Abstract Number: 2080 – 14P

A Novel Bonding Technique Usinga Polyfunctional Silyl-Reagent 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 20101

ABSTRACT
Reversed- phase LC columns have been improved by a puresilica, a new end-capping reagent, bonding technology and ahybrid silica particle et al. and are widely used now. Most ofreversed phase silica materials are monomerically or
polymerically bonded with alkyl chain, then end-capped withtrimethylsilane or hexamehyltrisiloxane et al. In this study,
polyfunctional silyl-reagent was synthesized withoctadecyltrimethoxylsilane and hexamethyldichlorotrisiloxan.
This reagent is called hexamethyloctadecyltetrasiloxane(HMODTS). C18 silica gel, which was bonded with this reagent
and finally end-capped with trimethylchlorosilane, wasevaluated to separate acidic and basic compounds. Stability ofthis phase was evaluated under both acidic and basic pH athigh temperature. This phase showed symmetrical peaks of
Pittcon 20102both acidic and basic compounds such as formic acid andamitriptyline. Especially a symmetric peak of amitriptyline wasobtained even if both acetonitrile and ammonium acetate wereused as a component of a mobile phase although most of C18columns showed a terrible tailing peak of amitriptyline at thesame conditions. Column life was more than 500 hours frompH 1.5 to pH 10 at 50 degree Celsius. A novel bonding
technique using a polyfunctional silyl-reagent could makeeffect of residual silanol groups the least.

Pittcon 20103

C18 silyl-reagent 1 (HMODTS) Patent pending(Sunniest C18)
Hexamethyloctadecyltetrasilane
Step 2

Step 1toluene＋ H2O＋80℃，4h

MeO

Si

OMeOMeHOSiOMeOMeHOMeCl

MeMe MeMe MeSiO

Si

OSiCl

toluene30℃，2hSi

OMeOMeMeCl

Pittcon 2010

MeMe
Si

O

MeMe
Si

O

MeSi

O

Si

OMeOMe4Bonding state of HMODTS on silica

Final TMS

HO
Si
O
Si
Si
Si

O

Si

Si
O
Si
OO
O

O

Si

Si

Si
Si O
Si O
O O

Si

O

O

OO

O

Si
OO

SiOOO

SiOO

Si

SiOO
O
SiOOO

SiOSi
Si
HOOO

Si
OSi
OOSiO OSiOSiO OHOSiOOSiSiSiO O
O O OOOSi O
O Si OSiOOOSiOOO

An Arm of HMODTS moves like a Geometrid caterpillar, so that a functionalgroup on the tip of the arm can bond with a silanol group which Is locatedanywhere.
Pittcon 2010

5

Characteristics of Sunniest C18
Used Silica gel:
12 nm, 340 m2/g, 5 μm
Carbon content after bonding HMODTS:
16.1%
Carbon content after final end-capping:
16.3%
Pittcon 20106

Evaluation of end-capping
Comparison of amitriptyline peak I
CH3OH, pH7.5, 40 ºC
Sunniest C18 （HMODTS)
1４

３

N(4)=11,000TF(4)=1.06

2Sunniest RP-AQUA
1４３2

N(4)=10,500TF(4)=1.07

Column size: 150 X 4.6 mmParticle size: 5 μm
Mobile phase:
CH3OH/20mM Phosphate buffer pH7.5=80/20
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Sample: 1 = Uracil
2 = Propranolol
CH3ONHCH3OH

1４

A company C183

2

1N(4)=8,950
TF(4)=1.76
４

B company C18３

2N(4)=10,000TF(4)=1.50

3 = Nortriptyline
NHCH34 = Amitriptyline
CH3NCH30246810121416Retention time/min

Pittcon 20107Evaluation of end-capping
Comparison of amitriptyline peak IICH3OH, pH6.0, 22 ºCSunniest C18 (HMODTS)
1４

N(4)=9,000
TF(4)=1.08

３2４Sunniest RP-AQUA
1N(4)=8,000
TF(4)=1.10

３2

1４

A company C18

N(4)=3,900TF(4)=2.92

321B company C18

N(4)=5,800
TF(4)=2.86

４３2Column size: 150 X 4.6 mm
Particle size: 5 μm
Mobile phase:
CH3OH/20mM Phosphate buffer pH6.0=80/20
Flow rate: 1.0 mL/minTemperature: 22 ºC
CH
Sample: 1 = Uracil
ONHCH
2 = Propranolol
33OH

3 = Nortriptyline

NHCH34 = Amitriptyline

CH3NCH30246810Retention time/min

1214Pittcon 20108Evaluation of end-capping
Comparison of amitriptyline peak III-A
CH3CN, pH7.0, 40 ºC

Column size: 150 X 4.6 mm
Particle size: 5 μm
Mobile phase:
CH3CN/20mM Phosphate buffer pH7.0=60/40
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Sample: 1 = Uracil
CH
2 = Propranolol
ONH
CHSunniest C18 (HMODTS)
1４３

N(4)=14,000TF(4)=1.16

2Sunniest RP-AQUA
1４３21A company C18
321N(4)=13,800
TF(4)=1.24

４33OH

B company C18４３23 = Nortriptyline

N(4)=4,000
TF(4)=4.33

NHCH3

4 = Amitriptyline

N(4)=4,300
TF(4)=5.24

CH3N
CH302

468

101214

Retention time/min

161820

Pittcon 201022249

Evaluation of end-capping
Comparison of amitriptyline peak III-B
ColumnTF

NColumn

TF

N

Sunniest C18(HMODTS)

1.16 14,000

Japanese company C1 C18

2.148,700

Sunniest RP-AQUA

1.24 13,800

P11.099,500

D15.19

M12.01 11,200

D22.19 14,200

Japanese company D1 C18

1.30 12,000

AT3.255,300

Japanese company D2 C182.928,000

S11.748,300

Japanese company D3 C18

2.706,100

W11.97 10,600

Japanese company E1 C18

0.99 11,400

WS21.59 10,100

Japanese company F1 C18

3.44

W3

1.33 10,000

Japanese company G1 C18

1.71 10,000

3,3006,700

Japanese company A1 C18 3.078,500

Japanese company G2 C18

2.15 11,500

Japanese company A2 C18 2.52

9,200Japanese company H1 C1811.12,100

Japanese company B1 C18 2.2350

Japanese company I1 C183.777,400

A13.285,900

Japanese company B2 C18 2.01 10,900
Japanese company B3 C18 7,75

Column size: 150 X 4.6 mm
Particle size: 5 μm
Mobile phase:
CH3CN/20mM Phosphatebuffer pH7.0=60/40
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Sample: Amitriptyline

CH3N
CH33,600

Pittcon 20101010090
807060(%)

Relative retention factor of ethylbenzene

Stability under acidic condition
Test condition
Column size: 150 x 4.6 mm
Mobile phase:
CH3CN/1.0% TFA, pH1=10/90
Flow rate: 1.0 mL/min
Temperature: 80 ºC

5040302010Sunniest C18 (HMODTS)
Sunniest RP-AQUA
Sunniest C8Company B C18Company D C18
Company E C18020406080100 120

Measurement condition
Column size: 150 x 4.6 mm
Mobile phase:
CH3CN/H2O=60/40
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Sample: 1 = Uracil
2 = Ethylbenzene

Time (h)Pittcon 201012

Relative plate number of butylbenzene (%)

Stability under basic pH condition at 50 ºC
Durable test condition
Column: Sunniest C18 HMODTS, 5 μm
150 x4.6 mm
Mobile phase: CH3OH/20mM Sodium
borate /10mM NaOH=30/21/49 (pH10)
Flow rate: 1.0 mL/min
Temperature: 50 ºC
Measurement condition
Sunniest C18 HMODTS

Pittcon 2010

Column: Sunniest C18 HMODTS, 5μm
150 x 4.6 mm
Mobile phase: CH3OH/H2O=75/25
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Sample: 1 = Butylbenzene

13

pH selectivity
23
1pH2.36543

pH7142653,540pH10251

61015
20
Retention time/min

Column: Sunniest C18 HMODTS, 5 μm,150 x 4.6 mm
Mobile phase:A1) 20mM Phosphoric acid pH2.3A2) 20mM Phosphate buffer pH7A3) 20mM Phosphate buffer pH10B) Acetonitrile
Time (min)030%B (%)
226
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Detection: UV@250 nm
Sample: 1= Thiamine HCl Vitamin B1
2 = Nicotinamide
3 = Nicotinic acid
4 = Pyridoxine HCl Vitamin B6
5 = Folic acid
25630
= Riboflavin Vitamin B2

Pittcon 201014

Comparison of 4 kinds of mobile phaseA41

N(4) =11,000
TF(4) =1.03 Column: Sunniest C18 HMODTS, 5 μm 150 x 4.6 mm
Mobile phase:A) CH3OH/20mM Phosphate buffer pH7.5 = 80/20B) CH3OH/20mM Phosphate buffer pH6.0 = 80/20C) CH3CN/20mM Phosphate buffer pH7.0 = 60/40
4N(4) =9,000 D) CH3CN/10mM Ammonium acetate pH6.8 = 40/60
TF(4) =1.06 Flow rate: 1.0 mL/min
Temperature: 40 ºC for A, C and D, 22 ºC for B
Sample: 1 = Uracil,
2 = Propranolol,
4 N(4) =12,500
3 = Nortriptyline,
4 = Amitriptyline,
TF(4) =1.04
5 = Toluene

32B

3

12C

1235

D

01223

4

4

6N(4) =12,100
TF(4) =1.09

810Retention time (min)1251416

Pittcon 2010

182015Comparison of amitriptyline peak using
mobile phase for LC/MS
N=12,200
TF=1.30
1

N=12,100
TF=1.21

N=12,000
TF=1.28

Sunniest C18 HMODTS
Sunniest RP-AQUA
N=5,100
TF=2.4

Sunniest C8Column size: 150 x 4.6 mm
Particle size: 5 μm
Mobile phase: CH3CN/10mM
Ammonium acetate
pH6.8=40/60
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Sample: Amitriptyline

N=8,500
TF=2.6JD3 C18

N=5,500
TF=2.1W3 C18CH3N

N=8,100
TF=2.0JB2 C18CH3JI1 C1802468101214

Retention time/minPittcon 201016

Bleeding Test I
Column size: 150 x 2.0 mm
Mobile phase:A) 0.1% acetic acidB) CH3CN
Gradient:
Time: 0min 3min 14.4min 18min 19min%B: 5% 5% 100% 100% 5%
Flow rate: 0.2 mL/min
Temperature: 40 ºC
Detection: Corona CAD

Company F C18
Area:960,000

Company B C18
Area: 1,150,000

Sunniest C18 HMODTS
Area: 94,000

Retention time/min

Pittcon 201017

Bleeding Test II
5.E+08

Column size: 150 x 2.0 mm
Mobile phase:A) 0.1% acetic acidB) CH3CN
Gradient:
Time: 0min 3min 14.4min 18min 19min%B: 5% 5% 100% 100% 5%
Flow rate: 0.2 mL/min
Temperature: 40 ºC
MS： ABI API-4000
Ionization: Turboionspray (cation)
Measurement mode:
Q1 Scan m/z 100-1000

5.E+084.E+08Company B C18
Intensity (cps)

4.E+083.E+083.E+082.E+082.E+081.E+08Sunniest C18 HMODTS

5.E+070.E+000510

1520

Time (min)Pittcon 201018

2 μm particle
Narrow particle distribution
Measured by Coulter Counter method
Conventional particle

Particle used in this study

D10:1.67 μm
D50: 2.09 μm
D90: 2.65 μm
D90/D10=1.59

D10:1.78 μmD50: 2.04 μm
D90: 2.44 μm
D90/D10=1.37(μm)

The narrowestparticle
distribution

20% volume was cut off from both sides respectively.
Pittcon 201019

Pressure (MPa)Column pressure using methanol or
acetonitrile and water

Concentration of organic solvent (%)

Column: Sunniest C18 HMODTS , 2 μm 50 x 2.0 mm
Mobile phase: CH3OH/H2O, CH3CN/H2O
Flow rate: 0.5 mL/min
Temperature: 40 ºC
Pittcon 201020

Evaluation of amitriptyline on 2 μm particle
A Methanol/20 mM phosphate buffer pH 7.5 = (80:20)
12N(4) =8,068
TF(4) =1.13

34

4

N(4) =7,670
TF(4) =1.50
Other C18

B Acetonitrilel/20 mM phosphate buffer pH 7.0 = (60:40
12

Comparison of plate number

N(4) =7,529
TF(4) =1.19

34
4

N(4) =3,068
TF(4) =4.26
Other C18

0.0

0.4

0.8

1.2

1.6

2.0
1.00

2.00

Comparison of tailing factor

Retention time (min)Column: Sunniest C18 HMODTS, 2 μm 50 x 2.0 mm, Temperature: 40 ºC
Sample: 1 = Uracil, 2 = Propranolol, 3 = Nortriptyline, 4 = Amitriptyline,
Pittcon 201021High throughput separation of analgesics
Sunniest C18 HMODTS
5 μm, 150 x 4.6 mm

12Mobile phase:N(4)=80,000 plate/m
3

4

CH3CN/0.1% Formic acid = 20/80
Flow rate: 1.0 mL/min for 150 x 4.6 mm ,0.6 mL/min for 50 x 2.0 mm
Temperature: 40 ºC
Detection: UV@230 nm
1NH

CH3
OAcetaminophen

HOCH3

2
N(4)=162,000 plate/mNO

3Sunniest C18 HMODTS

ON

CH3OOH

Antipyrine

AspirinO
CH32 μm, 50 x 2.0 mm
012345678910124

H2NH3COO

Ethenzamide

Retention time (min)Pittcon 201022

Conclusions
 Polyfunctional silyl-reagents were developed

using C18 silyl reagent and end-capping
reagent such as octadecyltrimethoxylsilane,hexamethyldichlorotrisiloxane(HMODTS).
 Functional group of HMODTS can bond with any
silanol groups on silica surface.
 There is the least effect of residual silanol groups
on proposed C18 stationary phase. And basic
compounds can be separated well without any
restriction concerned with a mobile phase.
 2 μm particle was applied and same result wasobtained.
Pittcon 201023