Evaluation of Porous Layer Thickness of Core Shell Particle forSeparation of Proteinswww.chromanik.co.jp

Norikazu Nagae, Tomoyasu Tsukamoto, Shun Kojima
ChromaNik Technologies Inc., 6-3-1 Namiyoke, Minato-ku, Osaka, JapanAbstract

The feature of superficially porous (core shell) particle used as a highly efficient material is existence of a core, a thin porous layer and narrow particle size distribution, which lead tohigher efficiency than totally porous particle. Recently a core shell particle with wide pore for biomacromolecular separations has developed by a few manufacturers. It has been said thatthin porous layer of core shell particle have an advantage for separation of large molecules such proteins because a diffusion coefficient becomes small to proportional to a molecularweight and a mass transfer speed also decreases. In this paper, thickness of porous layer of core shell particle was evaluated to separate proteins. 2 kinds of thickness of porous layer suchas 0.2 μm and 0.5 μm thickness were applied for separation of standard protein samples. On fast separation, 0.2 μm of porous layer showed sharper peaks than 0.5 μm of porous layer.
However at 80 degree Celsius and using 60 min gradient time program, 0.5 μm of porous layer showed much sharper peaks than 0.2 μm of porous layer. It was considered that 0.5 μm ofporous layer had a wider specific surface area than 0.2 μm of porous layer and this wider specific surface area leaded separation efficiency concerning the partition interaction on thestationary phase to be large.

Effect of temperature

Column:SunShell C8-30, 2.6 μm (30 nm) 100 (150) x 2.1 mm,Mobile phase: A) 0.1% TFA in waterB) 0.08 % TFA in Acetonitrile
2Gradient program: Time 0 min 15 min%B 20% 65%
Flow rate: 0.5 mL/min
Temperature: 25 oC 60 oC and 80 oC
1Detection: UV@215 nm
Sample:1 = Cytochrome C, 2 = Lysozyme, 3 = BSA,
4 = Myoglobin, 5 = Ovalbumin

Comparison of thickness of porous layer
100 x 2.1 mm80 oC, 5 min gradient150 mm L
342

3.4 μm

W0.5=0.87 sec

1345Sunshell C8-30, 3.4 μm
Specific surface area: 15m2/g

Sunshell C8-30HT 3.4 μm (0.2 μm)

50.5 μm

60 C

W0.5=0.93 sec

80 oC
22.6 μm

6810

Retention time/min

Effect of gradient time134515 min

150 x 2.1 mm
80 oC, 60 min gradient21

W0.5=8.0 sec
34

Sunshell C8-30HT
3.4 μm (0.2 μm)
W0.5=6.0 sec

35 minRetention time/min
Retention time/min

Core shell 3.4 um, 30 nm

0.5
0.4
0.3
0.2
0.1
0101001000

5Pore size distribution

Column:Sunshell C8-30HT, 3.4 μm (30 nm, 0.2 μm layer) 100 or 150 x 2.1 mm
SunShell C8-30, 2.6 μm (30 nm, 0.5 μm layer) 100 or 150 x 2.1 mm
Mobile phase: A) 0.1% TFA in water, B) 0.08 % TFA in Acetonitrile
Gradient program: Time 0 min 5 or 60 min%B 20%65%
Flow rate: 0.5 mL/minoTemperature: 80 C
Detection: UV@215 nm
Sample:1 = Cytochrome C, 2 = Lysozyme, 3 = BSA, 4 = Myoglobin, 5 = Ovalbumin

In case of fast separation using 5 minute gradient program,is better thanSunShell C8-30 2.6 μm(0.5 μm)

25 minCore shell 2.6 um, 30 nm

0.6SunShell C8-30, 2.6 μm
Specific surface area: 40m2/g

12Column:
SunShell C8-30, 2.6 μm (30 nm) 100 x 2.1 mm,
Mobile phase: A) 0.1% TFA in waterB) 0.08 % TFA in Acetonitrile
Gradient program: Time 0 min 15 or 25 or 35 min%B 20%
65%
Flow rate: 0.5 mL/min ,Temperature: 80 oC
Detection: UV@215 nm,
Sample:1 = Cytochrome C, 2 = Lysozyme, 3 = BSA,
4 = Myoglobin, 5 = Ovalbumin

Core shell 2.6 um, 16 nm

0.7

Pore Diameter (nm)Retention time/min

20.81SunShell C8-30 2.6 μm (0.5 μm)
4Desorption Dv (log d) (cc/g)

0.9

25 oC

o00.2 μm

In case of high resolution separation using 60 minutegradient program at 80 oC,
reverselyis better than