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

Norikazu Nagae1), Tomoyasu Tsukamoto1), Harald Dibowski2, Annette Dibowski2,Anders Grahn3), Ralf Jutvik3) and Vinay D. Gaintonde3)

1) ChromaNik Technologies Inc., 6-3-1 Namiyoke, Minato-ku, Osaka, Japan
www.dichrom.com
2) dichrom GmbH, Gräwenkolkstraße 66, 45770 Marl, Germany 3) Biotech AB, Box 133, 439 23, Onsala, Sweden

www.biotech-sweden.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 abased 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 and retention 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-solublecompound to compare with a conventional C18, while a core shell PFP could separate 3 kinds of isomers of cresol completely although a C18 could notseparate meta-cresol and para-cresol.
Different selectivity by different stationary phases was confirmed on core shell silica particles as well as fully porous silica particles.
0.5 mmTable 1. Characteristics of SunShell

0.5 mmCore2.6 mm

2.6 mmCore shell silica

Core1.6 mm

1 25437C1860 MPa

1.5 - 109150

4.5C860 MPa

1.5 - 9SunShell PFP

2.6

9150

4.5

Pentafluoropheny
l60 MPa

2-8

SunShell PhE

2.69150

4

Phenylethyl60 MPa

2-8

SunShell RP-AQUA

2.6

1690

4C2860 MPa

2 – 8a)SunShell C18-WP

2.6

1690

5C1860 MPa

1.5 - 106465

1 2,34Column dimension: 150 x 4.6 mm
Mobile 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

76SunShell PFP

1 275643SunShell C8275402468101214

SunShell C18

63161820

SunShell C18-WP (16nm)

Hydrogen bonding Hydrophobicity Steric selectivity

SunShell RP-AQUA（C28)
SunShell PhE(phenylethyl)

522

24

Retention time/min(Amylbenzene/Butylbenzene)(Triphenylene/o-Terphenyl)

C18-WP0.401.55

1.35

RP-AQUA

0.52

1.52

1.30

PhE

1.00

1.38

0.93

1PFP

1.00

1.31

2.38

1C8

0.32

1.46

1.08

C18

0.39

1.60

1.46

2214434C28

Figure 4. Separation of peptides

Column: C18; SunShell C18-WP, 2.6 mm 150 x 4.6 mm, C28; SunShell RP-Aqua, 2.6 mm 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 0% – 100% in 35 min
Flow rate: 1.0 mL/min, Temperature: 25 ºC, Detection: UV@210 nmSample:1 = Cytochrome C, 2 = Lysozyme, 3 = Myoglobin, 4 = Ovalbumin

Figure 5. Separation of proteins
1,23SunShell C18

213SunShell PFP

2101234Column:
SunShell C18, PFP 2.6 mm 150 x 2.1 mm
Mobile phase:
CH3OH/water=75:25 for SunShell C18
CH3OH/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

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

Conclusion

215Figure 9. Comparison between PFP and C18 (4)Separation of isomer of cresol

SunShell C82SunShell C18

324Column: C18; SunShell C18, PFP, 2.6 mm 150 x 4.6 mm

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

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

1OCH 3NHCH3ONH1OSunShell PFP
2NHOHCH 3NHCH3NCH 3435Retention time/min

Figure 6. Comparison between PFP and C18 (1)Figure 7. Comparison between PFP and C18 (2)2Separation of nucleic acid bases Change of retention of thymine at 40 oC(measurement every stop flow for 1 hour)2
540 ºC

25 ºC

134213540

12
3
Retention time/min

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

Retention time/min

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

21SunShell
PFP
0SunShell PFP

2,3,4SunShell C18, PFP 2.6 mm
150 x 2.1 mm
Mobile phase: CH3OH/water
3or
4 Waterbuffer=30/70
Flow rate: 0.3 mL/min
3420mM phosphate Temperature: 25 ºC
buffer pH7Detection: UV@250nm
Sample: 1 = Theobromine
3450mM phosphate
2 = Theophyline
buffer pH73 = Caffeine
4 = Phenol

21SunShell
PFPColumn: C18； SunShell C18-WP, 2.6 mm (16 nm) 150 x 4.6 mm
C28; SunShell RP-Aqua, 2.6 mm (16 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 0% – 100% in 35 min
Flow rate: 1.0 mL/min , Temperature: 25 ºC, Detection: UV@210 nm,Sample: Tryptic digest of myoglobin

Water

21SunShellPFPColumn dimension: 150 x 4.6 mm
Mobile phase: CH3CN/20mMH3PO4=10/90
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Detection: UV@250nm
Sample: 1 = 8-Quinolinol (Oxine)
2 = Caffeine

SunShell PhE(phenylethyl)

Figure 3. Comparison of oxine and caffeine

Relative retention of thymine (%)

1C282

4C18323SunShell
1C184
2

SunShell RP-Aqua

1Figure 2. Comparison of separation of standard samples

121(Caffeine/Phenol)

C182

1Table 2. Comparison of selectivity

SunShell C18-WP (16nm)

6Available
pH range

2.677

1 2,3154

3Maximumoperatingpressurea) This value is evaluated under 100% aqueous condition because SunShell RP-AQUA has reproducible retention
under 100% aqueous condition.

Figure 1. Schematic diagram of a core shell silica particle

1 2

Bonded phase

SunShell C8

SunShell C18

Porous silica

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%

Bonded phase

Pore
SpecificCarbon
Particle size
diameter surface area content(mm)2(nm)(m /g)(%)
2.69150
7N(4)=30,000
490807036050403020
100Initial1234Measurement number every stop flow

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

Figure 10. Reproducibility of retention under
100% aqueous condition

SunShell RP-AQUA

N(2)=37,900
1100Column: SunShell RP-AQUA,2.6 mm 150 x 4.6 mm
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.