A Novel End-capping for Reversed Phase for LC/MSSunShell and Sunniest column
Etsuko Shearer1 Norikazu Nagae2, and 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

Proposed end-capping
https://pyvot.tech/Distributor in the US

An End-capping has been recognized to be an important factor fora silica based reversed phase column. In this study, not onlybonding with an end-capping reagent but also conversion ofsilanol groups to siloxane bond by heating were evaluated as anend-capping.

End-capping with hexamethyldichlorotrisiloxane and TMS on C18 silica

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

C18

Comparison of 2 kinds of end-capping

Sunrise C18-SAC

Conventional end-capping

Final TMS
Silanol Activity
ControlTechnology
Silanol groups withhigh absorption
activity

HeatingC18

Stationary phase
C18

C18

C18

C18

End-capping layer
Change over tosiloxane bondHOSiEnd-capping
reagentOSi
SiOOOOSi

SiOSi

Si OO OSiO O SiBoth Sunniest double end-capping and Silanol
Activity Control

OSiOO OHOSiOOOSi OOSiSiOOSiSi
SiO O
HO OOOSiOO OO OOSi
OSiOSi
OOSi
Si
SiSi
OSi
O O
O Si
O Si O OOO O O O
OOOOO
SiOOO
Si

Si

Si O

SiSiSi

Stationary phase
O H

End-capping reagent moves like a Geometrid caterpillar, so that afunctional group on the tip of the arm can bond with a silanol groupwhich Is located anywhere.

O H

Non hydrated silanol group by influence of hydrophobicity of alkyl groups

C18

Hydrated silanol group without influence of alkyl groups

C18

C18

C18C18MorehydrophobicEnd-capping layer

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

We named this end-capping method as Sunniest double end-capping.

Stability under acidic pH condition

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

TIC of ＋Q1

10,000th

Column: SunShell C18 2.6 µm, 50 x 2.1 mm
Mobile phase: A) 0.1% trifluoroacetic acid pH 2.0B) Acetonitrile
Gradient program: Time (min)
03
3.1 5

9,000th8,000th7,000th6,000th
5,000th4,000th%B

1,000th
1st injection
1

2
34Retention time/min

90

Flow rate: 0.5 mL/min
Temperature: 40 oC
Detection: UV@270nm
Injection volume: 0.5 μL
Sample: 1=Benzydamin (0.5 mg/mL),
2=Ketoprofen (0.04 mg/mL),
3= Indomethacin (0.05 mg/mL)

3,000th
2,000th01010Column size: 50 x 2.1 mm
Mobile phase: A) 0.1% acetic acidB) Acetonitrile
Gradient program:

10Time (min)0157Brand L C18%B55100100

SunSell C18

Flow rate: 0.4 mL/min
Temperature: 40 ºC
MS： ABI API-4000
Ionization: Turboionspray (cation)
Measurement mode: Q1 Scan m/z 100-1000

5Simultaneous Analysis of Pesticide (LC/MS)

IDA measurement using SunShell C18, 2.6 μm 150 x 0.075 mm i.d.
and Nano LC/MS

Positive

Sample: Tryptic digest of BSA, 30 μg on column
Detection: QTRAP5500
Detection mode: IDA measurement
HPLC：Ultimate 3000 RSLC nano
Trap column：
Acclaim PepMap 100, 3 μm, 20 x 0.075 mm i.d.
Analytical column:
SunSehll C18, 2.6 μm, 150 x 0.075 mm i.d.
Mobile phase:
To trap column, 0.1% TFA (Sample load)
To anal. Column, A) 0.1% Formic acid,B) 0.1% Formic
acid/Acetonitrile=20/80
Gradient in 25 min

Sample No.
187

150

100

501Courtesy of a pharmaceutical company in JapanminColumn: SunShell C18 2.6 µm, 100 x 2.1 mm
Mobile phase: A) 0.5 mM Ammonium acetate in H2OB) 0.5 mM Ammonium acetate in CH3OH
A/B = 95/5 – 1 min – 50/50 – 14 min – 2/98 – 5 min – 2/98 – 0.1 min – 95/5 –（Equilibrating,10 min）, v/v
Flow rate: 0.2 mL/min
Temperature: 40 oC
Detection: LC/MS/MS (QTRAP®4500: ESI, MRM)
Injection volume: 5 µL (STD 10ppb)

Conclusion◆ Hexamethyldichlorotrisiloxane wasused 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 tosiloxane bonding by heating on C18silica.
◆ Stability under acidic pH condition wasimproved by a proposed end-capping.
After verification with the database, the sequence identification rate of BSA was over60%, which was a higher identification rate than conventional nano-columns.
*