A New Concept of End-capping for Reversed Phase Silica Material:
Conversion of Silanol Groups to Siloxane Bond by Heatingwww.bionikinc.com

Norikazu Nagae1, Etsuko Shearer2, Tomoyasu Tuskamoto1
1. ChromaNik technologies Inc. Namiyoke, Minato-ku, Osaka 552-0001Japan
2. BioNik Inc. 3397-19 Obuchi, Fuji, Shizuoka 417-0801 Japan*Corresponding author email: nagae@chromanik.co.jp

https://pyvot.tech/

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 peaktailing of a basic compound caused by a residual silanol group on the silica surface. An Endcapping 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 withtrimethylsilyl reagents (TMS). This end-capping method has been improved by various endcapping reagents with not only a monofunctional group but also a difunctional group, a highreaction 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, not only bonding with an end-capping reagent but also conversion of silanol groups
to siloxane bond by heating were tried as an end-capping. After bonding with C18 on a silica gel,a difunctional end-capping reagent like hexamethyldichlorotrisiloxane was used as a first endcapping, then trimethylchlorosilane was used as a second end-capping. These two end-capping
reactions were done at over 200 degree Celsius under a nitrogen atmosphere. The C18 with theabove end-capping was more hydrophobic and showed a longer retention time higher than theC18 with the above end-capping done at 40 degree Celsius lower temperature although both
C18s showed a good peak shape for a basic compound. Conversion of silanol groups to siloxanebond by heating made hydrophobicity high. The peak shape of a basic compound and stability
under both acidic and basic pH conditions were evaluated.

End-capping with hexamethyldichlorotrisiloxane and TMS on C18 silica

www.chromanik.co.jp

Another end-capping with heating on
C18 silica, reduce of silanol groups
Typical no end-capping C18

Final TMS

C18Sunrise C18-SAC

Silanol ActivityControl
Technology
Silanol groups withhigh absorption
activity

HeatingO

Si

Si

Si

SiOO

SiOO

Si

Si
SiEnd-capping layer

Both Sunniest end-capping and Silanol Activity ControlOSiHOOSiSiOOSiOO OOOSiSi

Stationary phaseOO OHOOOSiSiSiO O
O O OOOSi O
O Si OSiOOSiOSiOOOOOO

SiSiOOOSiOSiOSiSiSi O
Si OO OSiOSiOOOSiOOC18OOO

End-capping reagent moves like a Geometrid caterpillar, so thata functional group on the tip of the arm can bond with a silanolgroup which Is located anywhere.
We named this end-capping method as Sunniest end-capping.

Relationship between retention factor
and carbon loading

Comparison of amitriptyline peak

Carbon loading of only C18

7.0%
7.3%

Carbon loading
after end-capping

SunShell C18, 2.6 μm

10.4

7.31257.7%

Ascentis Express C18, 2.7 μm

9.78.0133

PoroShell C18 EC, 2.7 μm

9.08.5135

Cortecs C18, 2.7 μm

7.7

7.3113(not cut off C18
chain by heating)

Yes
10.6

No
9.5

*Base material, core shell silica 2.6 μm; column dimension, 150 x 4.6 mm; mobile phase,methanol/water=75/25; flow rate, 1.0 mL/min; sample, amylbenzene;

Accucore C18, 2.6 μm

7.4

8.8

130

Kinetex C18, 2.6 μm

5.44.9102oX C(X-40) oC

10% reduce
24681012141618202232

N4=16,700, TF4=1.20

a. Retention factor of amylbenzene, mobile phase; methanol/water=75/25, 40 oC.
b. Measured after C18 packing material was pulled out of a column.
c. Measured using C18 materials sintered at 600 degree Celsius for 8 hours.

Both Sunniest end-capping and Silanol Activity Control

24

N4=12,800, TF4=1.89
4O H

Non hydrated silanol group by influence of hydrophobicity of alkyl groups

O HHydrated silanol group without influence of alkyl groups

44123456SunShell C18 2.6 μm
Company A C18 2.7 μm

N4=5,200, TF4=2.61

Company P C18 2.6 μm

N4=5,300, TF4=2.73

Company T C18 2.6 μm

N4=4,800, TF4=3.12

Company W C18 2.7 μm

78

N4=3,400, TF4=3.24
9Regarding retention factor, a reversed result was obtained. Lesssilanol group made hydrophobicity of stationary phase high.

SunShell C18 showed the largest retention factor althoughcarbon loading was not the highest.
It is considered that less residual silanol group madehydrophobicity of a stationary phase high, then maderetention factor large.

C18C18C18End-capping layer

Morehydrophobic80
SunShell C1860

Company S C18

Durable test condition
Column size: 50 x 2.1 mm
Mobile phase:
CH3OH/20mM Sodium
borate/10mM NaOH=30/21/49(pH10)
Flow rate: 0.4 mL/min
Temperature: 50 ºC

Bleeding test using LC/MS＋Q1: 5.997 min to 7.999 min of Sample

TIC of ＋Q1

Company P C1840Company T C18
Company A C18

20

01,000 2,000 3,000 4,000 5,000 6,000

Measurement condition
Column size: 50 x 2.1 mm
Mobile phase:
CH3OH/H2O=70/30
Flow rate: 0.4 mL/min
Temperature: 40 ºCSample: 1 = Butylbenzene

Brand L C18

SunSell C18

Elution volume/mL

Column size: 50 x 2.1 mm
Mobile phase:A) 0.1% acetic acidB) CH3CN
Gradient:
Time: 0min 1min 5min 7min
%B: 5% 5% 100% 100%
Flow rate: 0.4 mL/min
Temperature: 40 ºC
MS： ABI API-4000
Ionization:
Turboionspray (cation)
Measurement mode:
Q1 Scan m/z 100-1000

Company S C18 2.7 μm

10Retention time/min

Column dimension, 150 x 4.6 mm; mobile phase,acetonitrile/10mM ammonium acetate pH6.8 = 40/60;
flow rate, 1.0 mL/min; temp.: 40oC; sample, 1=uracil,
2=propranolol, 3=nortriptyline, 4=amitriptyline.

CH3NCH3

Amitriptyline

Retention time/min

X oC was too high to keep carbon loading of initial C18. (X-40)
oC could keep carbon loading. As a result, carbon loading
after end-capping at (X-40) oC was higher than that at X oC.

C18

A basic compound shows no tailing on Sunrise C18-SAC
because hydrated silanol groups don’t make a basic
compound tailing as well as silica column on HILIC modeshows no tailing for a basic compound.

040Amylbenzene

0147.0%(cut off some C18
chains by heating)

Silanol activity control
Retention factor (k)Retention Carbon Specificfactor aloading b surface area b, c(%)(m2/g)(X-40) oC

C18SiO

Relative plate of butylbenzene /%X oC(more than 200 oC )Stationary phase
C18

Change over tosiloxane bondOSi

4

End-capping reaction
temperature

C18

C18

HOEnd-capping
reagent

100(reaction at two different temperature)

Conventional end-capping
C18

Stability under basic pH condition

Carbon loading and retention factor

Comparison of 2 kinds of end-capping

SunShell C18 showed the highest theoretical plate (N) andthe lowest tailing factor. Highly-effective end-capping
including heat treatment (silanol activity control) reduced
tailing for a basic compound. As a consequence, highefficiency for a reversed column was achieved by highlyeffective end-capping.

Conclusion
Hexamethyldichlorotrisiloxane was used as an end-capping reagent for a first end-capping step. Thentrimethylchlorosilane (TMS) was used as an end-capping reagent for a second end-capping step.
Silanol groups were changed to siloxane bonding by heating on C18 silica.
End-capping done by both bonding of a silylation agent and conversion from silanol groups tosiloxane bond leaded to not only high efficiency for basic compounds but also increase in retention.
Stability under both acidic and basic pH conditions increased due to proposed end-capping.