Evaluation of Porous Layer Thickness of Core
Shell Particle for Separation of ProteinsTomoyasu Tsukamoto, Norikazu Nagae
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 to higher 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 that thin porous layer of core shell particle have an advantage forseparation of large molecules such proteins because a diffusion coefficient becomes small to proportional to a molecular weight and a mass transfer speed also decreases. In this paper, thickness of porous layer ofcore shell particle was evaluated to separate proteins.
2 kinds of thickness of porous layer such as 0.2 μm and 0.5 μm thickness were applied for separation of standard protein samples. One was 3.4 μm particle size, 0.2 μm porous layer and 15 m2/g specific surface area
and another was 2.6 μm, 0.5 μm and 15 m2/g. Separation was achieved using a gradient elution with 0.1% trifluoroacetic acid and acetonitrile including 0.08% trifluoroacetic acid.
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 of porous layer had the 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 the stationary phase to be large. Better separation of proteins contributes not only the thin porous layer but also the large surface area.

2 kinds of coreshell particles

Pore size distribution

Prototype particle

SunShell particle

コア0.2 mm

0.5 mm

2.6 mm
3.4 mmParticle size: 2.6 μm
Thickness of porous layer: 0.5μm
Specific surface area: 40 m2/g

Particle size: 3.4 μm
Thickness of porous layer: 0.2 μm
Specific surface area: 15 m2/g

Figure 2. Pore distribution of core shell particles
Measurement : Nitrogen adsorption method

Figure 1. Schematic diagram of a core shell silica particles

Effect of gradient time
195000Influence of temperature
2Column:
SunShell C8-30, 2.6 μm (30 nm) 150 x 2.1 mm,Mobile phase: A) 0.1% TFA in water B) 0.08 % TFA in Acetonitrile
Gradient program: Time 0 min 35 or 45 or 60 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

145000The differences of thickness of porous layer and particle size wereoffset each other at 60 oC, so that the peak width was same.

3

160 oC and 15 min gradient

4 W0.5=2.5 sec
W0.5=2.5 sec

5Prototype 3.4 μm (0.2 μm)
SunShell C8-30 2.6 μm (0.5 μm)

95000Time 0 min%B 20%

Time 0 min%B 20%

45000

W0.5=4.8 sec

35min
65%

80 oC and 35 min gradient

45min
65%

Time 0 min%B 20%

Prototype 3.4 μm (0.2 μm)
SunShell C8-30 2.6 μm (0.5 μm)

60min
65%

05105101520253035Figure 3. Chromatogram of protein mixture

404512W0.5=6.0 sec

The difference of peak width more than we expected wasobserved under the long gradient time conditions,

32025Figure 4. Comparison of chromatogram under the different temperature
Column: SunShell C8-30, 2.6 μm (30 nm, 0.5 μm layer) 100 x 2.1 mm, Sunshell C8-30, 3.4 μm (30 nm, 0.2 μm layer) 100 x 2.1 mm (prototype)
Mobile phase: A) 0.1% TFA in water B) 0.08 % TFA in Acetonitrile
Gradient program: Time 0 min 15 or 35 min%B20%
65%
Flow rate: 0.5 mL/min , Temperature: 60 or 80 oC, Detection: UV@215 nm, Sample:1 = Cytochrome C, 2 = Lysozyme, 3 = BSA, 4 = Myoglobin, 5 = Ovalbumin

Influence of gradient time
W0.5=8.0 sec

15Surface area works well for separation.

Retention time/min-50000There was no difference between 0.2 μm
and 0.5 μm of porous layer at 80 oC. Thesechromatograms showed difference of only
particle sizeW0.5=4.2 sec

Influence of gradient time 2524Sunshell C8-30, 3.4 μm

180 oC and 5 min gradientIn case of fast separation, 0.2 μm ofporous layer showed better separation
than 0.5 μm of porous layer.

W0.5=0.87 sec
W0.5=0.93 sec
345SunShellC8-30, 2.6 μm
0351015202530

3540

Prototype 3.4 μm (0.2 μm)

54SunShell C8-30 2.6 μm (0.5 μm)
2.6 μm coreshell silica with 0.5 μm porous layer showed
better separation than 3.4 μm with 0.2 μm porous layer.

W0.5=4.8 sec

There is no difference between 0.2 μm and0.5 μm of porous layer at 80 oC

W0.5=4.2 sec
80 oC and 35 min gradient24262830323436384042

Prototype 3.4 μm (0.2 μm)

Figure 5. Comparison of chromatogram under long gradient time
Column: SunShell C8-30, 2.6 μm (30 nm, 0.5 μm layer) 150 x 2.1 mm, Sunshell C8-30, 3.4 μm (30 nm, 0.2 μm layer) 150 x 2.1 mm (prototype)
Mobile phase: A) 0.1% TFA in water B) 0.08 % TFA in AcetonitrileGradient program: Time 0 min
60 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

Which is better ?
SunShell particle

SunShell C8-30 2.6 μm (0.5 μm)
05101512025Retention time/minFigure 6. Comparison of chromatogram under short gradient timeColumn: SunShell C8-30, 2.6 μm (30 nm, 0.5 μm layer) 100 x 2.1 mm, Sunshell C8-30, 3.4 μm (30 nm, 0.2 μm layer) 100 x 2.1 mm (prototype)
Mobile phase: A) 0.1% TFA in water B) 0.08 % TFA in Acetonitrile
Gradient program: Time 0 min 5 or 35 min%B20%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

Prototype particle

Conclusions
Particle size: 2.6 μm
Thickness of porous layer: 0.5 μm
Specific surface area: 40 m2/g

Particle size: 3.4 μm
Thickness of porous layer: 0.2 μm
Specific surface area: 15 m2/gIt is said that thin layer of porous layer is suitable for separation of large biomolecules
such as proteins. At more than 60 degree C, however, there is little difference ofefficiency between 0.2 μm and 0.5 μm of porous layer.
Separation of proteins using 2.6 μm of particle and 0.5 μm of porous
1 layer is better than
one using 3.4 μm of particle and 0.2 μm of porous layer at 80 degree C and 35min
gradient time because of a small particle.

0.5 μm thickness porous layer core shell silica showed betterseparation of proteins than 0.2 μm thickness porous layer core shellsilica under the long time gradient condition.
In fast separation, the peak shape for 0.2 μm porous layer core shellsilica was better than 0.5 μm porous layer core shell
It is important to choice the column which is fit to use for your purpose.