Evaluation of C30 Phase Bonded on
Superficially Porous Silica
www.chromanik.co.jp

Norikazu Nagae, Tomoyasu Tsukamoto and Shun Kojima
ChromaNik Technologies Inc. Namiyoke, Minato-ku, Osaka Japan 552-0001*Corresponding author email: nagae@chromanik.co.jp

A long alkyl group like C30 (triacontyl group) phase has been known to be more suitable than a conventional C18 phase for separationof hydrophobic structurally related isomers such as vitamin E or vitamin K1. In this study, separation factor of beta-tocopherol andgamma-tocopherol which were structurally related isomers was evaluated to vary both a pore diameter of the superficially porous
silica and a ligand density of the C30 group. Regarding a pore diameter, 12 nm showed the largest separation factor of beta andgamma-tocopherol among 10nm, 12 nm and 16 nm. Regarding a ligand density, the higher a ligand density, the larger a separation
factor of beta and gamma-tocopherol. However, when a ligand density was too high, much high hydrophobicity caused peak tailingand a drop of theoretical plate. The most suitable ligand density existed for the highest resolution. Finally separation of cis and transvitamin K1 was compared and the same result as separation of beta and gamma-tocopherol was obtained.

The problem of totally porous C30 column
1Company A C30, 5 μm

324

1Company B C30, 5 μm

Table 1: Physical properties and separation factor and resolution of vitamin EColumn dimension, 250 x 4.6 mm;
mobile phase, methanol/water = 97/3;
flow rate, 1.0 mL/min;temperature, 30 oC;
detection, UV295 nm;
sample,1 = δ-tocopherol,
HO

2 3O

2 = γ-tocopherol,

4HOO

1

Conventional C18, 5 μm

2, 3HO

1015

20

2530

HO35O

Retention time / min

Separation factor of β/γ-tocopherol

Evaluation of C30 phases bonded on Core shell silica
Bonding method of C30 phase
1) Triacontyltrichlorosilane was bonded on core shell silica at reflux in toluene.
2) As an end-capping, trimethylchlorosilane was reacted at reflux in toluene.
Table 2: Physical properties and separation factor of vitamin E and K1
241215241115230216110714280314220713081112

2.6

2.6

2.6

2.6

2.6

2.6

2.6

0.5

0.50.5

0.5

0.5

0.5

0.5

Specific surface area (m /g)

112

106

96.1

79.6

79.6

73.5

73.5

Pore volume a (mL/g)

0.287

0.286

0.288

0.299

0.299

0.285

0.285

Average pore diameter a (nm)

10.2

10.8

12.0

15.0

15.0

15.5

15.5

Carbon loading of only C30 (%)

6.59

5.48

6.08

4.43

5.40

4.10

5.20

Carbon loading including end-capping (%)

7.56

6.57

7.04

5.30

6.05

4.70

5.86

Thickness of porous layer (μm)
Core shellsilicaa2

Ligand (C30) density (μmol/m2)

1.791.55

1.91

1.64

2.03

1.64

2.11

Separation factor of β/γ-tocopherol b

1.0625

1.05701.06401.0376

1.0626

1.04151.0629Separation factor of cis /trans vitamin K1 c

1.08561.08291.09441.0692

1.1054

1.07581.1174

Volume of 1.5 nm thickness inside pore d (μL/m2)

1.279

1.292

1.313

1.350

1.350

1.355

1.355

Ligand (C30) density e (mol/L)

1.400

1.200

1.455

1.215

1.500

1.208

1.555

a: Measured by Quantachrom Autosorb.
b: Mobile phase, methanol/water=97/3; temperature, 25 oC.
c: Mobile phase, methanol/water=96/4; temperature, 25 oC.
d: Postulated a pore as a cylinder with a same diameter, listed as
the volume of per square meter.
e: Ligand density in the volume of 1.5 nm thickness inside of pore.

r1 (5 nm)

1.5 nm

24

68

1.9

3.7

3.0

Theoretical plate

16,5006,100

15,800

USP tailing factor

1.02

1.63

1.04

Separation factor of β/γ-tocopherol

1.048

1.062

---

Resolution of β/γ-tocopherol

1.30

0.85

---

31214

1.0600Batch: 081112
Ligand density: 2.11 μmol/m2
N3=15,500
TF3=1.34

4

11.0550
1.0500Batch: 220713
Ligand density: 1.64 μmol/m2
N3=18,100
TF3=1.25

3
21.04504

11.0400

0.5

11.5

2

3

2

C30 ligand density (mol/L)
1.0700Batch: 280314
Ligand density: 2.03 μmol/m2
N3=14,000
TF3=1.06

4

1
10.2 - 12.0 nm of pore

1.065015.0 - 15.5 nm of pore

1.06003Batch: 110714
Ligand density: 1.64 μmol/m2
N3=18,700
TF3=1.11

2

1.05504
1Too highhydrophobicity
to perform agood peak.

1.05001.0450
1.04000121.0350
00.5

11.5

22.5

34Retention time / min567

Figure 2: Comparison of theoretical plate and USP tailing factor.

C30 ligand density (μmol/m2)

Column dimension, 100 x 2.1mm; mobile phase, acetonitrile/water = 60/40; flow rate,
0.3 mL/min; temperature, 30 oC; peak, 1 = uracil, 2 = ethylbenzoate, 3= acenaphthene,
4 = butylbenzene.

Figure 1: Relationship between 2 kinds ofligand density and separation factor.

Company C core shell C3035 oC

25 oCSeparation factor:1.042

Separation factor:1.130

20 oC

Separation factor:1.061

oSeparation factor:1.080

trans0510cis

15

Retention time / min20

Figure 4: Separation of vitamin K1 isomers.

15 C
25

Vitamin K1 isomers (trans and cis)
Separation factor

Separation factor:1.031

Separation factor:1.094

Separation factor:1.167

Table 3: Separation factor and resolution of vitamin K1

Separation factor:1.024

30 oC

Retention time / minFigure 3: Separation of vitamin E.

Column, C30 batch# 230216, 2.6 mm 150 x 3.0 mm; mobile phase,methanol/water = 97/3; flow rate, 0.43 mL/min; temperature, 25 oC;
detection, UV295 nm, sample, 1 = δ-tocopherol, 2 = γ-tocopherol,
3 = β-tocopherol, 4 = α-tocopherol.

215.0 - 151 nm of pore

Separation factor:1.065

3Separation factor of β/γ-tocopherol: 1.064
Resolution of β/γ-tocopherol: 2.01

18

Ligand density (μmol/m2)

10.2 -12.0 nm of pore

1.0650

Separation factor:1.042

101320

C30 batch# 230216 (SunShell C30)

4020

18

*Regarding a ligand density, number of moles per volume is considered to be better than number ofmoles per surface area. The higher a ligand density, the larger the separation factor of β/γ-tocopherol.
However, more than 2 μmol/m2 of ligand density caused the low theoretical plate and the tailing peak.

1213

Carbon loading (%)

1.070001.5 nm

r2 (7.5 nm)

per unit area

Separation of Vitamin E and K1 isomers
C30 batch# 230216(SunShell C30)

300

Pore diameter (nm)1.0350

Separation factor of β/γ-tocopherol

Batch number
Particle diameter (μm)

Conventional C18, 5 μm

200

*When the ligand (C30) density is too high, peak tailing occurs more and atheoretical plate is low. High ligand density on the particle surface is one ofreasons of a low theoretical plate and a tailing peak.

O

4 = α-tocopherol.
5Company B C30, 5 μm

300

3 = β-tocopherol,
40Company A C30, 5 μm
Specific surface area (m2/g)

030

24Resolution

C30 batch#230216

Company C
C30

C30 batch#230216

Company C
C3035 oC

1.042

1.024

1.23---30 oC

1.065

1.031

1.94---o25 C

1.094

1.042

2.88

0.91

20 oC

1.130

1.061

3.33

1.41

15 oC

1.167

1.080

3.79

1.78
OVitamin K1 (trans)

H 3C
H
H 3C

Vitamin K1 (cis)cis6Retention time / min8HCH3trans

10H

CH 3

CH 3

CH 3O

CH 3

CH 3OOCH3

CH 3

HCH3

H

CH 3

HColumn, C30 batch# 230216 (SunShell C30), 2.6 mm 100 x 2.1 mm, company C core shell C30, 2.6 mm 100 x 2.1 mm; mobile phase, methanol/water= 96/4; flow rate, 0.35 mL/min; temperature is described in a figure; detection, UV250 nm; sample, vitamin K1 isomers (trans and cis).

Conclusion* C30 phase could separate β-tocopherol and γ-tocopherol although C18 phase could not separate such isomers.
* The higher C30 ligand density, the larger separation factor of β-tocopherol and γ-tocopherol. However, high hydrophobicity on the particle surface made packing state bad. As a result, morethan 2 μmol/m2 of ligand density caused a low theoretical plate and a tailing peak.
* C30 phase bonded inside a pore with 12 nm diameter showed not only high ligand density per volume but also no too high hydrophobicity on the particle surface in order to perform goodseparation (high theoretical plate and no tailing). Proposed C30 phase (batch# 230216) showed better separation of vitamin K1 isomers than company C C30.