A Novel Bonding Technique Using a Polyfunctional Silyl-Reagent for
Reversed-Phase Liquid Chromatography--- A NEW APPROCH !!!! IINorikazu Nagae1), Tomoyasu Tsukamoto1), Anders Grahn2), Ralf Jutvik2) and Vinay D. Gaitonde2)
1) ChromaNik Technologies Inc., 6-3-1 Namiyoke, Minato-ku, Osaka, Japan2) Biotech AB, Box 133, 439 23, Onsala, Sweden

Abstract

Reversed- phase LC columns and Sorbent development has experienced
chromatographic performance improvement in terms of ULTRA puresilica, New End capping Reagents and chemistries, along withimprovement in Bonding technology, innovative hybrid silica particle andhigh speed fused core Sorbents as well as sub 2 micron high efficiency
sorbents and columns. These new innovative contributions are wellrecognized and accepted in HPLC work practices.
In regards to Bonding chemistries, most of reversed phase silica
materials are monomerically or polymerically bonded with alkyl chain,then end-capped with trimethylsilane or hexamehyltrisiloxane etc. In thisstudy, polyfunctional silyl-reagent was synthesized withoctadecyltrimethoxylsilane and hexamethyldichlorotrisiloxan. This reagent
is called hexamethyloctadecyltetrasiloxane (HMODTS – C18). We are able
to bond the Chromatographic Silica particles with this reagent and finallyend-cap with trimethylchlorosilane .
The resultant Sorbent was evaluated for chromatographic performancefor resolving acidic and basic compounds in various Mobile phasecompositions and Buffers. Stability of this phase was evaluated under bothacidic and basic pH at high temperature. This phase showed symmetricalpeaks of both acidic and basic compounds such as Formic acid andAmitriptyline. Interestingly enough we observed a symmetric peak of
Amitriptyline in Mobile phase consisting of Acetonitrile and Ammonium
acetate compositions. Majority of the C18 columns showed a long tailingpeak of Amitriptyline under similar conditions. We observed that theColumn life was more than 500 hours from pH 1.5 to pH 10 at 50 degree
Celsius. A novel bonding technique using a polyfunctional silylreagent(HMODTS – C18) could make effect of residual silanol groups theleast and hence offer a tailing free chromatographic performance as well asendurance at elevated temperatures.

Patent pending

Step 2

Step 1

Final TMS

toluene+ H O

Me MeMe MeMe Me+2Cl

80℃, 4hSiO

SiO

SiCltoluene

HOSiO30℃, 2h

Si OMeMeO

HOOMe

HOSiOMeOMe

SiOMeOMe

MeMeMeMe
SiOMe
SiO OMe
SiOMeMe
SiClO

CH3OH, pH7.5, 40 ºC
4Sunniest C18 （ HMODTS)

1324Sunniest RP-A QUA

132

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

TF

N12,500

Develosil ODS-UG-5

Column

2.14

8,700

4

1 A company C1832
B company C1810224NHSunniest RP-AQUA

1.1212,800

Gemini-NX C18

1.09

9,500Acclaim PolarAdvantage II

5.19

3,300

Hiber Purospher STAR RP-18e

2.01

11,200Acclaim 120 C18

2.19

14,200Inertsil ODS-4

1.30

12,000ACE C18

3.25

5,300

Inertsil ODS-SP

2.92

8,000

Ascentis C18

1.74

8,300

Inertsil ODS-3

2.70

6,100

Atlantis T3 C18

1.97

10,600L-column ODS

1.56

10,400

SunFire C18

1.59

10,100Shim-pack VP-ODS

3.44

6,700

X-Bridge C18

1.33

10,000

TSKgel ODS-100V

1.71

10,0008101214NH16

Cadenza 5CD-C18

3.07

8,500

TSKgel ODS-100Z

2.15

11,500

Unison US-C182.529,200

Wakopak Navi C18-5

11.1

2,100

CAPCELLPAK C18 MG II

2.01

10,900YMC-Pack Pro C18

3.77

7,400

CAPCELLPAK C18 MG III

7.75

3,600

ZORBAX Eelipse Plus C18

3.28

5,900

NCH3706050

Sunniest C18 (HMODTS)
Sunniest RP-AQUA
Sunniest C8
B company C18
D company C18F company C184030200204060

80100120

Time (h)N(4) =11,000
TF(4) =1.03

Column: Sunniest C18 (HMODTS), 5 mm 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
D) CH3CN/10mM Ammonium acetate pH6.8 = 40/60
4 N(4) =9,000
Flow rate: 1.0 mL/min
TF(4) =1.06
Temperature: 40 ºC for A, C and D, 22 ºC for B
Sample: 1 = Uracil,
2 = Propranolol,
N(4) =12,500
4 TF(4) =1.04
3 = Nortriptyline,
4 = Amitriptyline,
5 = Toluene
N(4) =12,100

2

1 B company
C18
32

CH3

4 = Amitriptyline

0 2C H3BC132

1 23D

51 234

4.E+084.E+083.E+08Company B C183.E+08
2.E+082.E+08

TF(4) =1.09

1.E+08Sunniest C18 HMODTS

5.E+072468101214161820

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

0.E+00Retention time (min)05101520Time (min)Loading capacity comparison of amitriptyline

Stability of retention under 0.1% formic acidmobile phase
Sunniest C18, 5 mm (HMODTS)
tR=4.25 min

InitialAfter 12 hours

tR=4.24 min

After elution of pH 10mobile phasetR=4.29 min

After 12 hours

tR=4.25 min

6NHC H3OH

3 = Nortriptyline
NHN(4)=4,300
TF(4)=5.24

CH34

4 = Amitriptyline

8 10 12 14 16 18 20 22 24

Retention time/min

CH3N

CH3

Stability under basic pH condition at 50 ºC

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+08504CH 3O

N(4)=4,000

4 TF(4)=4.33

120

Durable test condition
Column: Sunniest C18 (HMODTS), 5 mm
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

100
80
60
40
20Sunniest
SunniestC18C18 (HMODTS)

00200

400

600

800

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

Time (h)5.E+08Intensity (cps)3N(4)=13,800
TF(4)=1.24

Bleed test using LC/MS4A

4C H3Test 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

80

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

1 A company
C182 3Comparison of 4 kinds of mobile phase12

1 32CH3N90

43Sunniest RP-A QUAOH100CH 31

3 = Nortriptyline

TF(4)=1.50

Column size: 150 X 4.6 mm
Particle size: 5 mm
Mobile phase:
CH3CN/20mM Phosphate buffer pH7.0=60/40
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Sample: 1 = Uracil
2 = Propranolol

Sunniest C18 (HMODTS)

4 N(4)=10,000

36O

N(4)=8,950
TF(4)=1.76

Stability under acidic condition

Column 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

CH3CN, pH7.0, 40 ºC

C H3

Relative plate number of butylbenzene (%)

N1.04

Relative retention of ethylbenzene (% )TF

Evaluation of endend-capping
Comparison of amitriptyline peak IIColumn size: 150 X 4.6 mm
Particle size: 5 mm
Mobile phase:
CH3OH/20mM Phosphate buffer pH7.5=80/20
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Different
Sample: 1 = Uracil
condition
2 = Propranolol

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

Particle size: 5 mmPore diameter: 12nm
Surface area: 340 m2/g

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.

Evaluation of endend-capping
Comparison of amitriptyline peak I

Evaluation of endend-capping
Comparison of amitriptyline peak IIII-BColumn

SiSiOSi OOSi
O SiSiSi OSiSi OOO O
Si O SiO OH
OSi
Si OOOO Si
SiO Si OSi
SiOO
HO O
SiOO
SiOOO
O OOSiOSiOSiOOSiSi
SiSiOOOSiO Si
OOO SiOO O O OOOO Si OOOOOOOOSi
SiSiORetention time/min

Sunniest C18 (HMODTS)

Bonding state of HMODTS on silica

Novel C18 silyl
silyl--reagent (HMODTS)

Sunniest C18, 5 mm(HMODTS)

Condition
Column dimension: 150 x 4.6 mm
Mobile phase: Acetonitrile/0.1%
formic acid (30:70)
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Detection: UV@250nm
Sample: Amitriptyline (0.27mg/mL)
Injection Volume:0.2 mL

Condition
Column dimension: 150 x 4.6 mm
Mobile phase: Acetonitrile/0.1%
formic acid (30:70)
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Detection: UV@250nm
Sample: Amitriptyline (0.27mg/mL)
Injection Volume:0.2, 1, 2, 4 and 8 mL

8 mL
4 mL
2 mL
1 mL
0.2 mL

N=8,752

Conclusion
 Polyfunctional silyl-reagents were developedusing 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 silanolgroups on proposed C18 stationary phase. Andbasic compounds can be separated well without
any restriction concerned with a mobile phase.
 Stable retention can be obtained by proposed
C18 in acidic, low-ionic-strength mobile

phases such as a mixture of 0.1% formic acidand acetonitrile. Furthermore under a suchcondition less tailing peaks was obtained.

CH3Conventional C18, 5 mm

N

tR=6.59 min

InitialAfter 12 hours

tR=5.24 min

CH345 min

Conventional C18, 5 mm

6N=8,423

CH3N

After 2 hours

CH3tR=6.16 min

After elution of pH 10 mobile phase
After 12 hours

tR=7.67 min

tR=5.15 min

45 min6Only 5 ug of amitriptyline injection (0.2 mL) allows peak shape to be good for any RP-columns.

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