Evaluation of Optimized C30 Phase for
Separation of Structurally Related Isomers
Etsuko Shearer1, Norikazu Nagae2 and Tomoyasu Tsukamoto2
http://www.bionikinc.com

1 BioNik Inc. 3397-19 Obuchi, Fuji, Shizuoka 417-0801 Japanwww.chromanik.co.jp

2 ChromaNik Technologies Inc. Namiyoke, Minato-ku, Osaka 552-0001 Japan

A long alkyl group like C30 (triacontyl group) phase has been known to be more suitable than a conventional C18 phase for separation ofhydrophobic structurally related isomers such as vitamin E or vitamin K1. However, in many cases a C30 column shows a tailing peak. This tailing isconsidered to be a reason why C30 ligand density is too high. We have reported optimization for a pore diameter of the superficially porous silica anda ligand density of C30 phase. In this study, the optimized C30 phase was evaluated to compare with C18 phase or 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 notseparate them. The higher temperature, 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 separate them.
This C30 phase was applied for separation of some structural related isomers, and showed higher resolution of isomers than a C18 phase.
Separation factor of β/γ-tocopherol

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

15.0 - 15.5 nm of pore
1.06001.0550

106

96.1

79.6

79.6

73.5

73.5

0.287

0.286

0.288

0.299

0.299

0.285

0.285

10.2

10.8

12.0

15.0

15.0

15.5

15.5

6.59

5.48

6.08

4.43

5.40

4.10

5.20Carbon 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

1.04501.04004

1.0350
0.5

1

1.5

2

Separation factor of β/γ-tocopherol b

1.0625

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

r1 (5 nm)

1.5 nmRetention time / min

32Figure 3: Separation of vitamin E

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

4Column, 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 E0123
4Retention time / min

567SunShell C30, 2.6 μm
150 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)

2.5

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

1

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.

2 3Too highhydrophobicity
to perform agood peak.

1.0500C30 ligand density (μmol/m2)

112

Pore volume a (mL/g)
Average pore diameter a (nm)

C30 batch# 230216(SunShell C30)

10.2 - 12.0 nm of pore

1.06500Specific surface area a (m2/g)

Carbon loading of only C30 (%)

11.0700

Separation factor of β/γtocopherol

1.064

Resolution of β/γ-tocopherol

2.01*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.

per unit area

Comparison of C30 phases
C30 batch# 230216 (SunShell C30)

Company C core shell C30

Separation factor:1.042

Table 3: Separation factor and resolution of vitamin K135 C30 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

HResolution

H 3C

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

05

cis

10

1520trans

15 oC

1.167

1.080

3.79

1.78

025268

H

CH 3

CH 3

H

CH 3

HO

Vitamin K1 (cis)OOCH 3HCH3CH 3CH 310*SunShell C30 showed 2 times longer retention time and over 2 times higher
resolution than company C core shell C30.

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; flowrate, 0.35 mL/min; temperature is described in a figure; detection, UV250 nm; sample, vitamin K1 isomers (trans and cis).

Applications of C30 phase2CH 3cis
4Retention time / minH

CH3CH3trans

Separation factor:1.167

O
H 3C

Separation factor
Separation factor:1.065

Vitamin K1 (trans)

Vitamin K1 isomers (trans and cis)

Separation factor:1.024

oSample: Linseed oil

12
1

1=a-carotene,
CH
H3
H3C

CH3 H HH

SunShell C30 150 x 4.6 mm

H

H

CH3H

CH3CH3H

H

H

CH3H

H

H

H H3C

CH3H3C

2=b-carotene

SunShell C18

CH3H

H3C

SunShell C30

CH3 H

H

H

H

CH3H

CH3CH3H

H

HH

CH3H

H

H H3C

CH3HH3CSunShell C18 150 x 4.6 mm
024681012Retention time / minFigure 5: Separation of Carotenes
152025Column 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.

30354045

Retention time / min

Sample: Peanut oil

SunShell C30 150 x 4.6 mmb) 2-propanol

SunShell C18 150 x 4.6 mma) methyl tert-butyl ether

15202530354045Conclusion

H50

Retention time / min20

Figure 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 oC;a) 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.

253035404550

Retention time / minFigure 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.

5560

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 high hydrophobicity
on the particle surface in order to perform good
separation (high theoretical 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 of carotenes
than C18 phase.
C30 phase showed better separation for oilsample and shorter analysis time using methyl
tert-butyl ether as mobile phase than using 2propanol.
C30 phase showed different selectivity
comparing with C18 phase when oil samples
were separated.