Abstract Number: 1990 - 6P

A Novel Bonding Technique Usinga Polyfunctional Silyl-Reagent for
Reversed-Phase Liquid
Chromatography
Norikazu Nagae Ph.D, Kouji Yamamoto, Chiaki Kadota
ChromaNik Technologies Inc.
Email: info@chromanik.co.jp
Website: http://chromanik.co.jp
Pittcon 20091

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 hexamethyltrisiloxane et al. In this study,
polyfunctional silyl-reagent (I) was synthesized withoctadecyltrimethoxylsilane and hexamethyldichlorotrisiloxane,
polyfunctional silyl-reagent (II) was synthesized withoctadecyltrichlorolsilane and bis(triethoxysilyl)ethane. The
former reagent is called hexamethyloctadecyltetrasiloxane(HMODTS). Silica gel, which was bonded with these reagentsand finally end-capped with trimethylchlorosilane, wasPittcon 20092

evaluated to separate acidic and basic compounds. Stability ofthese phases was evaluated under acidic and basic pH at hightemperature. These phases showed symmetrical peaks of bothacidic 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 C18
columns show a terrible tailing peak of amitriptyline at thesame conditions. In the case of silyl-reagent (II), column life
was more than 250 hours from pH 2 to pH 8 at 80 degree
Celsius. A novel bonding technique using a polyfunctional silylreagent could make effect of residual silanol groups the least.

Pittcon 20093

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

Step 1toluene＋ H2O＋80℃，4h

MeO

SiOMe
OMe

HOSiOMeOMe

HOMeClMeMe MeMe MeSiOSi

O

SiCl

toluene30℃，2h

SiOMe
OMe

MeCl

Pittcon 2009

MeMe
SiO

MeMe
SiO

MeSiO

Si

OMeOMe4Bonding state of HMODTS on silica

Final TMS

HOSiOSiSiSiOSiSiOSiOOOOSiSiSiSi OSi OO OSiOOOOOSiOOSiOOOSiOOSiSiOOOSiOOOSiOSiSiHOOOSiOSiOOSiO 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 20095Characteristics of Sunniest C18
Used Silica gel:
12 nm, 340 m2/g, 5 mmCarbon content after bonding HMODTS:16.1%
Carbon content after final end-capping:
16.3%
Pittcon 20096Evaluation of end-capping
Comparison of amitriptyline peak I
CH3OH, pH7.5, 40 ºC
Sunniest C18 （HMODTS)
1４

３

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

2Sunniest RP-AQUA
1４３
2

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

Column size: 150 X 4.6 mm
Particle size: 5mm
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

1４A company C1832

1N(4)=8,950
TF(4)=1.76
４B company C18
３2N(4)=10,000
TF(4)=1.50

3 = Nortriptyline
NH

4 = Amitriptyline
CH3N0246810Retention time/min

1214CH3CH316Pittcon 20097Evaluation of end-capping
Comparison of amitriptyline peak II
CH3OH, pH6.0, 22 ºC
Sunniest C18 (HMODTS)
1４

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

３2

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

３21４

A company C18N(4)=3,900
TF(4)=2.92

321B company C18３202N(4)=5,800
TF(4)=2.86４Column size: 150 X 4.6 mm
Particle size: 5mm
Mobile phase:
CH3OH/20mM Phosphate buffer pH6.0=80/20
Flow rate: 1.0 mL/min
Temperature: 22 ºC
CHSample: 1 = Uracil
O
NH
CH
2 = Propranolol
33OH

3 = Nortriptyline

4 = Amitriptyline

NHCH3N46

8

10Retention time/min

12CH3CH314

Pittcon 20098

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

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

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

2Sunniest RP-AQUA
1４３21A company C183212４3OH

４３468101214Retention time/min

3 = Nortriptyline

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

B company C1820

3

N(4)=13,800
TF(4)=1.24

1618NH

4 = Amitriptyline

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

20

Pittcon 200922CH3CH3NCH3249

Evaluation of end-capping
Comparison of amitriptyline peak III-BColumn

TFNColumn

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

D15.19M1

2.01 11,200

D2

2.19 14,200

Japanese company D1 C18

1.30 12,000

AT

3.25

5,300

Japanese company D2 C18

2.928,000

S11.748,300

Japanese company D3 C18

2.706,100

W11.97 10,600

Japanese company E1 C18

0.99 11,400

WS2

1.59 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 11,500Japanese company A2 C18 2.529,200

Japanese company H1 C1811.12,100

Japanese company B1 C18 2.2350

Japanese company I1 C183.77

7,400

A13.285,900

Japanese company B2 C18 2.01 10,900
Japanese company B3 C18 7,75Column size: 150 X 4.6 mm
Particle size: 5 mm
Mobile phase:
CH3CN/20mM Phosphatebuffer pH7.0=60/40
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Sample: Amitriptyline

CH3N

CH3

3,600

Pittcon 200910

Change of water solvation
Why does peak tailing occur under different conditions although thesame column is used?
HHOHCH3OH+ H
N
CH3H OHHHO
HCH3OH+ H
N
CH3O H
HH OHMethanol 22ºC

Methanol 40ºC

Water solvation volume on a cation changes
under conditions such as methanol oracetonitrile, 40ºC or 22ºC.

H

CH3+ H O HN
CH3O H
HAcetonitrile 40ºC
The less water solvation, the more peak tailing.
Pittcon 2009

11

100

Test conditions
Column size: 150 x 4.6 mm
Mobile phase:
CH3CN/1.0% TFA, pH1=10/90
Flow rate: 1.0 mL/min
Temperature: 80 ºC90
8070(%)

Relative retention factor of ethylbenzene

Stability under acidic conditions

605040Sunniest C18 (HMODTS)

30Sunniest C8Sunniest RP-AQUA
Company B C1820Company D C18
Company E C1810020406080100 120

Time （ｈ）
Pittcon 2009

Measurement conditions
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

12Separation of standard samples I
Chelating sample

Neutral samples
2315405101517262025

30

35

Retention time/min

Column: Sunniest C18 HMODTS, 5mm 150 x 4.6 mm
Mobile phase: CH3OH/H2O=75/25
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Pressure: 5.4 MPa
Sample: 1 = Uracil,
2 = Caffeine,
3 = Phenol,
4 = Butylbenzene
5 = o-Terphenyl
6 = Amylbenzene
7 = Triphenylene
Hydrophobicity： a（Amylbenzene/Butylbenzene）
Hydrogen bonding capacity：a（Caffeine/Phenol）
Steric selectivity a（Triphenylene/o-Terphenyl）

1.58
0.42
1.41

Pittcon 2009

0246

8

Retention time/min

Column: Sunniest C18HMODTS, 5mm 150 x 4.6 mm
Mobile phase:
CH3CN/20mM H3PO4=10/90
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Detection: UV@250nm
Sample: 1 = 8-Quinolinol
2 = Caffeine

13Separation of standard samples IIAcidic samples

Pyridine and Phenol
3a（Pyridine/Phenol） = 0.37

12321024680

Retention time/min

2468Retention time/min

Column: Sunniest C18
HMODTS, 5mm 150 x 4.6 mm
Mobile phase: CH3OH/H2O=30/70
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Detection: UV@250nm
Sample: 1 = Uracil
2 = Pyridine
3 = Phenol

Column: Sunniest C18
HMODTS, 5mm 150 x 4.6 mmMobile phase: CH3CN/0.1% H3PO4=2/98
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Detection: UV@210nm
Sample: 1 = Formic acid
2 = Acetic acid
3 = Propionic AcidPittcon 200914

Separation of basic compounds using
mobile phase for LC/MS
4Column: Sunniest C18 HMODTS, 5mm 150 x 4.6 mm
Mobile phase:
CH3CN/10mM Ammonium acetate pH6.8=40/60
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Sample: 1 = Uracil，

1
3

2 = Propranolol，

CH 3O

NHCH 3OH

23 = Nortriptyline，
NHCH3

4 = Amitriptyline
CH3N012347678910CH3Retention time/min

Pittcon 200915

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: 4.6x150 mmParticle size: 5mm
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/min

Pittcon 200916

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 200917

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+0005101520

Time (min)Pittcon 200918

Synthesis of Octyl group (C8,HMOTS)
Separation of amitriptyline
4AColumn: Sunniest C8 HMOTS, 5mm 150 x 4.6 mm
N(4)=11,700 Mobile phase:A) CH3CN/20mM Phosphate buffer pH7.0=60/40
TF(4)=1.09B) CH3CN/10mM Ammonium acetate pH6.8=40/60
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Sample: 1 = Uracil，3

12

2 = Propranolol，
4B

O

N(4)=11,200
TF(4)=1.21

13CH 3NH

CH 3OH3 = Nortriptyline，

2

NH

CH3

4 = Amitriptyline
CH30123456N

7CH3Retention time/min

Pittcon 200919

C18 silyl-reagent 2 Patent pending(SunArmor C18)

End-capping

Bond with silica gel

H2 OHCOH2HOSi2SiOHOHSiC
H2 O OHO2O SiSiOOOCC2 SiSi2OSiSi2 SiOH2H2OOOOOOCCOOOSiOSiSiSi22OSiOO O
O O OOSiOSiSiOOOSiSiOSiOOSiSi OOOSiOO OSiOOOOO SiOOOOHOXXSiX

H2COSi2X X

Si O
SiOX XSiX

H2C2H2CX X

Si XX

2SiXXXOO

Used Silica gel: 12 nm, 340 m2/g, 5 mm
Pittcon 200920

Stability at 80ºC-pH 2R e la tive p la te n u m b e r（ ）R ela tive re te n tio n tim e （ ）/ %1201008060Conditions
SunArmor C18, 5mm 150 mm x 4.6 mm i.d..

40Mobile phase: ACN/ 0.1% trifluoroacetic acid pH2.0=40:60
Flow rate: 1.0 mL/min, Temperature: 80 oC

2000100

200

300
Time / hour

Pittcon 2009

400

500

600

21

Stability at 80ºC-pH 8.0

R e la tive p la te n u m b e r（ ）R e la tive re te n tio n tim e （ ）/ %120
1008060

Conditions
SunArmor C18, 5mm 150 mm x 4.6 mm i.d..

40Mobile phase:
ACN/ 20 mM Ammonium acetate + TEA pH8.0=40:60

20Flow rate: 1.0 mL/min, Temperature: 80 oC00100200300400500

Time / hour
Pittcon 200922

Conclusions
 Polyfunctional silyl-reagents were developed

using C18 silyl reagent and end-capping
reagent such as octadecyltrimethoxylsilane,hexamethyldichlorotrisiloxane andoctadecyltrichlorolsilane,bis(triethoxysilyl)ethane.
 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.
Pittcon 200923