Development of Core Shell Particle with Large Pore for Separation ofPeptides and Proteinswww.chromanik.co.jp

1)
1)
2)
3)
3)Norikazu Nagae , Tomoyasu Tsukamoto , Alessandro Baldi , Anders Grahn , Ralf Jutvik

1) ChromaNik Technologies Inc., 6-3-1 Namiyoke, Minato-ku, Osaka, Japan
2) Protea Biosciences, 955 Hartman Run Rd. Morgantown, WV 26507 USA
3) Biotech AB, Box 133, 439 23, Onsala, Sweden

proteabio.com

www.biotech-sweden.com

Abstract
More than 10 kinds of core shell particle column have been available recently because 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.
Moreover, large molecules such peptides or proteins have been watched as medicine in recent years. Silica gel with 30 nm pores has generally
been used for separation of proteins.
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 and30nm pore was developed. As a novel bonding chemistry, hexa-functional C18 reagent with two sets of trichlorosilane was applied. It is consideredthat this reagent makes acidic stability high because of six positions of siloxane at most. Acidic stability was evaluated under 0.1 % formic acidsolution/acetonitrile as a typical LC/MS mobile phase condition at 70 degree Celsius. It was confirmed that the hexa-functional C18 was stable formore than 1000 hours. The developed materials bonded with hexa-functional C18, C8 and C4 were evaluated to separate not only standards ofpeptides and proteins but also tryptic digest of a protein using UV and Mass spectrometry detectors.

CoreDesorption Dv (log d) (cc/g)

0.5 μm
2.6 μmTable 1. Pore distribution of core shell particle

0.90.5 μm
2.6 μmCore1.6 μm

Porous silica
Particle diameter: 2.6 μm, Core diameter: 1.6 μm, Thickness of porous
silica: 0.5 μm, Pore volume: 0.30 mL/g, Specific surface area: 90 m2/g and40 m2/g, Pore diameter: 16 nm and 30 nm.

0.8Core shell 16 nm

Porediameter

Surfacearea

Carbonloading

Ligand Surface
coverageEndcapping

0.5

SunShell C18-WP

16 nm90 m2/g5%2.5 mmol/m2

Yes

0.4

SunShell HFC18-16

16 nm

90 m2/g

2.5%1.2 mmol/m2

Yes

0.3

SunShell HFC18-30

30 nm

40 m2/g

1.3%1.2 mmol/m2

Yes

Amplus C18-16

16 nm

90 m2/g

2.5%1.2 mmol/m2

Yes

Amplus C18-30

30 nm

40 m2/g

1.3%

1.2 mmol/m2

Yes

Amplus C8-30

30 nm

40 m2/g

1.2%2.5 mmol/m2

Yes

Amplus C4-30

30 nm

40 m2/g

0.9%

3 mmol/m2

Yes

0.70.6

Core shell 30 nm

0.20.10110

Pore Diameter (nm)Measurement : Nitrogen adsorption method

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

Relative retention (%)

100

SunShell C18-WP

SunShell HFC18-16

Conventional tri-functional C18

Novel hexa-functional C18

TMSOSiTrimethylchlorosilane (TMS)SiO

SiO（X: Cl, OCH3, OC2H5)O

Figure 3. Schematic
diagram of novel reagent

SiO

Si O O
OSiSiOOO

SiSiO

OSiSiO OO

SiSiO

Si OOOSi
Si O OSi OO OOOOSiSiSiSiSi
HO OOSiOO

Si SiOOOOSiOOSi

Si
SiOO OOSiOOOOSi

Si O

OOO

Si OO

Si OOOSiOOSiSiOOSi OO OSi

O O SiOSiOO OHOSiOOOOSi
Si
Si
HO O
OO
OO O
O O
SiOSiOOSi
SiSi
O O
O Si
Si O O O
OOOO
O

Si O

SiOOSiSi

OSi
Si

SiFigure 4. Schematic diagram of the state of bonding onsilica surface

Cytochrome C

Myoglobin

0.1% formic acid, pH2.6
70 ºC

4020SunShell C18-WP0200400600

800

1000

Durable test condition
Column : SunShell HFC18-16 2.6mm, 50 x 2.1 mm
Mobile phase: CH3CN/0.1% formic acid, pH2.6=40/60
Flow rate: 0.4 mL/min
Temperature: 70 ºC Measurement condition
Mobile phase: CH3CN/H2O=60/40
Flow rate: 0.4 mL/min
Temperature: 40 ºC
Sample: 1 = Uracil, 2 = Butylbenzene

Figure 6. Comparison of separation of peptides
Column: SunShell HFC18-16, 2.6 μm (16 nm) 150 x 4.6 mm
SunShell C18-WP, 2.6 μ m (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:
Flow rate: 1.0 mL/min , Temperature: 25 ºC,Sample: Tryptic digest of cytochrome C

Detection: UV@210 nm

Column:Amplus C18-16, 2.6 μm (16 nm) 100 x 2.1 mm
Mobile phase: A) 0.1% TFA in waterB) 0.1 % TFA in Acetonitrile
Gradient program: Time 0 min 1 min 6 min%B 20% 20% 95%
Flow rate: 0.50 mL/min
Temperature: 40 oC
Detection: UV@214 nm
Injection volume: 10 μL
Concentration: 0.10 μg/μL each protein
Sample:1. Angiotensin I
2. Somatostatin
3. Glu-1-fibrinopeptide

SunShell HFC18-16SunShell HFC18-30

Amplus C8-30

Figure 9. Separation of standard peptides

Amplus C4-30

Retention time/min

60

Figure 5. Stability of HFC18 underLC/MS mobile phase conditionSi

Hexamethydichlorotrisiloxane
SiOO

SunShell HFC18-16

Time (h)HOO

95% line

800TMS+1000

Figure 2. Pore distribution of core shell particle

Figure 1. Schematic diagram of a core shell silica particle

Sunniest
Bonding
Technology

100

Retention time/min

Column:Amplus C8-30, 2.6 μm (30 nm) 150 x 2.1 mm
Mobile phase: A) 0.1% TFA in waterB) 0.1 % TFA in Acetonitrile
Gradient program: Time 0 min 20 min%B 22% 70.5%
Flow rate: 0.35 mL/min
Temperature: 40 oC
Detection: UV@214 nm
Injection volume: 10 μL
Concentration: 0.01 μg/μL each protein
Sample:1. Angiotensin I
2. Ribonuclease A
3. Cytochrome C
4. Lysozyme
5. Transferrin
6. Bovine Serum Albumin
7. Myoglobin
8. Carbonic Anhydrase

Figure 7. Separation of peptides
Column dimension: 150 x 4.6 mm
Mobile phase: A) 0.1% TFA in water, B) 0.1 % TFA in Acetonitrile
Gradient program: Time 0 min 3 min 40 min%B 5%5%50%
Flow rate: 1.0 mL/min
Temperature: Ambient
Detection: UV@210 nm
Sample: Tryptic digest of cytochrome C and myoglobin

Amplus C18-30

Figure 11. Separation of ribonuclease A/B
Column:Amplus C8-30, 2.6 μm (30 nm) 100 x 2.1 mm
Mobile phase: A) 0.1% Formic acid in waterB) 0.1 % Formic acid in Acetonitrile
Gradient program: Time 0 min 10 min%B 17.5% 17.5%
Isocratic separation
Flow rate: 0.50 mL/min
Temperature: 60 oC
Detection: MS,Injection volume: 10 μL
Concentration: 0.10 μg/μL each protein
Sample:1. Ribonuclease B
2. Ribonuclease A

Amplus C8-30

Figure 10. Separation of standard proteinsAmplus C4-30

Conclusion
Retention time/min

Figure 8. Separation of proteins
Column dimension: 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*Regarding the pore size distribution, 30nm core shell particle was wider than 16 nm one.
*A novel Hexa-functional C18 phase showed not only high acidic stability for more than
1000 hours under typical LC/MS mobile phase condition but also different selectivity of
peptides from a conventional tri-functional C18.
*30 nm C8 phase showed the best separation of ovalbumin in the three kinds of 30 nm
phase.