Novel end-capping method with silyl-reagent
including ethylene chain
N. Nagaea, T. Tsukamotoa, R. Koyamaa, E. Shearerb[a] ChromaNik Technologies Inc. Namiyoke, Minato-ku, Osaka Japan[b] BioNik Inc. Obuchi, Fuji, Shizuoka Japanwww.bionikinc.com

www.chromanik.co.jp

Trimethylsilyl reagents (TMS) have been widely used as end-capping reagents for silica-based reversed-phase LC packing. Recently, difunctional silyl reagents such as
hexamethyldichlorotrisiloxane have been used as end-capping reagents to reduce residual silanol groups on silica gel surface. Some brands of C18 end-capped withhexamethyldichlorotrisiloxane show an excellent peak shape for strongly basic compounds.
As a new concept, 1,2-bis(dimethylchlorosilyl)ethane, which was bonded the methyl groups of two trimethylchlorosilane reagents, was thought to function as a denser endcapping reagent than trimethylchlorosilane. In this study, we evaluated a reversed-phase packing with both 1,2-bis(dimethylchlorosilyl)ethane as an end-capping reagent and ahigh temperature reaction to convert the silanol groups into siloxane bonds. Core-shell silica was used as the silica base material for the evaluation of the two bonding
techniques described above. The proposed core shell C18 phase was compared with two other core shell C18 phases for stability under both acidic and basic pH conditions
and inertness towards acidic, basic and metal chelating compound. An ethylene chain is expected to provide high stability of its packing material under basic pH condition
and 1,2-bis(dimethylchlorosilyl)ethane as an end-capping reagent contains an ethylene chain in the molecule, so that the proposed core shell C18 column was compared withhybrid type core shell C18 columns which were known to be more stable under basic pH conditions than a conventional C18 column.
As a result, the proposed C18 column not only exhibited almost same stability as hybrid type core shell C18 columns under basic pH conditions, but also showed much higher
stability than hybrid type core shell C18 columns under acidic pH conditions. Regarding inertness towards basic and metal chelating compounds, the proposed core shell C18column exhibited almost the same peak shape as hybrid type core shell C18 columns. However, for formic acid as an acidic compound, the proposed core shell C18 column
showed a symmetrical peak while hybrid type core shell C18 columns showed a terrible tailing peak.

Novel end-capping method【As a new concept】

Conventional end-capping
C18

C18

C18

C18

C18

End-capping layer

1,2-Bis(chlorodimethylsilyl)ethane╋High temperature end-capping
C18

C18

C18

C18

C18

End-capping layer* A high temperature reaction (more than 200 oC) causes
the silanol groups to change the siloxane bonds.

ArmorShell C18: 2.6 μm of core shell silica bonded with trifunctional C18 and end-capped using the above novel end-capping method.
C18 of Company A and Company B uses hybrid core-shell silica in which ethylene chains are bonded on the silica surface. (Kinetex EVO C18 and PoroShell HPH C18)

Comparison of a peak shape【Oxine】【Amitriptyline】【Pyridine】

Company A Hybrid C18, 5 μm

314Company A Hybrid C18, 5 μm

1223【Formic acid】

Company A Hybrid
2C18, 5 μm

Company A HybridC18, 5 μm 32

TF=1.31

TF＝2.03

TF=6.48

113Company B Hybrid C18, 2.7 μm3212Company B Hybrid C18, 42.7 μm

1

TF＝1.34

TF=1.29

22Company B HybridC18, 2.7 μm
TF=5.24

1

ArmorShell C18, 2.6 μm

3231

14

ArmorShell C18, 2.6 μm

TF＝1.32

2

ArmorShell C18, 2.6 μm

TF=1.24

1

12Company B HybridC18, 2.7 μm

332ArmorShell C18,2.6 μm

TF=1.29102480646

80

2Retention time/min

Retention time/min

Condition (pyridine)
Column dimension: 150 x 4.6 mm
Mobile phase: CH3OH/H2O=30/70
Flow rate: 1.0 mL/minTemperature: 40 ºC
Detection: UV@250nm
Sample: 1 = Uracil, 2 = Pyridine, 3 = Phenol

4ONHNHOHOCH3CH3NHCH3ONN

46Condition (formic acid)
Column dimension: 150 x 4.6 mm
Mobile phase: CH3CN/0.1% H3PO4=2/98
Flow rate: 1.0 mL/min*Hybrid C18s showed a very poor peak
Temperature: 40 ºC
Detection: UV@210nm
shape for formic acid. It is doubted that
Sample: 1 = Formic acid (red)some amines as a by-product2 = Acetic acidremained on the surface of packing
3 = Propionic Acidmaterials.

NN
CH3

2Retention time/min

33N

0Condition (oxine)
Column dimension: 150 x 4.6 mm
Mobile phase: CH3CN/20mM H3PO4=10/90
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Detection: UV@250nm
NSample: 1 = 8-Quinolinol (Oxine)
OCH
OH
2 = Caffeine H C
NCH3

NH

6Retention time/min

Condition (amitriptyline)
Column dimension: 150 x 4.6 mm
Mobile phase: Acetonitrile/20mM phosphate buffer pH7.0=60/40
Flow rate: 1.0 mL/min
Temp.: 40oC
Detection: UV@250nm
Sample: 1=Uracil, 2=Propranolol, 3= Nortriptyline, 4=Amitriptyline

OHO

2CH3

Comparison of stability under acidic and basic pH conditions【Acidic pH】【Basic pH】
Durable test condition
Column size: 50 x 2.1 mm
Mobile phase: CH3CN/1.0% TFA, (pH 1)=10/90
Flow rate: 0.4 mL/min
Temperature: 80 ºC

7 to10 times more stable

Relative retention factor/%

10090%

80

Measurement condition
Column size: 50 x 2.1 mm
Mobile phase: CH3CN/H2O=60/40
Flow rate: 0.4 mL/min,Temperature: 40 ºC
Sample: 1 = Uracil (t0), 2 = Butylbenzene

60● ArmorShell C18● SunShell C18● Prominert Biphenyl● Company A hybrid C18● Company B hybrid C1840200050100150

Elution time/hConclusion
Conclusion

200250300*The proposed C18 (ArmorShell C18) is over 7 times morestable under acidic pH condition than company A and B
C18s at a 10% reduction in retention factor.

120

Durable test condition
Column size: 50 x 2.1 mm
Mobile phase: CH3OH/10mM Ammonium bicarbonate (pH10.5)=30/70
Flow rate: 0.8 mL/min
Temperature: 60 ºC

100

Relative plate/%

12080● ArmorShell C18● Company A hybrid C18● Company B hybrid C1860Measurement condition
Column size: 50 x 2.1 mm
Mobile phase: CH3CN/H2O=60/40
Flow rate: 0.2 mL/min
Temperature: 40 ºCSample: 1 = Butylbenzene

402000100200300

400500600700*The proposed C18 (ArmorShell C18) exhibits similar stability
to company A and B C18s under basic pH condition.

Elution time/h✓ The proposed end-capping method improved not only a peak shape but also stability under both acidic and basic pH conditions.
✓ The ethylene chains in or on the silica gel are known to improve stability under basic pH condition. But the proposed end-capping reagent including an ethylenegroup improved stability under not only basic pH but also acidic pH condition. It is considered that this result was not due to the effect of the ethylene chain, which
has been said so far, but due to the increase in the efficiency or density of end-capping.
✓ Company A states that a silyl reagent with dimethylamine as a functional group was used in the patent examples. So, it is considered that the dimethylamine
remaining on the silica surface adsorbed formic acid, causing severe tailing of the formic acid peak.