Selectivity of stationary phases with alkyl, phenyl and pentafluorophenylgroups on core shell particle
www.chromanik.co.jp

Norikazu Nagae1), Tomoyasu Tsukamoto1), Scott Silver2)

1) ChromaNik Technologies Inc., 6-3-1 Namiyoke, Minato-ku, Osaka, Japan
2) Innovations United, 300 East 57th Street, Suite 11J, New York, NY 10022 USA

www.innovationsunited.com

Abstract

Brand columns packed with superficially porous particles have been available for some time. The superficially porous media
or so called core-shell media offers significant improvements such as higher efficiency and lower pressure drop for existing
HPLC operations without having to replace existing HPLC systems with UHPLC systems.
In this study, a 2.6 μm core-shell silica with a non-porous core approximately 1.6 μm in diameter and a superficially porous
layer of 0.5 μm was used as a based material. Core-shell silicas bonded with C18, C28, phenylethyl and pentafluorophenyl(PFP) groups were evaluated for hydrogen bonding capacity, hydrophobicity, steric selectivity and both peak shape andretention of oxine as a metal chelating compound.
A core-shell C28 with long chain ligands was suitable for separation of both high polar compounds using 100% aqueous
mobile phase and a fat-soluble compound to compare with a conventional C18, while a core shell PFP could separate 3 kindsof isomers of cresol completely although a C18 could not separate meta-cresol and para-cresol. Different selectivity bydifferent stationary phases was confirmed on core shell silica particles as well as fully porous silica particles.
0.5 mmCore2.6 mm

2.6 mmCore shell silica

Core1.6 mm

SunShell C18
SunShell C8
SunShell PFPSunShell Phenyl
SunShell P-AQUA
SunShell C18-WP
SunShell HFC18-16
SunShell HFC18-30
SunShell C8-30
SunShell C4-30

Particle diameter: 2.6 mm, Core diameter: 1.6 mm, Thickness of porous
silica: 0.5 mm, Pore volume: 0.30 mL/g, Specific surface area: 150 m2/g,Pore diameter: 9 nm, The ratio of porous silica volume: 77%

Figure 1. Schematic diagram of a core shell silica particle

1 23
1 2,36

SunShell RP-AQUA（C28)

SunShell Phenyl645

1 2,3476

SunShell PFP
71 256431Column dimension: 150 x 4.6 mmMobile phase: CH3OH/H2O=75/25
Flow rate: 1.0 mL/min
Temperature: 40 ºCSample:
1 = Uracil, 2 = Caffeine,
3 = Phenol, 4 = Butylbenzene,
5 = o-Terphenyl, 6 = Amylbenzene,
7 = Triphenylene

5,7SunShell C8

202

4

68

10

1214

SunShell C18

6316

18

20

C18C8

Sunniest
Bonding
Technology

1.5 - 10
1.5 - 92-8
1.5 - 92 - 8a)
1.5 - 102-9
2-9
2-92-8

Pentafluoropheny l

Phenylhexyl
C28
C18C18C18
C8C4Hexamethydichlorotrisiloxane

2224

Retention time/minSiSiXXXSiSiXXO

SiOSiOXO（X: Cl, OCH3, OC2H5)Si O
OOSiOOSiOOOSiOSiOOSiOOOSiSiSiO

Si OOSi
Si O OSi OOOOSiOOSiOSiSiOSi
O OO O
HO O
Si O
Si Si
SiOOOOOO

Schematic diagram of the state ofbonding on silica surface

Schematic diagram of reagent

2

SunShell C18-WP (16nm)(Amylbenzene/Butylbenzene)(Triphenylene/o-Terphenyl)

0.40

1.55

1.35

RP-AQUA

0.52

1.52

1.30

PhEenyl

1.00

1.58

1.01

PFP1.00

1.31

2.38

C80.32

1.46

1.08

C180.39

1.60

1.46

21SunShell RP-Aqua

122121SunShell Phenyl (phenylhexyl)
SunShell PFP

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
Sample: 1 = 8-Quinolinol (Oxine)
2 = Caffeine

SunShell C82

1SunShell C18Figure 4. Comparison of oxine and caffeine

Figure 3. Comparison of separation of standard samples
Sample: Tryptic digest of myoglobin

TMS+

Trimethylchlorosilane (TMS)

1(Caffeine/Phenol)

C18-WP

754

7
4.5
4.5
545
2.5
1.3
1.2
0.9

Hydrogen bonding Hydrophobicity Steric selectivity

754150150150
15090909040
4040

Table 2. Comparison of selectivity

SunShell C18-WP (16nm)

699991616
1630
30
30

Available
pH range

Bonded phasea) This value is evaluated under 100% aqueous condition because SunShell RP-AQUA has reproducible
retention under 100% aqueous condition.

7543

2.6
2.6
2.6
2.6
2.6
2.6
2.6
2.6
2.6
2.6

Hexa-Functional C18 has six functional groups.
This HFC18 is much more stable under acidic condition.

Bonded phase

Particle PoreSpecificCarbon
diameter surfacesize
contentarea (m2/g) (%)
(mm)
(nm)

Porous silica

1 2Figure 2. What is HFC18?

Table 1. Characteristics of SunShell
0.5 mmSample: Tryptic digest of cytochrome C
SunShell HFC18-16

SunShell HFC18-16

SunShell
HFC18-30

Column: C18; SunShell C18, PFP, 2.6 mm 150 x 4.6 mm

2,3,4SunShell Mobile phase: CH3CN/10mM Phosphate buffer pH7.0 = 80/20
Flow rate: 1.0 mL/min
C18Temperature: 40 ºC

SunShell HFC18-30

SunShell C18-WP

OOSunShellC4-30
SunShell C4-30

Figure 6. Separation of proteins

1,23SunShell C18

213SunShell PFP

2101234Retention time/min

5Column:
SunShell C18, PFP 2.6 mm 150 x 2.1mm
Mobile phase:
CH3OH/water=75:25 for SunShell
C18CH3OH/water=60:40 for SunShell
PFP
Flow rate: 0.3 mL/min
Temperature: 25 ºC
Detection: UV@250nm
Sample: 1 = o-Xylene
2 = m-Xylene
3 = p-Xylene

Figure 8. Comparison between PFP and C18 (3)Separation of isomer of xylene

Conclusion

SunShell C183

1 2 3SunShell PFPColumn:
SunShell C18, 2.6 mm 150 x 4.6 mm
SunShell PFP, 2.7 mm 150 x 4.6 mm
Mobile phase: CH3OH/H2O=40/60
Flow rate: 1.0 mL/min
Temperature: 25 ºC
Sample: 1 = p-Cresol
2 = m-Cresol
3 = o-Cresol

Separation of nucleic acid bases
40 ºC

25 ºC

1253

380
70
60

5430
20
100

123Retention time/min

4Column: SunShell RP-AQUA, 2.6 mm 75 x 4.6 mm
Mobile phase: 10mM Phosphate buffer pH7.0
Flow rate: 1.0 mL/min
Temperature: 40 ºC and 25 ºC
Sample: 1 = Cytosine, 2 = Uracil, 3 = Thymidine,
4 = Uridine, 5 = Thymine

Figure 9. Comparison between PFP and C18 (4)Separation of isomer of cresol

20Initial

1234

Measurement number every stop flow

SunShell RP-Aqua showed more than 97% ofreproducibility for retention using 100% aqueous
buffer as a mobile phase.

Figure 10. Reproducibility of retention under
100% aqueous condition

NHCH3OH

3CH3NHCH3NCH34Figure 7. Comparison between PFP and C18
2SunShell RP-AQUAN(2)=37,900

N(4)=30,000
435040O

SunShell PFP

110090421NH

1

Change of retention of thymine at 40 oC(measurement every stop flow for 1 hour)
Relative retention of thymine(%)

Figure 5. Separation of peptides

CH3NH

Column:SunShell HFC18-30, 2.6 mm (30 nm) 150 x 4.6 mm,
SunShell C8-30, 2.6 mm (30 nm) 150 x 4.6 mm,
SunShell C4-30, 2.6 mm (30 nm) 150 x 4.6 mm,
Mobile phase: A) 0.1% TFA in water,B) 0.1 % TFA in Acetonitrile
Gradient program: Time 0 min 15 min%B 20% 65%
Flow rate: 1.5 mL/min ,Temperature: Ambient
Detection: UV@210 nm,
Sample:1 = Cytochrome C, 2 = Lysozyme,
3 = BSA, 4 = Myoglobin, 5 = Ovalbumin

SunShell C8-30
Column: SunShell HFC18-16, C18-WP, 2.6 mm (16 nm) 150 x 4.6 mm
SunShell HFC18-30, C8-30, C4-30, 2.6 mm (30 nm) 150 x 4.6 mm
Mobile phase: A) 0.1% TFA in Acetonitrile/water(10:90)
B) 0.1 % TFA in Acetonitrile
Gradient program: %B 5% – 5% in5 min and -50% in 35 min
Flow rate: 1.0 mL/min , Temperature: 25 ºC,
Detection: UV@210 nm.

Sample: 1 = Uracil, 2 = Propranolol, 3 = Nortriptyline, 4=Amitriptyline

1SunShell
C8-30

Column: SunShell RP-AQUA,
2.6 mm 150 x 4.6mm
Mobile phase:
20mM Phosphate buffer
pH6.0
Flow rate: 1.0 mL/min
Temperature: 25 ºC
Detection: UV@250nm
Sample: 1 = 5’-GDP
2 = 5’-ATP
3 = 5’-ADP
4 = 5’-AMP

Figure 11. Separation of nucleotides*Hydrogen bonding, hydrophobicity and steric selectivity of alkyl groups, phenyl group and pentafluorophenyl group were evaluated.
*Pentafluorophenyl showed highest hydrogen bonding and highest steric selectivity and much different from C18 group. Pentaflorophenyl group showedmuch longer retention time for a polar compounds and could separate isomers better than C18 group.
*C28 group showed reproducible retention under 100% aqueous condition.