Evaluation of Optimized C30 Phase for Separationof Structurally Related Isomers
Tomoyasu Tsukamoto, Norikazu Nagae and Shun Kojima
ChromaNik Technologies Inc. Namiyoke, Minato-ku, Osaka Japan 552-0001

www.chromanik.co.jp

Separation factor of β/γ-tocopherol*Corresponding author email: tsukamoto@chromanik.co.jp
A long alkyl group like C30 (triacontyl group) phase has been
known to be more suitable than a conventional C18 phase forseparation of hydrophobic structurally related isomers such asvitamin E or vitamin K1. However, in many cases a C30 column
shows a tailing peak. This tailing is considered to be a reason
why C30 ligand density is too high. We have reported
optimization for a pore diameter of the superficially porous
silica and a ligand density of C30 phase [1]. In this study, theoptimized C30 phase was evaluated to compare with C18 phaseor the other C30 phase.
Regarding separation of cis and trans-vitamin K1, the optimized
C30 phase showed better separation than the other C30 phase
while a C18 phase could not separate them. The highertemperature, the worse separation of isomers. The optimized
C30 phase could separate cis and trans-vitamin k1 at over 30
degree Celsius although the other C30 phase couldn’t.
This C30 phase was applied for separation of some structural
related isomers, and showed higher resolution of isomers thanconventional C18 phase.

Optimization 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 1: Physical properties and separation factor of vitamin E and K1Batch numberCore shellsilica241215241115230216110714280314220713081112

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

0.5

0.5

0.5

0.5

0.5

Specific surface area a (m2/g)

112

106

96.1

79.6

79.6

73.5

73.5

aPore volume (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

1
10.2 - 12.0 nm of pore

1.0650

C30 batch# 230216(SunShell C30)

15.0 - 15.5 nm of pore
1.06001.05501.04503

1.04004

1.0350
00.5

1

1.5

2

2.5

C30 ligand density (μmol/m2)

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

6.59

5.48

6.08

4.43

5.40

4.10

5.20

Retention time / min

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 b1.0625

1.05701.06401.0376

1.0626

1.04151.0629Volume 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 thevolume of per square meter.
e: Ligand density in the volume of 1.5 nm thickness inside of pore.

r1 (5 nm)

1.5 nm11234Retention time / min

56Column, C30 batch# 230216, 2.6 µm 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.

Table 2: Separation factor of vitamin E

7SunShell C30, 2.6 μm150 x 3.0 mm

Figure 2: 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.

1.5 nmr2 (7.5 nm)

40Figure 3: Separation of vitamin E.

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

2per unit area

Applications of C30 phase2

1=a-carotene,2
135 oC

Separation factor:1.042

CHH3

Separation factor:1.024

H3CCH3 H

HHCH3HH

H

CH3HHCH3H

CH 3H

H

H

H H3C

CH3H
H3C

SunShell C30 150 x 4.6 mm
2=b-caroteneoSeparation factor:1.065

30 C

Separation factor:1.031

CH3H

H3C

CH3 H

H

CH3H

H

H

CH3CH3H

H

H

H

CH3H

H

H H3C

CH3H
H3C

SunShell C18 100 x 2.1 mm

Separation factor:1.094

25 oC

Separation factor:1.042

Separation factor:1.130

20 oC

Separation factor:1.061

SunShell C30 100 x 2.1 mm
024681012Figure 5: Separation of Carotenes
transSeparation factor:1.167

trans15 oCcisSunShell C18 150 x 4.6 mm

Column size: 100 x 2.1 mm, Mobile phase: 80/20=CH3CN/CHCl3
Flow rate: 0.2 mL/min, Temperature: 30 ºC, Detection:UV450nm
Sample: 1=a-carotene, 2=b-carotene

Separation factor:1.080
cis

1505101520

25030

24Retention time / min

6820

2530

3540

4510Sample : Peanut Oil

Retention time / minFigure 4: Separation of vitamin K1 isomers.
Column, C30 batch# 230216 (SunShell C30), 2.6 µm 100 x 2.1 mm, company C core shell C30, 2.6 µm 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).
Vitamin K1 isomers (trans and cis)
Separation factor

SunShell C30 150 x 4.6 mmb) 2-propanol

Table 3: Separation factor and resolution of vitamin K1
OVitamin K1 (trans)

Resolution

H 3C
H
H 3C

C30 batch#230216

Company C
C30

C30 batch#230216

Company C
C30

35 oC

1.042

1.024

1.23---

30 oC

1.065

1.031

1.94

---

25 oC

1.094

1.042

2.880.91

20 oC

1.130

1.061

3.33

1.41

15 oC

1.167

1.080

3.79

1.78

H

CH3

Vitamin K1 (cis)HCH 3CH 3CH 3OOa) methyl tert-butyl ether

O
CH3

SunShell C18 150 x 4.6 mm

CH 3

H
CH3

HCH 3H

CH 3

CH 3

15

202530

3540

45

1.064

Resolution of β/γ-tocopherol

2.01

Conclusion

Sample : Linseed oil

Company C core shell C30

Separation factor of β/γtocopherol*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.

1C30 batch# 230216 (SunShell C30)

2Too highhydrophobicity
to perform agood peak.

1.05003

Carbon loading of only C30 (%)

Comparison of C30 phases

1.070050Figure 6: Chromatogram of linseed oil using MTBE or 2-propanol
Column dimension, 150 x 4.6 mm; flow rate, 1.0 mL/min; temperature, 25 oCa) mobile phase : A:acetonitrile,, B: methyl tert-butyl ether (MTBE) B% 5 to 50 in 55 minb) mobile phase : A:acetonitrile,, B:2-propanol B% 10 to 50 in 55 min

2025303540

455055Figure 7: Separation of oil samples
Column dimension, 150 x 4.6 mm; flow rate, 1.0 mL/min; temperature, 25 oC
mobile phase : A:acetonitrile,, B:MTBE B% 10 to 50 in 55 min

60

C30 phase could separate β-tocopherol andγ-tocopherol although C18 phase could notseparate such isomers.
C30 phase bonded inside a pore with 12 nm
diameter showed not only high ligand density
per volume but also no too highhydrophobicity on the particle surface inorder to perform good separation (hightheoretical plate and no tailing). Proposed
C30 phase (batch# 230216) showed better
separation of vitamin K1 isomers thancompany C C30.
C30 phase showed both longer retention timeof carotenes and better separation ofcarotenes than C18 phase.
C30 phase showed better separation for oilsample and shorter analysis time using
methyl tert-butyl ether as mobile phase thanusing 2-propanol.
C30 phase showed different selectivity
comparing with C18 phase when oil samples
were separated.