthine
C18 with TMS
Sunreise C1832

1= Theobromine 2= Theophylline

43 times longer

N4OO

345678NH

N
CH33=Caffeine
H3C2HNN

3Sunrise C18-SAC

1H3CONH

N
CH3

2

C18 with heattreatment
1CH3HN
O0OO

191011

CH34= Phenol
OHNNN

N
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
Pittcon 200911

Separation of antidepressants
1=Uracil

2=Propranolol

3=Nortriptyline

4=Amitriptyline

5=Toluene
CH3HNOCH3ONHONHCH3OH4NHCH3N

2CH3CH33C18 with TMS Sunrise C18

1Retention increase by ionexchange interaction

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

51
5

C18 with heat treatment Sunrise C18-SAC
Acetonitrile/20m phosphate buffer pH6.0 (80:20)
420510152032530

35404550556065Time (min)Pittcon 200912

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

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

5Column: C18 with TMS, Sunrise C18,
C18 with heat treatment, Sunrise C18SAC 150 x 4.6 mm
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Sample: 1=Uracil, 2=Propranolol,
3=Nortriptyline, 4=Amitriptyline,
5=Toluene

15Acetonitrile/20mM phosphate buffer pH6.0 (80:20)

05101520

4322530
Time (min)35Pittcon 2009

40

45

50

5560

65

13

Separation of basic compounds withammonium acetate: Effect of pH
12pH 2.751 2pH 3.51 2pH 4.143

Only acetic acid1pH 5.0Column: C18 with heat treatment,
Sunrise C18-SAC 150 x 4.6 mm
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=12,5002354

N=11,500

1 2pH 5.9

14345

N=11,500

2pH 6.803

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

5101520Time (min)52530

Pittcon 2009

N=11,500354014Separation of basic compounds withammonium acetate: Effect of salt concentration
1200 mM
1100 mM
150 mM

232543

N=12,00054

N=12,50025314

N=13,000

225 mM

3110 mM

Column: C18 with heat treatment,
Sunrise C18-SAC 150 x 4.6 mm
Mobile phase:
Acetonitrile/ ammonium acetate pH4.1 (70:30)
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Sample: 1=Uracil, 2=Toluene, 3=Propranolol,
4=Nortriptyline, 5=Amitriptyline

542302

N=13,500

468105412 14 16Time (min)1820

Pittcon 20092224

N=12,00026

15

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]
Toluenelog k = -Propranolol

Nortriptyline

Amitriptyline

N (ami)zBzV
1log[ A] + B log[ A] + log r +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

Pittcon 200916

Effect of salt of buffer to retention timeRetention time (min)

2520

eisTr

ate
rm

fo
m

m

iu
mon

am

am

mon

iu

iu macci trate

at
e
ph
mo
n

am

am

mon

iu

m

phos

fo
um

di
So

Procainamide

etat
e

e
at
rm

et

at

te
mac

ci
Sodiu

um
di

SoSodium

phos

phtraate

15
1050N-Acetylprocainamide
Pittcon 2009

Column: C18 with heat treatment,
Sunrise C18-SAC 150 x 4.6 mm
Mobile phase:
Methanol / 20mM buffer pH6.8(70:30)Flow rate: 1.0 mL/min
Temperature: 40 ºC
Sample: 1=Procainamide,
2=N-Acetylprocainamide
17SodiTrisp
So hosphdiat
So ume
c
diit
um
ra
S
ac te
odam
mo ium eta
teniu
fo
rm
maam
phos t e
moniu ph aam
mo m c teitrniu
at
am
me
mo
ac
etniu
at
me
fo
rm
at
e

2.4
2.2
2
1.8
1.6
1.4
1.21umAsymmetry (USP tailing factor)

Effect of salt of buffer to peak asymmetry

Procainamide
N-Acetylprocainamide

Pittcon 2009

Column: C18 with heat treatment,Sunrise C18-SAC 150 x 4.6 mm
Mobile phase:
Methanol / 20mM buffer pH6.8(70:30)Flow rate: 1.0 mL/min
Temperature: 40 ºC
Sample: 1=Procainamide,
2=N-Acetylprocainamide
18

Separation of metal chelating compound
1

Other pure ODS
1Column: Other pure ODS 5mm

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

2Temperature: 40 ºC

1

B: 50mM formicacid

1

Sample: 1=8-Quinolinol，2=Caffeine22
2

C: 50mM aceticacid

110246810 12Time (min)14160246810 12 14Time min)161820Pittcon 20092219

Separation of nucleic acid bases
1HNO

N

2
HN

NH2OO

N
H

CH3

HNOOH

OH
H

HO

3OH

H3CHON
H

4Column:C18 with heat treatment,
Sunrise C18-SAC 150 x 4.6 mm
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

NONNH2＊05

10Retention time / min

15Pittcon 200920

【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 be tailing.
 Furthermore, ion-exchange interaction was recognized
and retention of basic compounds could be changedby pH or salt concentration in a 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 a silica support.

Pittcon 200921