A New Concept of End-capping for Reversed Phase Silica Material:
Remaining of Water-Solvated Silanol Groups
Etsuko Shearer1, Norikazu Nagae2, Tomoyasu Tuskamoto2
1. BioNik Inc. 3397-19 Obuchi, Fuji, Shizuoka 417-0801 Japan
2. ChromaNik technologies Inc. Namiyoke, Minato-ku, Osaka 552-0001Japan*Corresponding author email: nagae@chromanik.co.jp

www.bionikinc.com

www.chromanik.co.jp

A reversed phase column like C18 has been widely used in various fields such as pharmaceutical, medical, environmental, industrial and food area. Especially silica
based C18 column has showed high performance although an end-capping must be done enough in order to decease a peak tailing of a basic compound caused by aresidual silanol group on the silica surface. An End-capping has been recognized to be an important factor for a silica based reversed phase column. In the beginning
residual silanol groups after C18 bonding were reduced by bonding with trimethylsilyl reagents (TMS). This end-capping method has been improved by various end-capping
reagents with not only a monofunctional group but also a difunctional group, a high reaction temperature or a long reaction time. In the past ten years some column
manufacturers have developed the unique method that positive charge added on the silica surface suppressed influence of residual silanol groups against a basic compound.
In this study, an end-capping was tried by only heating as an unprecedented method. Silica gel bonded with only octadecyl group was applied heating at over 200 degree Celsius
under a nitrogen atmosphere. After heating C18 silica gel was kept under the solution of methanol/water=50/50 at 50 degree Celsius for 2 hours. Then an activity of residual
silanol groups were evaluated to separate basic compounds. The C18 silica gel heated under the optimum conditions showed a symmetrical peak of a basic compound such as
amitriptyline, although it included silanol groups. It was considered that heat treatment controlled silanol activity against a basic compound. It was also observed that retentionof basic compounds was more than ten times longer to compare with a conventional end-capped C18 stationary phase. Furthermore effect of both pH and salt concentration ofa mobile phase for retention of samples was studied. As a result, an ion exchange interaction between a silanol group and a basic compound definitely occurred on the proposed
C18 stationary phase. Heating could control silanol activity against a basic compound. Consequently residual silanol groups controlled its activity could change selectivity of basic
compounds without peak tailing.

Separation of basic compounds withammonium acetate: Effect of pHExperiment

1H

Column A
Column BOO

Isolated Silanol
HOSiSiVicinal Silanol
OSiPyridine peak
60%
CH3OH/H2O

Geminal Silanol

Our hypothesis

3.41a)

2.67b)%C: 16.8%
12.11Consequencec)• Decrease density of alkyl groups and make hydrophobicitylow.
• Control activity of silanol groups near only hydrophobic site

%C: 19.5%

05Retention Time / min1200 mM
1100 mM
150 mM

Bonded waterOHSiOOOOO

SiOO

Si3OSi１b)

OHO

3 C18 with heat treatment

05Si－H2O

SiSiO+H2O

Silanol groupNH

303540 min

23214

3

N=12,00054

N=12,500

254

N=13,000

225 mM354

N=13,500

232Column: C18 with heat treatment,
Sunrise C18-SAC 150 x 4.6 mm
Mobile phase:
Acetonitrile/ ammonium acetate (70:30)
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Sample: 1=Uracil, 2=Toluene, 3=Propranolol,
4=Nortriptyline, 5=Amitriptyline

54

68

1054

12 14 16 18 20 22
Retention time / min24

N=12,000

26

N

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

ToluenePropranolol

2.0

2.5

3.0log [buffer concentration]

1520log k = −3=Nortriptyline

4=Amitriptyline

Nortriptyline

Amitriptyline

N (ami)zBz
V1log[ A] + B log[ A] + log r +log K ABzAzAVm z A

A：ion in eluent，B：ion of sample， k：retention of B，K：selection coefficient
ZA, ZB：charge，Vr, Vm：resin volume and mobile phase volume in column

5=Toluene

OO

SiNH

CH3OH

4NH

23CH3NCH3Conclusion

CH3

C18 with TMS Sunrise C18

1High temp.

5Retention increase by ionexchange interaction

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

Siloxane

Warped siloxane

25CH3OLow temp.

2=Propranolol

20

N=11,500

CH3OSiOOHNO
Si

10Retention time / minN
H

Separation of antidepressants
1=UracilOHO②

Change of silanol groups by heat treatment
OHHNSunrise C18-SACc)

O

C18 without heattreatment②
１

Desorption ofsilanol group(800 C)O

Mobile phase: 30% CH3OH/H2O
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Sample: 1=Uracil②=Pyridine
3=PhenolOH

Sunrise C18a)

Desorption ofbonded water(400 C)H
HOOOO

HOSiSiH

１HSiOOOHOO

H

H

C18 with TMSDesorption ofabsorbed water(105  150 C)H

15

5Relationship between bufferconcentration and retention

3②H

103log kO

420

Comparison of pyridine peak shape

HAbsorbed water

5 N=11,500

Separation of basic compounds with ammonium
acetate: Effect of salt concentration

0Variation of water on silica surface

510 mM
15An ammonium ion makes a peak shapeof a basic compound sharp.

23

1

10

N=11,500

430

Column (b)+TMS%C: 18%d)54

pH 6.8

Column: a) 14%, b) 17%, C) 19%
C18 with heat treatment,d) 17% with end-capping (TMS)
150 x 4.6 mm
Mobile phase:
Methanol/25mM Phosphate buffer pH7.5(50:50)
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Sample: 1=Procainamide,
2=N-Acetylprocainamide%C: 14.8%

N=12,500

1 21

Comparison of carbon content• Peak tailing of a basic compound is caused by the state ofsilanol groups, not by existence of silanol groups on silica
surface.
• Silanol groups near a strong hydrophobic site don’t occur
an ion-exchange interaction etc. rapidly.

543pH 5.9– Column: 14%: 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 solutionO

N=13,000

41 2pH 5.0• Evaluation

HColumn: 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

Only acetic acid533

pH 4.1– Temperature: 200 C, Treatment time: 48 hours– As reference, prepare C18 phase without heat treatment

OH1 2• Heat treatment in a vacuumColumn C18.00

O– Carbon content: 14%, 17%, 19%– As reference, prepareC18 phase with end-capping (TMS)

15.11

H

1 2pH 3.5

• Bonded Octadecylsilane

O54

3– Pore diameter: 12 nm, Specific surface area: 340 m2/g, Particle size: 5 µm

14.03

O

Si

13.72

O

Si2pH 2.7

13.08

Si

• Silica gel

N [amitriptyline]

Form of silanol groups and peak shape ofpyridine

1C18 with heat treatment Sunrise C18-SAC5• Condensation of silanol group starts from near 100 C.

Acetonitrile/20m phosphate buffer pH6.0 (80:20)• Vacuum heat treatment makes an amount of silanol group low bywater deprivation and siloxane produces.
• Under 400 C, siloxane reverts silanol groups by rehydration.
• Over 400 C, rehydration do not occur.

20Control of silanol group

5101520

32530
3540
Retention time / min415

Prevention of tailing for basic compoundO

heatHOOOSiSiSiHOH

SiIncrease of retention of polar and basic compounds, in spiteof no change of retention of a neutral compound

2

Change toSiloxane

35HHOOSi

41HH

Siloxane away fromhydrophobic site is rehydrated.
Consequently silanol groupproduces and it is unaffectedby hydrophobic site.

Acetonitrile/20mM phosphatebuffer pH3.0 (50:50)

2 3

Active silanolgroups for basic
compounds

45

5055

60

65

Control of retention by mobile phase pH

After heat treatment
Siloxane near hydrophobic site(C18) is kept at the same stateand no silanol goup produces
by rehydration.

◆ Residual silanol group in proposed C18 phase

4SiOOOSiSiO
SiAcetonitrile/20mM
phosphate buffer pH4.5(50:50)

Column: 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

H1Si

5

Acetonitrile/20mM phosphate buffer pH6.0 (80:20)
234made retention of polar compounds including
basic compounds large.
◆ Silanol group controlled its activity by heattreatment did not make peak of basic compounds
be tailing.
◆ Furthermore, ion-exchange interaction wasrecognized and retention of basic compounds
could be changed by pH or salt concentration in amobile phase.
◆ Proposed C18 phase was effective to separation ofa metal 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.