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 tobe more suitable than a conventional C18 phase for separation ofhydrophobic structurally related isomers such as vitamin E or vitamin
K1. In this study, separation factor of beta-tocopherol and gammatocopherol which were structurally related isomers was evaluated tovary both a pore diameter of the superficially porous silica and aligand density of the C30 group. Regarding a pore diameter, 12 nm
showed the largest separation factor of beta and gamma-tocopherol
among 10nm, 12 nm and 16 nm. Regarding a ligand density, thehigher a ligand density, the larger a separation factor of beta andgamma-tocopherol. However, when a ligand density was too high,much high hydrophobicity caused peak tailing and a drop oftheoretical plate. The most suitable ligand density existed for thehighest resolution. Finally separation of cis and trans-vitamin K1 wascompared and the same result as separation of beta and gammatocopherol was obtained.

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.

241115230216110714280314220713081112

Particle diameter (μm)

2.6

2.6

2.6

2.6

2.6

2.6

2.6

Thickness of porous layer (μm)

0.5

0.5

0.50.5

0.5

0.5

0.5

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

Ligand (C30) density (μmol/m2)

1.79

1.55

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

r1 (5 nm)

1.5 nm04

per unit area

2 3HOO43 = β-tocopherol,HO

1

Conventional C18, 5 μm

2, 3O

4 = α-tocopherol.
HO4O

Separation factor of β/γ-tocopherol

Company B C30, 5 μm

1.07001.060011.0500

1.04001015202530

Retention time / minFigure 1: separation of vitamin E.

Column dimension, 250 x 4.6 mm; mobile phase, methanol/water = 97/3; flow rate, 1.0 mL/min; temperature, 30 oC; detection, UV295 nm.

Table 1: Physical properties and separation factor and resolution of vitamin E
Company A C30, 5 μm

Company B C30, 5 μm

Conventional C18, 5 μm

Specific surface area (m2/g)

300

200300

Pore diameter (nm)132013Carbon loading (%)

182018Ligand density (μmol/m2)

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

---

*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.

41035
Separation factor of β/γ-tocopherol

50.5

11.5

32

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

41

10.2 - 12.0 nm of pore

1.065032

15.0 - 15.5 nm of pore

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

2

1.05504

Too highhydrophobicity
to perform agood peak.

1.05001.04501.040010

1.0350
00.5

1

1.5

2

C30 ligand density (μmol/m2)

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

2.5

1214C30 batch# 230216 (SunShell C30)

Company C core shell C30

Separation factor:1.042

35 oC

Separation factor:1.024

Separation factor:1.065

30 oC

Separation factor:1.031

Separation factor:1.094

25 oC

Separation factor:1.042

20 oC

Separation factor:1.061

15 oC

Separation factor:1.080

Separation factor:1.130

5transcis

10

15

Retention time / min

2025030

2transcis

4

68

10

Retention time / min

34
Retention time / min

Column, 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).

Table 3: Separation factor and resolution of vitamin K1
Vitamin K1 isomers (trans and cis)

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

3
2

1.0350

0Batch: 081112
Ligand density: 2.11 μmol/m2
N3=15,500
TF3=1.34

4

1.05501.045012Column, 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.

03215.0 - 151 nm of pore

10Figure 5: Separation of vitamin K1 isomers.

10.2 -12.0 nm of pore

1.06508Figure 4: Separation of vitamin E.

O

2 = γ-tocopherol,

16Retention time / min

1.5 nmr2 (7.5 nm)

2Separation factor:1.167

HO434241215

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 volumeof per square meter.
e: Ligand density in the volume of 1.5 nm thickness inside of pore.

sample,1 = δ-tocopherol,

3

Separation factor of β/γ-tocopherol: 1.064
Resolution of β/γ-tocopherol: 2.01
2Batch numberCore shell silica Specific surface area a (m2/g)

12

1

C30 batch# 230216(SunShell C30)

Table 2: Physical properties and separation factor of vitamin E and K1

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

Separation of Vitamin E and K1 isomers

567Figure 3: Comparison of theoretical plate and USP tailing factor.

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.

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

Separation factor

OResolution

C30 batch#
230216Company C
C30

C30 batch#
230216Company C
C30

35 oC

1.042

1.024

1.23

---

30 oC

1.065

1.031

1.94

---

25 oC

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

Vitamin K1 (trans)

H 3C
HH 3CHCH3

Vitamin K1 (cis)HCH 3CH 3CH 3O

CH 3CH 3OOCH3CH 3HCH3HCH 3HConclusion* C30 phase could separate β-tocopherol and γ-tocopherol although C18phase could not separate such isomers.
* The higher C30 ligand density, the larger separation factor of β-tocopheroland γ-tocopherol. However, high hydrophobicity on the particle surface madepacking state bad. As a result, more than 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 theparticle surface in order to perform good separation (high theoretical plateand no tailing). Proposed C30 phase (batch# 230216) showed better
separation of vitamin K1 isomers than company C C30.