ChromaNik Seminar in Italy and Hungary
1) About ChromaNik Technologies (10 min)
2) Feature of Core Shell Particle and SunShell Bonding
technology (30 min)
3) Comparison of Core Shell C18 Columns (Accucore, Ascentis
Express, Cortecs, Kinetex, PoroShell and SunShell) (20 min)
4) Applications related Food, Proteins and Other (20min)
ChromaNik Technologies Inc.
Founded in December, 2005 by Norikazu Nagae
who worked for Nomura Chemical (Develosil) for
22 years.
Main products:
Sunrise C18-SAC, C18, C28 since 2007
Sunniest C18, RP-AQUA, C8, etc. since 2008
SunShell C18, C8, PFP, HILIC-Amide etc. since 2011
ChromaNik Technologies Inc.
Osaka
Address:
6-3-1 Namiyoke, Minato-ku, Osaka,
Japan
ChromaNik Technologies Inc.
Sales by ChromaNik
Annual Sales (EURO)
€ 1,200,000
€ 1,000,000
€ 800,000
€ 600,000
€ 400,000
€ 200,000
€0
2006 2007 2008 2009 2010 2011 2012 2013
Sunrise C18-SAC
Silanol activity controlled C18
■ Silanol activity control technology
ChromaNik developed the technique that decreased
only silanol groups with high absorption activity to a
basic compound and remained effective sailnol
groups on the stationary phase. Silanol activity control
and no end-capping led the existence of silanol
groups with high hydration which created a new and
unique reversed-phase separation mode including
hydrogen bond and ion-exchange interaction.
Furthermore, silanol activity controlling, then endcapping technique improved a peak shape of a basic
compound exceedingly.
■ Separation of basic compounds with
ammonium acetate: Effect of salt
concentration(Sunrise C18-SAC)
1
2
Column size: 4.6x150 mm
Mobile phase: 70/30=CH3CN
/acetate buffer (pH4.1)
Flow rate: 1.0 mL/min
Temperature: 40 C
Sample:
1 = uracil
2 = toluene
3 = propranolol
4 = nortriptyline
5 = amitriptyline
5
3
4
200mM
2
1
5
3
4
100mM
2
1
3
5
4
50mM
2
1
3
Silanol groups with
high absorption
activity
Changeover to
siloxane bond
Silanol Activity
Control
Technology
O
H
Si
H
H
25mM
1
Si
O
O
O
O
O
O
O
Si
Si
Si
Si
Si
Si
H
O
Si
5
4
2
3
10mM
Si
0
2
4
6
8
10
12
14
16
18
retention time / min
Silanol activity control
5
4
20
22
24
26
Sunniest C18
Special end-capping,
as a result high stability
Final TMS
HO
Si
O
Si
Si
Si
O
Si
O
Si
O
Si
O
O
Si
O
O
O
O
Si
O
O
Si
O
Si
O O Si
O
Si
Si
O
O OH
O
Si
Si
Si
O
O
O
O
Si
O
Si
Si
Si
HO O
O O
OO O
O O
Si
O
Si
O
Si
O
Si O
O Si O O Si O
O
Si O O
O
Si
O
O
O
O
O
Si
Si O
SiO
O O
O
Si
O
Si
O O
Si O
An Arm of HMODTS moves like a Geometrid caterpillar, so that a functional group
on the tip of the arm can bond with a silanol group which Is located anywhere.
7
SunShell Core shell column
M. Biba et al. / Journal of Pharmaceutical and Biomedical Analysis 96 (2014) 54–57
100 mM TEAA in water pH7 (mobile phase A)
100 mM TEAA in acetonitrile (mobile phase B)
Kinetex C18 showed terrible
peaks after 100 injections.
SunShell C18 showed good
peaks after 300 injections.
2) Feature of Core Shell Particle and
SunShell Bonding Technology
History of Core Shell Silica
A dominant patent
was materialized at
1967 and 1968.
(1) J.J. Kirkland, F.A. Truszkowski, C.H. Dilks, and G.S. Engel, J. Chromatogr.,
A 890, 3–13 (2000).
(2) J.J. Kirkland, T.J. Langlois, and J.J. DeStefano, Am. Lab. 39, 18–21 (2007).
10
Current Trends in HPLC Column Usage
LCGC Europe Jan 1, 2012 By: Ronald E. Majors
Table 14: Types of columns that will be tried in future.
Type of Column
Will Try in Future (%)
Superficially porous particle
54
Porous, sub 2 mm
49
Hybrid
34
Monolith, silica-based
31
Monolith, polymer-based
30
CoreShell (Solid Core) Column
 ACE UltraCore Super C18
 Accucore C18
 Aeris PEPTIDE XB-C18
 Amplus C18-30
 Ascentis Express C18
 BioShell C18
 Brownlee SPP C18
 BlueShell C18
 Capcell Core C18
 CORTECS C18
 COSMOCORE C18
 HALO C18
 Kinetex C18
 Meteoric Core C18
 NUCLEOSHELL C18
 Poroshell C18
 Raptor ARC18
 SpeedCore C18
 SunShell C18
 Coresep 100
12
2 kinds of typical core shell particles
Monolayer porous silica structure
Multilayer porous silica structure
using layer-by-layer method
Particle distribution of A company core shell
Coulter counter
Volume (%)
D90/D10=1.12
Particle size (mm)
14
Schematic Diagram of Core Shell silica
Monolayer structure
0.5 mm
0.5 mm
2.6 mm
(SunShell)
2.6 mm
Core
Core
1.6 mm
Porous silica
Porous silica
Particle diameter: 2.6 mm, Core diameter: 1.6 mm,
Thickness of porous silica: 0.5 mm
Pore volume: 0.30mL/g, Specific surface area: 150 m2/g,
Pore diameter: 9 nm
The ratio of porous silica volume: 77%
Van Deemter Equation
H
B term
C term
A term
u
A term : Eddy diffusion(dp is particle diameter)
B term : Longitudinal diffusion
(Dm is diffusion coefficient)
C term : Mass transfer
1. F. D. Antia and C. Horvath, J. Chromatogr., 435 (1988) 1-15.
16
Comparison of Plate Height Plots
18
Plate Height, um
16
Fully porous 5 um
14
Fully porous 3 um
12
Fully proous 1.8 um
10
SunShell 2.6 um
8
Column: C18, 50 x 4.6 mm
Mobile phase:
Acetonitrile/water=(60/40)
Temperature: 25 oC
Sample : Naphthalene
6
4
2
0
0
5
10
Mobile Phase Velocity, mm/sec
15
Comparison of Back Pressure for High Throughput Columns
45
Sunniest C18-HT 2.0 um
Pressure, MPa
40
Brand A C18 1.9 um
35
Brand B C18 1.8 um
30
Brand C C18 1.7 um
Brand D C18 2.6 um
25
SunShell C18 2.6 um
20
15
Column dimension: 50 x 2.1 mm
Mobile phase: Acetonitrile/water=(70/30)
Temperature: 25 oC
10
5
0
0
0.5
1
Flow rate, mL/min
18
1.5
Impedance time t0/N2
When back pressure is constant,
t0(no retained time) is proportional to N2(square plate).
t0 ∝ N2
t0 = A・N2
A = t0/N2
A is an impedance time.
Plate (N)
Column Length
Back pressure
Flow rate
t0
10,000
15 cm
10 MPa
1.0 mL/min
100 S
20,000
30 cm
20 MPa
1.0 mL/min
200 S
20,000
30 cm
10 MPa
0.5 mL/min
400 S
If a back pressure is same 10 MPa, t0 shows 4 times value when Plate becomes 2 times.
Desmet et al. Anal. Chem. 77, 4058 (2005).
19
Kinetic plot analysis at 40 MPa.
1000
t0/N2 (nS)
5 mm
3 mm 2 mm
Calculated values were
plotted for 1.0, 2.0, 3.0 , 5.0
μm fully porous particle .
1.0 mm
100
An experimental values were
plotted for 2.6 μm core shell
particle.
10
Core Shell 2.6 mm
t0=10000 s
t0=1000 s
t0=100 s
t0=10 s
1
3,162,278
316,228
1,000,000
100,000
N
40 MPa
t0=1 s
31,623
10,000
3,162
This figure means that we can
separate faster using Core
shell than fully porous and
core shell has higher plate
than fully porous at the same
analysis time (t0).
The curves for particulate columns were obtained by assuming
η=0.00046 Pa s, φ=700, Dm=2.22x10-9 m2/s, Knox equation, h=0.65ν1/3+2/ν+0.08ν, Dp,
totally porous 1.0, 2.0, 3.0 , 5.0 μm, core shell 2.6 mm.
Why does a 2.6 mm core shell particle show the same performance
as a sub 2 mm particle?
Narrow particle distribution
Sunniest, 2 mm
Wide particle distribution (Conventional silica gel D90/D10=1.50)
D10: 1.75 mm
D50: 2.01 mm
D90: 2.31 mm
D90/D10=1.32
Company F, 2 mm
SunShell, 2.6 mm
D10: 1.67 mm
D50: 2.09 mm
D90: 2.65 mm
D90/D10=1.59
D10: 2.46 mm
D50: 2.63 mm
D90: 2.82 mm
D90/D10=1.15
Flow of mobile phase
Narrow particle distribution (core shell silicaD90/D10=1.15)
4
0.5
0.5
11
22 (mm)
Comparison of Particle Size Distribution
4
4
Packing state of core shell and fully porous silica
The size distribution of a core shell (SunShell) particle is much narrower than that of a conventional
fully porous particle, so that the space among particles in the column reduces and efficiency increases
by reducing Eddy Diffusion (multi-path diffusion) as the A term in Van Deemter Equation.
Difference of diffusion in column at longitudinal direction
A solute diffuses both outside a particle and in a pore.
Fully porous silica
Core shell silica
Surface ratio of core shell silica is around 30%.
Cores block the path of diffusion of
a solute.
B term decreases to 70%.
22
HETP at low flow rate
20
Column: SunShell C18 2.6 mm, 50 x 2.1 mm
totally porous 2 mm 50 x 2.1 mm
Mobile phase: Acetonitrile/water=(60/40)
Sample : Naphthalene
Plate height (μm)
16
Core shell 2.6 um
2 times
12
Fully porous 2 um
8
4
Almost same HETP
0
0
0.2
0.4
0.6
Flow rate (mL/min)
23
0.8
1
Short diffusion path by thin porous silica layer
18
Fully porous 5 um
16
1.8 μm
Fully porous 3 um
Fully proous 1.8 um
Plate Height, um
14
2.6 μm
Core Shell 2.6 um
12
10
8
6
4
2
0
0
24
2
4
6
8
Mobile Phase Velocity, mm/sec
Comparison of Performance by Plate/Pressure
Plates
Pressure(MPa)
Plate/pressure
Sunniest C18 –HT 2.0 mm
9,900
16.7
593
Brand A C18 1.9 mm
7,660
16.3
470
Brand B C18 1.8 mm
10,100
19.6
515
Brand C C18 1.7 mm
11,140
32.0
348
SunShell C18 2.6 mm
9,600
9.7
990
Sunniest C18 –HT 2.0 mm
Brand A C18 1.9 mm
Brand B C18 1.8 mm
Brand C C18 1.7 mm
SunShell C18 2.6 mm
0
5,000
10,000
0
10
20
30
0
Column: 50 x 2.1 mm C18, Mobile phase: Acetonitrile/water=(70/30), Temperature: 25 oC
250
500
750
1000
Comparison of retention between fully
porous silica C18 and core shell silica C18s
Fully porous silica C18
Sunniest C18 5 μm
Core shell silica C18
SunShell C18 2.6 μm
Core shell silica C18
Kinetex C18 2.6 μm
340 m2/g
150 m2/g
Effective 200 m2/g
1.5 g (510 m2)
2.7 g (405 m2)
2.7 g
Specific surface area
Packing weight in the column
Retention
time (tR)
1 = Uracil
1.70
2 = Caffeine
1.90
3 = Phenol
2.17
4 = Butylbenzene
13.35
5 = o-Terphenyl
19.19
6 = Amylbenzene
19.96
7 = Triphenylene
24.35
2 3 SunShell C18 2.6 μm 5
1
4
5
10
Retention
time (tR)
1.35
1.47
1.65
10.01
14.24
15.09
20.33
4
7
5
6
15
20
Retention time/min
25
Retention
factor (k)
0
0.09
0.22
6.41
9.55
10.18
14.06
Retention
time (tR)
1.36
1.49
1.61
6.19
8.15
8.75
9.44
Retention
factor (k)
0
0.10
0.18
3.55
4.99
5.43
5.94
Mobile phase: Methanol/water(75:25)
Temperature: 40oC
Column dimension: 150 x 4.6 mm
Flow rate: 1.0 mL/min
6
2 3 Sunniest C18 5 μm
1
0
Retention
factor (k)
0
0.12
0.28
6.85
10.29
10.74
13.32
7
30
Characteristics of SunShell (1)
Core shell silica
Bonded phase
Pore
diameter
(nm)
Specific surface
area
(m2/g)
Carbon
content
(%)
Bonded phase
End-capping
Available pH
range
9
150
7
C18
Sunniest endcapping
1.5 - 10
90
150
150
5.5
4.5
4.5
C18
C8
Sunniest endcapping
Pentafluorophenyl
TMS endcapping
1.5 - 10
1.5 - 9
2-8
SunShell Phenyl
SunShell RP-AQUA
SunShell C18-WP
2.6
2.6
2.6
2.6
9
9
9
9
16
16
150
90
90
5
4
5
Phenylhexyl
C28
C18
Sunniest endcapping
Sunniest endcapping
1.5 - 9
2 - 8a)
1.5 - 10
SunShell HFC18-16
2.6
16
90
2.5
C18
Sunniest endcapping
1.5 – 9
SunShell HFC18-30
2.6
30
40
1.3
C18
Sunniest endcapping
1.5 – 9
SunShell C8-30
SunShell C4-30
2.6
2.6
30
30
40
40
1.2
0.9
C8
C4
Sunniest endcapping
1.5 – 9
1.5 – 9
Particle size
(mm)
SunShell C18
SunShell C18 5 μm
SunShell C8
SunShell PFP
2.6
5
2.6
Sunniest endcapping
Sunniest endcapping
Sunniest endcapping
a) This value is evaluated under 100% aqueous condition because SunShell RP-Aqua has reproducible retention under
100% aqueous condition.
Sunniest (SunShell) Bonding Technology
Trifunctional silyl-reagent,
(Octadecyltrichlorosilane)
Difunctional silyl-endcapping reagent
(Hexamethyldichlorotrisiloxane)
Second silyl-endcapping reagent
(above + Trimethylchrolosilane(TMS))
High reaction temperature for endcapping
C18 silyl-reagent (HMODTS)
Hexamethyloctadecyltetrasilane
Step 2
Step 1
toluene
+ H2O
+
80oC,4h
MeO
Si
OMe
OMe
HO
Si
OMe
OMe
HO
Me
Cl
MeMe MeMe Me
Si
O
Si
O
Si
Cl
toluene
30oC,2h
Si
OMe
OMe
Me
Cl
MeMe
Si
O
MeMe
Si
O
Me
Si
O
Si
OMe
OMe
29
Bonding state of HMODTS on silica
Final TMS
HO
Si
O
Si
Si
O
Si
O
Si
O O Si
O
Si
Si
O
O OH
O
Si
Si
Si
O
O
O
O
Si
O
Si
O
Si
Si
Si
HO O
O
O
Si
O
O
O
O
Si
O
Si
OO
O
Si
O
O
Si
Si
Si
Si
O
O
O
Si
O
O Si
O
Si
O
O
O
O
O
O
O
O
O
O
O
Si
O
O
O
Si
O
Si
O
O
Si O Si
SiO
O O
Si
Si O
O O
An Arm of HMODTS moves like a Geometrid caterpillar, so that a functional group
on the tip of the arm can bond with a silanol group which Is located anywhere.
30
Comparison of amitriptyline peak
1
2
4
3
SunShell C18
TF=1.20
PoroShell C18 EC
TF=1.89
Kinetex C18
TF=2.61
Accucore C18
TF=2.73
Cortecs C18
TF=3.12
Ascentis Express C18
TF=3.24
0
1
2
3
4
5
6
7
8
9
10
Retention time/min
Mobile phase: Acetonitrile/10mM ammonium acetate pH6.8=(40:60)
Column dimension: 150 x 4.6 mm, Flow rate: 1.0 mL/min, Temp.: 40oC
Sample: 1=Uracil, 2=Propranolol, 3= Nortriptyline, 4=Amitriptyline
CH3
N
CH3
Amitriptyline
Stability under acidic pH condition
Relative retention/%
100
▲
Durable test condition
Column size: 50 x 2.1 mm
Mobile phase: CH3CN/1.0% TFA,
pH1=10/90
Flow rate: 0.4 mL/min
Temperature: 80 ºC
▲
80
60
SunShell C18
Company S C18
40
Company T C18
Company P C18
20
Company A C18
0
0
20
40
60
Time/h
80
100
120
Measurement condition
Column size: 50 x 2.1 mm
Mobile phase: CH3CN/H2O=60/40
Flow rate: 0.4 mL/min
Temperature: 40 ºC
Sample: 1 = Uracil
2 = Butylbenzene
Stability under basic pH condition
Relative plate of butylbenzene /%
100
80
60
SunShell C18
Durable test condition
Column size: 50 x 2.1 mm
Mobile phase:
CH3OH/20mM Sodium
borate/10mM NaOH=30/21/49
(pH10)
Flow rate: 0.4 mL/min
Temperature: 50 ºC
Company S C18
40
Company P C18
Company T C18
20
Company A C18
0
0
1,000 2,000 3,000 4,000 5,000 6,000
Elution volume/mL
Measurement condition
Column size: 50 x 2.1 mm
Mobile phase: CH3OH/H2O=70/30
Flow rate: 0.4 mL/min
Temperature: 40 ºC
Sample: 1 = Butylbenzene
Bleeding test using LC/MS
+Q1: 5.997 min to 7.999 min of Sample
TIC of +Q1
Brand L C18
SunSell C18
Column size: 50 x 2.1 mm
Mobile phase:
A) 0.1% acetic acid
B) CH3CN
Gradient:
Time: 0min 1min 5min 7min
%B: 5% 5% 100% 100%
Flow rate: 0.4 mL/min
Temperature: 40 ºC
MS: ABI API-4000
Ionization:
Turboionspray (cation)
Measurement mode:
Q1 Scan m/z 100-1000
Examples of transfer (isocratic separation)
ACE C18, 5 mm 250 x 4.6 mm
HPLC
N(4)=19,313
3
1
2
SunShell C18, 2.6 mm 100 x 4.6 mm
3
1
0
4 5
2
2
4
6
8
10
12
14
16
18
20
22
N(4)=20,287
5
4
2
24
Retention time/min
2
1
1
N(4)=20,287
1/3 of analysis time
3
0
5
4
3
4
5
Sample: 1 = Benzydamine
2 = Ketoprofen
3 = Naproxen
4 = Indomethacin
5 = Ibuprofen
6
Retention time/min
UHPLC
3
1
1.8 mL/min
2
0
1
4
2
5
4
3
1
N(4)=24,124
1.0 mL/min
2
5
3
N(4)=22,424
Analysisi time decreases to 1/6 to compare
with a 5mm column sized 250x4.6mm.
4
Column:
Brand F C18, 5 mm 250 x 4.6 mm
SunShell C18, 2.6 mm 100 x 4.6 mm
Mobile phase:
CH3CN/20mM Phosphoric acid = 45/55
Flow rate: 1.0 mL/min,
1.8 mL/min at the lowest chromatogram
Temperature: 25 ºC
Pressure: 9.5 MPa for Brand F C18 5 mm
13.4 MPa for SunShell C18 2.6 mm
Detection: [email protected] nm
5
6
Retention time/min
35
HPLC: Hitachi LaChrom ELITE
(using 0.25 mm i.d. tubing)
UHPLC: Jasco X-LC
Retention comparison of cation between
C18 and PFP phases
2,3,4
SunShell C18
Column: SunShell C18, PFP, 2.6mm 150 x 4.6 mm
Mobile phase: CH3CN/10mM Phosphate buffer pH7.0 = 80/20
Flow rate: 1.8 mL/min
Temperature: 25 ºC
Sample: 1 = Uracil, 2 = Propranolol, 3 = Nortriptyline, 4 =Amitriptyline
1
1
SunShell PFP
3
2
36
4
Separation of isomers of cresol
1,2
SunShell C18
3
1 2 3
SunShell PFP
Column:
SunShell C18, 2.6 mm 150 x 4.6 mm
SunShell PFP, 2.6 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 nucleotides
2
SunShell RP-AQUA
N(2)=37,900
1
N(4)=30,000
4
3
Column:
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: [email protected]
Sample: 1 = 5’-GDP,
2 = 5’-ATP,
3 = 5’-ADP,
4 = 5’-AMP
Summary
A core shell particle makes value of all terms of
Van Deemter Equation, A , B and C be low.
Sunniest (SunShell) bonding technology can not
only decrease an effect of residual silanol groups
but also increase column stability under both
acidic and basic pH conditions.
3) Comparison of Core Shell C18 Columns
(Accucore, Ascentis Express, Cortecs,
Kinetex, PoroShell and SunShell) (20 min)
Comparison data of 6 kinds of
core shell C18 columns
Column name
1. Company P C18, 2.6 mm: Kinetex C18
2. Company T C18, 2.6 mm: Accucore C18
3. Company W C18, 2.7 mm: Cortecs C18
4. Company A C18, 2.7 mm: PoroShell C18 EC
5. Company S C18, 2.7 mm: Ascentis Express C18
6. SunShell C18, 2.6 mm
ChromaNik Technologies Inc.
Comparison of standard samples
Column:
Company P C18, 2.6 μm 150 x 4.6 mm (26.1 Mpa, 30,800 plate )
Company P C18
6
4
Company T C18, 2.6 μm 150 x 4.6 mm (22.7 Mpa, 31,600 plate)
26.1 MPa
Company W C18, 2.7 μm 150 x 4.6 mm (18.5 Mpa, 23,300 plate)
7
Company A C18, 2.7 μm 150 x 4.6 mm (30.6 Mpa, 30,200 plate)
5
k6=7.4
Company
T
C18
Company S C18, 2.7 μm 150 x 4.6 mm (22.2 Mpa, 31,800 plate)
N6=31,600
6
4
22.7 MPa
SunShell C18, 2.6 μm 150 x 4.6 mm (21.8 Mpa, 31,900 plate)
Mobile phase: CH3OH/H2O=75/25
7
k6=7.7
5
Company W C18 Flow rate: 1.0 mL/min
4
N6=23,300
6
18.5 MPa
Temperature: 40 ºC
Sample: 1 = Uracil, 2 = Caffeine, 3 = Phenol, 4 = Butylbenzene
7
k6=9.0
5
5 = o-Terphenyl, 6 = Amylbenzene, 7 = Triphenylene
Company A C18
N6=30,200
7
2
113
1
2
3
12
3
1 2
3
k6=5.4
N6=30,800
5
0
2
4
4
6
7
6
SunShell C18
21.8 MPa 4
8
Hydrogen bonding Hydrophobicity Steric selectivity
30.6 MPa
5
2 k6=9.7
1 3 N6=31,800
2
3
1
6
4
5
6
(Amylbenzene/Butylbenzene)
(Triphenylene/o-Terphenyl)
0.48
1.54
1.20
0.35
1.56
1.50
Company W C18
0.38
1.59
1.32
Company A C18
0.42
1.57
1.25
Company S C18
0.44
1.60
1.31
SunShell C18
0.39
1.60
1.46
Company P C18
Company S C18
Company T C18
22.2 MPa
7
k6=10.4
N6=31,900
10
12
14
16
Retention time/min
(Caffeine/Phenol)
18
20
22
24
Comparison of pyridine
3
2
1
Company P C18
Column:
Company P C18, 2.6 mm 150 x 4.6 mm
Company T C18, 2.6 mm 150 x 4.6 mm
Company W C18, 2.7 mm 150 x 4.6 mm
Company A C18, 2.7 mm 150 x 4.6 mm
Company S C18, 2.7 mm 150 x 4.6 mm
SunShell C18, 2.6 mm 150 x 4.6 mm
Mobile phase: CH3OH/H2O=30/70
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Detection: [email protected]
Sample: 1 = Uracil
2 = Pyridine
3 = Phenol
3
2
1
Company T C18
3
Company W C18
2
1
3
2
Company A C18
1
3
2
Company S C18
1
2
3
SunShell C18
1
0
1
2
3
4
5
Retention time/min
6
7
8
9
10
11
12
13
Comparison of oxine, metal chelating
compound
1
2
TF=1.24
Company P C18
2
Company T C18
1 TF=3.94
2
1
TF=1.56
Company W C18
2
1
TF=2.07
Company A C18
2
1
Company S C18
TF=2.04
2
1
TF=1.21
0
1
2
3
4
Retention time/min
5
Column:
Company P C18, 2.6 mm 150 x 4.6 mm
Company T C18, 2.6 mm 150 x 4.6 mm
Company W C18, 2.7 mm 150 x 4.6 mm
Company A C18, 2.7 mm 150 x 4.6 mm
Company S C18, 2.7 mm 150 x 4.6 mm
SunShell C18, 2.6 mm 150 x 4.6 mm
Mobile phase: CH3CN/20mM H3PO4=10/90
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Detection: [email protected]
Sample: 1 = 8-Quinolinol (Oxine)
2 = Caffeine
SunShell C18
6
7
8
9
10
Comparison of formic acid
1
TF=1.47
TF=1.86
3
2
Company P C18
Company T C18
3
2
TF=1.79 1
TF=1.45
3
2
1
Company W C18
1
2
Company A C18
3
2
TF=3.21
TF=1.46
0
1
3
1
Company S C18
1
3
2
SunShell C18
2
3
Retention time/min
4
5
6
Column:
Company P C18, 2.6 mm 150 x 4.6 mm
Company T C18, 2.6 mm 150 x 4.6 mm
Company W C18, 2.7 mm 150 x 4.6 mm
Company A C18, 2.7 mm 150 x 4.6 mm
Company S C18, 2.7 mm 150 x 4.6 mm
SunShell C18, 2.6 mm 150 x 4.6 mm
Mobile phase: CH3CN/0.1% H3PO4=2/98
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Detection: [email protected]
Sample: 1 = Formic acid
2 = Acetic acid
3 = Propionic Acid
Summary of standard samples
Pressurea
Retentionb
SunShell C18
○21.8
10.4
Ascentis Express C18
○22.2
PoroShell C18 EC
PlateC
Pyridine
Oxine
Formic acid Point
◎31,900
◎
◎
◎
14
9.7
◎31,800
△
△
×
7
×30.6
9.0
◎30,200
◎
△
◎
10
Cortecs C18
◎18.5
7.7
×23,300
×
○
△
6
Accucore C18
○22.7
7.4
◎31,600
×
×
△
6
Kinetex C18
△26.1
5.4
◎30,800
×
◎
◎
10
a. Mobile phase; methanol:water=75:25, 40 oC, 1mL/min, 150 x 4.6mm
b. Retention factor of amylbenzene
c. Theoretical plate of amylbenzene
◎: 3 point, ○: 2 point, △: 1 point, ×: 0 point
Particle size distribution
12
SunSehll C18 (2.54 μm)a
a
Ascentic Express C18 (2.52 μm)
Accucore C18 (2.53 μm) a
Kinetex C18 (2.34 μm)a
PoroShell C18 (2.34 μm)a
Cortecs C18 (2.77 μm) a
Number/%
10
8
6
4
2
0
1
2
Particle diameter/μm
4
*Measured using Beckman Coulter Multisizer 3 after C18 materials were sintered
at 600 degree Celsius for 8 hours. The value measure by Coulter Counter method
is smaller than the real value because a porous material includes an electrolyte
solution and the resistance value decreases.
a. Median particle size
8
Characteristics
Carbon
loading (%)
Specific surface Pore volume a
area a (m2/g)
(mL)
Pore diameter a
(nm)
7.3 (7)b
125 (150)b
0.261
8.34 (9)b
8.0
133 (150)b
0.278
8.20 (9)b
PoroShell C18 EC
8.5 (8)b
135 (130)b
0.414
12.3 (12)b
Accucore C18
8.8 (9)b
130 (130)b
0.273
8.39 (8)b
Cortecs C18
7.3 (6.6)b
113
0.264
9.32
Kinetex C18
4.9
(12 effective)b
102
(200 effective)b
0.237
9.25 (10)b
SunShell C18
Ascentis Express C18
a. Measured after C18 materials were sintered at 600 degree Celsius for 8 hours.
The measured value of each sintered core shell silica is considered to be smaller
than that of the original core shell silica.
b. Value written in each brochure or literature
All data were measured in ChromaNik laboratory.
Effective surface area
Specific surface area
Loading capacity of amitriptyline I
Mobile phase: Acetonitrile/20mM phosphate buffer pH7.0=(60:40)
Column dimension: 150 x 4.6 mm, Flow rate: 1.0 mL/min, Temp.: 40oC
CH3
N
CH3
3
16000
1
4
TF=1.18
2
SunShell C18
(core shell)
14000
TF=1.42
Theoretical plate
12000
10000
8000
6000
100 times
SunShell C18
Company A C18
Sunniest C18 3um
Company P C18
Company T C18
Compnay W C18
Company S C18
TF=2.43
TF=3.21
2000
T C18
(core shell)
W C18
(core shell)
TF=3.17
0.01
Sunniest
C18 3μm
(fully porous)
P C18
(core shell)
TF=1.25
4000
0
0.001
A C18
(core shell)
0.1
1
10
Sample weight/mg
0
Theoretical plate was calculated by 5σ method
using peak width at 4.4% of peak height.
S C18
(core shell)
TF=4.38
1
2
3
4
5
6
7
Retention time/min
8
9
Sample: 1=Uracil, 2=Propranolol,
3= Nortriptyline, 4=Amitriptyline
10
Loading capacity of amitriptyline I
Mobile phase: Acetonitrile/20mM phosphate buffer pH7.0=(60:40)
Column dimension: 150 x 4.6 mm, Flow rate: 1.0 mL/min, Temp.: 40oC
16000
14000
TF=2.09
5 μg
Theoretical plate
12000
10000
8000
6000
100 times
SunShell C18
Company A C18
Sunniest C18 3um
Company P C18
Company T C18
Compnay W C18
Company S C18
T C18
(core shell)
4000
TF=3.21
0.3 μg
2000
0
0.001
0.01
0.1
1
10
Sample weight/mg
0
Theoretical plate was calculated by 5σ method
using peak width at 4.4% of peak height.
1
2
3
4
5
6
7
Retention time/min
Sample: 1=Uracil, 2=Propranolol,
3= Nortriptyline, 4=Amitriptyline
8
9
10
Loading capacity of amitriptyline II
Mobile phase: Acetonitrile/10mM ammonium acetate pH6.8=(40:60)
Column dimension: 150 x 4.6 mm, Flow rate: 1.0 mL/min, Temp.: 40oC
14000
4
1
Theoretical plate
12000
10000
8000
6000
Sunshell C18
Company A C18
Company P C18
3
2
30 times
TF=1.20
SunShell C18
(core shell)
TF=1.89
A C18
(core shell)
Company T C18
Company W C18
Company S C18
T C18
(core shell)
TF=2.73
4000
W C18
(core shell)
TF=3.12
2000
0
0.001
P C18
(core shell)
TF=2.61
S C18
(core shell)
TF=3.24
0.01
0.1
1
Sample weight/mg
10
0
1
2
3
4
5
6
7
8
9
10
Retention time/min
Sample: 1=Uracil, 2=Propranolol,
3= Nortriptyline, 4=Amitriptyline
Loading capacity of amitriptyline III
Mobile phase: Acetonitrile/0.1% formic acid=(30:70)
Column dimension: 150 x 4.6 mm, Flow rate: 1.0 mL/min, Temp.: 40oC
4.5
Company S C18
USP tailing factor
4
3.5
USP tailing factor
Company T C18
3.6
Company P C18
Company A C18
3
2.9
Company W C18
2.4
SunShell C18
2.5
7 times
1.8
2
1.3
1.5
1
0.001
4
0.01
0.1
5 min
6
1
Sample weight/μg
In the case of using acetonitrile /0.1% formic acid as a mobile phase, amitriptyline peak shows more tailing because a
loading capacity decreases in an acidic, low-ionic-strength mobile phase.
Stability under acidic pH condition
Relative retention/%
100
▲
▲
▲
▲
80
60
SunShell C18
Company S C18
Company W C18
Company T C18
Company P C18
Company A C18
40
20
0
0
20
40
60
Time/h
80
100
120
Durable test condition
Column size: 50 x 2.1 mm
Mobile phase: CH3CN/1.0% TFA,
pH1=10/90
Flow rate: 0.4 mL/min
Temperature: 80 ºC
Measurement condition
Column size: 50 x 2.1 mm
Mobile phase: CH3CN/H2O=60/40
Flow rate: 0.4 mL/min
Temperature: 40 ºC
Sample: 1 = Uracil
2 = Butylbenzene
Stability under basic pH condition
Relative plate of butylbenzene /%
100
80
SunShell C18
60
Company S C18
Durable test condition
Column size: 50 x 2.1 mm
Mobile phase:
CH3OH/20mM Sodium
borate/10mM NaOH=30/21/49
(pH10)
Flow rate: 0.4 mL/min
Temperature: 50 ºC
Company P C18
40
Company T C18
Company A C18
20
0
0
1,000 2,000 3,000 4,000 5,000 6,000
Elution volume/mL
Measurement condition
Column size: 50 x 2.1 mm
Mobile phase: CH3OH/H2O=70/30
Flow rate: 0.4 mL/min
Temperature: 40 ºC
Sample: 1 = Butylbenzene
Summary of stability
Acidic condition Basic condition
pH 1
pH 10
pH range written in each
brochure
SunShell C18
◎
◎
1.5 - 10
Ascentis Express C18
○
○
2-9
Cortecs C18
○
not tested
2-8
PoroShell C18 EC
△
△
2-9
Accucore C18
△
△
1 - 11
Kinetex C18
△
△
1.5 - 10
Summary
SunShell C18 showed good peaks and the
highest stability.
The value described in the brochure is not
necessarily a true value.
4) Applications related Foods, Proteins and
Other (20min)
Separation of Oolong tea
Column: SunShell C18 2.6 μm, 75 x 4.6 mm
Mobile phase:
A) 0.1% Phosphoric acid
B) CH3CN
Gradient program
Time
0 min 7.5 min
4
%B
Flow rate: 1.0 mL/min,
Temperature: 25 ºC
Detection: [email protected] nm
Sample: Oolong tea
1
OH
2
OH
OH
O
7
2
5
OH
6
3
O
H
OH
5
Catechin
OH
OH
H
O
OH
OH
O
OH
H
9
OH
OH
O
4
5
6
H
H
O
OH
3
OH O
OH
OH
OH
Retention time/min
OH
Gallocatechin gallate
Epigallocatechin gallate l
OH O
2
OH
OH
H
O
1
H
OH
OH
OH
0
O
OH O
Epicatechin
8
OH
OH
OH
OH O
OH
Epicatechin gallate
N
Caffeine
OH
7
H
N
CH3
H
O
H
89
O
O
OH
OH
OH
OH
H
OH
CH3
N
N
H
Epigallocatechin
OH
1
OH
O
H
OH
H
OH
Gallocatechin
H3C
OH
OH
O
O
4
OH
OH
OH
H
OH
H
OH
6
25%
3
OH
OH
OH
2%
10 min
25%
OH
OH
Catechin gallate
Amino Acids derivatized with OPA and FMOC
(o - Phthalaldehyde Solution, Fluorenyl Methyl Chloro Formate)
5
7
13
10
8
15
16
14
6
11
4
3
1
2
12
17
9
0
2
4
6
8
10
Retention time (min)
12
14
16
18
Column: SunShell C18 2.6 μm, 150 x 2.1 mm
Mobile phase: A) 10mM Na2PO4 + 10mM Na2B4O7 + 0.5mM NaN3 (pH7.8)
B) Acetonitrile/Methanol/Water (45/45/10 %V)
Time(min)
%B
0
5
0.4
5
12.8
50
13.8
100
Flow rate: 0.61 mL/min, Temperature: 40 ºC, Detection: [email protected] nm
Sample: 1=Aspartic acid, 2=Glutaminc acid, 3=Serine, 4=Histidine, 5=Glycine, 6=Threonine, 7=Arginine,
8=Alanine, 9=Tyrosine, 10=Valine, 11=Methionine, 12=Tryptophan, 13=Pnehylalanine, 14=Isoleucine,
15=Leucine, 16=Lysine, 17=Proline
Trp, XIC 205.0900±0.0100
Phe, XIC 166.0900±0.0100
Ile,Leu, XIC 132.0000±0.1000
Tyr, XIC 182.0000±0.2000
Met, XIC 150.0610±0.0010
LC/MS of Amino
acids
Arg, XIC 175.1210±0.0010
Val, XIC 118.0000±0.2000
Lys, XIC 147.1160±0.0010
His, XIC 156.0800±0.0100
Pro, XIC 116.0730±0.0010
Glu, XIC 148.0600±0.0100
Ala, XIC 90.0560±0.0010
Cys, XIC 122.0280±0.0010
Thr, XIC 120.0670±0.0100
Gln, XIC 147.0790±0.0100
Gly, XIC 76.0406±0.0010
Asp, XIC 134.0470±0.0010
Ser, XIC 106.0500±0.0100
Asn, XIC 133.0630±0.0010
0
10
Retention time/min
20
Column: SunShell RP-AQUA 2.6 μm, 2.1x150mm
Mobile phase:
A) 5 mM HFBA,
B) 5 mM HFBA in CH3CN / H2O (9/1)
%B 0% to 20% in 20 min
Flow rate: 0.2 mL / min
Temperature: 40 oC
Detection: MS (NanoFrontier LD) ESI Positive,
Extracted ion chromatogram (EIC)
HPLC: LaChrom Ultra
Separation of organic acids
SunShell RP-AQUA 2.6 μm,
Column dimension: 150 x 4.6 mm
Mobile phase: 0.025 M KH2PO4, pH2.5
Flow rate: 1.5 mL/min and 1.0 mL/min
Column pressure: 32 MPa for SunShell and 14 MPa for Sunniest
Temperature: 25 oC
Detection: [email protected]
Sample: 1 = Oxalic acid, 2 = Tartaric acid, 3 = Formic acid,
4 = Malic acid, 5 = Lactic acid, 6 = Acetic acid,
7 = Diglycolic acid, 8 = Maleic acid, 9 = Citric acid,
10 = Succinic acid, 11 = Fumaric acid.
8
1.5 mL/min
3
2
11
4
1
56
9
7
10
Retention time/min
8
Sunniest RP-AQUA 3 μm,
1.0mL/min
2
3
4
11
1
5
6
7
9
10
Retention time/min
S company
Separation of organic acids
3
2
Core shell
Ascents Express
4
C18 AQ
5
2.7 μm,
6
11
9
7
10
1
8
3
Retention time/min
2
SunShell RP-AQUA
11
4
2.6 μm,
9
7
1
6
5
8
10
Retention time/min
Column dimension: 150 x 4.6 mm
Mobile phase: 0.1% H3PO4
Flow rate: 1.0 mL/min
Temperature: 40 oC
Detection: [email protected]
Sample: 1 = Oxalic acid,
2 = Tartaric acid,
3 = Formic acid,
4 = Malic acid,
5 = Lactic acid,
6 = Acetic acid,
7 = Diglycolic acid,
8 = Maleic acid,
9 = Citric acid,
10 = Succinic acid,
11 = Fumaric acid.
SunShell HILIC-Amide, 2.6 μm
For Hydrophilic Interaction Chromatography
Characteristics of SunShell HILIC-Amide
Core shell silica
SunShell HILIC-Amide
Amide (USP L68)
Particle
size
Pore
diameter
Specific
surface area
Carbon
content
Bonded
phase
Endcapping
Maximum operating
pressure
Available pH
range
2.6 μm
9 nm
150 m2/g
3%
Amide
no
60 MPa or 8,570 psi
2-8
Stationary phase of HILIC-Amide
R: Hydrophilic group
Stationary phase of SunShell HILIC-Amide consists of AMIDE and HYDROPHILIC GROUP, so that this
stationary phase is more polar than an individual group. High speed separation is leaded by core
shell structure that derives high efficiency and fast equilibration.
Separation of nucleic acid bases
2
1
SunShell
HILIC-Amide
4
3
5
Column:
SunShell HILIC-Amide 2.6 μm : 100 x 4.6 mm,
Ascentis Express OH5 2.6 μ m : 100 x 4.6 mm
Ascentis Express HILIC 2.6 μ m : 100 x 4.6 mm,
Mobile phase: Acetonitrile :
20 mM ammonium acetate(pH4.7) =8:2
Flow rate: 1.0 mL/min
Temperature: 40 oC
Detection: [email protected] nm,
Sample:
1. thymine, 2. uracil, 3. uridine, 4. cytosine, 5. cytidine
H
O
2
O
HO
H
OH
H
NH2
OH
H3C
O
NH
NH
O
H
HO
H
OH
H
O
N
N
O
N
H
N
H
4
3
1
O
Ascentis Express OH5
2
4
1
H
N
HN
O
O
N
H
N
NH2
5
3
O
OH
H
5
Ascentis Express HILIC
Retention time/min
Separation of water soluble vitamins
2
3
Column:
SunShell HILIC-Amide 2.6 mm : 100 x 4.6 mm,
Mobile phase: Acetonitrile :
25 mM phosphate buffer (pH2.5) =8:2
Flow rate: 1.0 mL/min
Temperature: 40 oC
Detection: [email protected] nm,
Sample:
1.Nicotinic acid, 2. ascorbic acid, 3. pyridoxine,
1
Retention time/min
O
OH
OH
H
HO
H
OH
HO
OH
O
N
OH
1. Nicotinic acid
HO
O
2. Ascorbic acid
H3C
N
3. Pyridoxine
Separation of artificial sweeteners
2
Column:
SunShell HILIC-Amide 2.6 mm : 100 x 4.6 mm,
Mobile phase: Acetonitrile :
25 mM phosphate buffer (pH2.5) =8:2
Flow rate: 1.0 mL/min ,
Temperature: Ambient
Detection: [email protected] nm,
Sample:
1. Aspartame, 2. Saccharin, 3. Acesulfame K,
1
3
0
0.5
1
O
1.5
2
2.5
Retention time/min
O
NHH CH3
O
NH2
H
O
OH
1. Aspartame
2. Saccharin
3. Acesulfame K
Separation of Glycosides
1
3
2
4
0
0.5
1
1.5
Column:
SunShell HILIC-Amide 2.6 mm : 100 x 4.6 mm,
Mobile phase: Acetonitrile :
25 mM phosphate Ammonium (pH4.9) =8:2
Flow rate: 1.0 mL/min
Temperature: Ambient
Detection: [email protected] nm
Sample:
1. Helicin, 2. Salicin, 3. Arbutin 4. Rutin
2
2.5
OH
OH
OHH
OH
OH
H
H
H
H
O
O
OH
O
OHH
OH
OH
H
H
H
H
O
O
OH
OHH
OH
OH
H
H
H
H
O
O
OH
O
O
CH3
H
OH
O
O
H
OH
H
O
O
H
H
H
OH
H
OH
H
OH
OHH
H
OH
OH
OH
1. Helicin
2. Salitin
3. Arbutin
4. Rutin
OH
Separation of Melamine and cyanuric acid
2
Column:
SunShell HILIC-Amide 2.6 mm : 100 x 4.6 mm,
Mobile phase: acetonitrile :
5 mM phosphate Buffer (pH6.9) =75:25
Flow rate: 1.0 mL/min ,
Temperature: 40 oC
Detection: [email protected] nm,
Sample:
1. Cyanuric acid, 2. Melamine,
1
0
0.5
1
1.5
2
2.5
O
NH
O
NH2
NH
NH
O
1. Cyanuric acid
N
NH2 N
N
NH2
2. Melamine
List of phases for separation of high molecular
weight compounds
Particle size,
thickness of
porous layer
Pore
diameter
Surface
area
Carbon
loading
2.6, 0.5 μm
16 nm
90 m2/g
5%
2.5 μmol/m2
Yes
SunShell HFC18-16 2.6, 0.5 μm
16 nm
90 m2/g
2.5%
1.2 μmol/m2
Yes
SunShell HFC18-30 2.6, 0.5 μm
30 nm
40 m2/g
1.3%
1.2 μmol/m2
Yes
SunShell C18-WP
C18 Surface
Endcoverage capping
SunShell C8-30
2.6, 0.5 μm
30 nm
40 m2/g
1.2%
2.5 μmol/m2
Yes
SunShell C4-30
2.6, 0.5 μm
30 nm
40 m2/g
0.9%
3 μmol/m2
Yes
30 nm
15 m2/g
2.5 μmol/m2
Yes
SunShell C8-30-2
(Prototype, sell
from 2015)
3.4, 0.2 μm
72
0.5%
Desorption Dv (log d) (cc/g)
Pore size distribution of core shell particle
0.9
0.8
Core shell 30nm
0.7
0.6
Core shell 16nm
0.5
0.4
0.3
0.2
0.1
0
1
10
100
Pore Diameter (nm)
73
1000
What is HFC18?
Hexa-Functional C18 has six functional groups.
This HFC18 is much more stable under acidic condition.
Sunniest
Bonding
Technology
TMS
Hexamethydichlorotrisiloxane
+
Trimethylchlorosilane (TMS)
Si
Si
O
(X: Cl, OCH3, OC2H5)
Schematic diagram of reagent
O
O
Si
O
Si
Si O O
O
Si
Si
O
O
O
O
O
Si
O O
Si
O
O
Si
Si
O
Si
O
Si O
Si
Si O O
Si
Si O
O O
O
O
O
Si
HO O
O
Si
O
O
Si
Si
O
Si
Si
Si
O
O
Si
O
O
O
Schematic diagram of the state of
74bonding on silica surface
O
O
O
Si O
O O
Stability of HFC18 under LC/MS mobile
phase condition
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
Relative retention (%)
100
95% line
80
60
40
0.1% formic acid, pH2.6
70 ºC
20
0
0
200
400
600
Time (h)
800
1000
Measurement condition
Mobile phase: CH3CN/H2O=60/40
Flow rate: 0.4 mL/min
Temperature: 40 ºC
Sample: 1 = Uracil
2 = Butylbenzene
Comparison of separation of peptides 1
SunShell HFC18-16
(1.2 μmol/m2)
SunShell C18-WP
(2.5 μmol/m2)
Column: SunShell HFC18-16, 2.6 mm (16 nm) 150 x 4.6 mm,
SunShell C18-WP, 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:
Flow rate: 1.0 mL/min , Temperature: 2576ºC,
Sample: Tryptic digest of myoglobin
Detection: [email protected] nm,
Comparison of separation of peptides 2
SunShell HFC18-16
SunShell C18-WP
Column: SunShell HFC18-16, 2.6 mm (16 nm) 150 x 4.6 mm,
SunShell C18-WP, 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:
Flow rate: 1.0 mL/min , Temperature: 2577ºC,
Sample: Tryptic digest of cytochrome C
Detection: [email protected] nm,
Separation of standard proteins
Column:
SunShell C8-30, 2.6 mm (30 nm) 150 x 2.1 mm,
Mobile phase: A) 0.1% TFA in water
B) 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: [email protected] 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
78
Separation of Ribonuclease A/B
Column:
SunShell HFC18-30, 2.6 mm (30 nm) 100 x 2.1
mm,
Mobile phase: A) 0.1% TFA in water
B) 0.1 % TFA in Acetonitrile
Gradient program: Time 0 min 20 min
%B 22% 70.5%
Flow rate: 0.50 mL/min ,
Temperature: 40 oC
Detection: [email protected] nm,
Injection volume: 10 μL,
Concentration: 0.10 μg/μL each protein,
Sample:
1. Ribonuclease B
2. Ribonuclease A
79
Separation of Ribonuclease A/B
MS Detection
Column:
SunShell HFC8-30, 2.6 mm (30 nm) 100 x 2.1 mm,
Mobile phase: A) 0.1% Formic acid in water
B) 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
80
Separation of Lipase
MS Detection
Column:
SunShell HFC8-30, 2.6 mm (30 nm) 100 x 2.1 mm,
Mobile phase: A) 0.1% Formic acid in water
B) 0.1 % Formic acid in Acetonitrile
Gradient program: Time 0 min 10 min
%B 15% 60%
Flow rate: 0.50 mL/min ,
Temperature: 60 oC
Detection: MS,
Injection volume: 10 μL,
Concentration: 0.10 μg/μL each protein,
Sample:
1. Lipase Impurity
2. Lipase
81
Separation of Proteins at 80 oC
Column:
SunShell C8-30, 2.6 μm (30 nm) 100 x 2.1 mm,
Mobile phase: A) 0.1% TFA in water
B) 0.08 % TFA in Acetonitrile
Gradient program: Time 0 min 35 min
%B 20% 65%
Flow rate: 0.5 mL/min ,
Temperature: 80 C
Detection: [email protected] nm,
Sample:1 = Cytochrome C, 2 = Lysozyme,
3 = BSA, 4 = Myoglobin, 5 = Ovalbumin
○
2
SunShell C8-30
3
1
4
5
10
15
5
20
25
Retention time/min
82
Separation of proteins Effect of temperature
Column:
340000
SunShell C8-30, 2.6 μm (30 nm) 100 x 2.1 mm,
Mobile phase: A) 0.1% TFA in water
B) 0.08 % TFA in Acetonitrile
Gradient
program: Time 0 min 15 min
290000
%B 20% 65%
Flow rate: 0.5 mL/min
80℃
25 oC
150 mm L
Temperature:
25 oC 60 oC and 80 oC
240000
Detection: [email protected] nm
Sample:1 = Cytochrome C, 2 = Lysozyme, 3 = BSA,
4190000
= Myoglobin, 5 = Ovalbumin
140000
60 oC
90000
40000
80 oC
-10000
0
2
4
6
8
10
12
Retention time/min
83
Separation of Proteins Effect of gradient time
390000
Column:
SunShell C8-30, 2.6 μm (30 nm) 100 x 2.1 mm,
Mobile phase: A) 0.1% TFA in water B) 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: [email protected] nm,
Sample:1 = Cytochrome C, 2 = Lysozyme, 3 = BSA, 4 = Myoglobin,
35 min
25 min
15 min
5 = Ovalbumin
340000
290000
240000
190000
140000
Time 0 min
%B 20%
15min
65%
90000
Time 0 min
%B 20%
25min
65%
40000
Time 0 min
%B 20%
35min
65%
-10000
0
5
10
15
Retention time/min
20
25
84
Comparing of Protein Separation
Comparison of thickness of porous layer
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 35 min
%B 20% 65%
Flow rate: 0.5 mL/min , Temperature: 80 oC Detection: [email protected] nm,
Sample:1 = Cytochrome C, 2 = Lysozyme, 3 = BSA, 4 = Myoglobin, 5 = Ovalbumin
2
1
3
5
4
Prototype 3.4 μm (0.2 μm)
SunShell C8-30 (0.5 μm)
3
0
5
10
15
5
4
13
15
17
19
21
Retention time/min
20
25
23
25
85
Separation of Proteins Comparison of thickness of porous layer
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
%B 20%
65%
Flow rate: 0.5 mL/min , Temperature: 60 or 80 oC, Detection: [email protected] nm,
Sample:1 = Cytochrome C, 2 = Lysozyme, 3 = BSA, 4 = Myoglobin, 5 = Ovalbumin
Thickness of porous layer and particle size are
offset each other at 60 oC, so that the weak
width is same.
2
3
1
W0.5=2.5 sec
4
60
oC
and 15 min gradient
5
W0.5=2.5 sec
Prototype 3.4 μm (0.2 μm)
SunShell C8-30 2.6 μm (0.5 μm)
W0.5=4.8 sec
80
oC
and 35 min gradient
There is no difference between 0.2 μm and
0.5 μm of porous layer at 80 oC
W0.5=4.2 sec
Prototype 3.4 μm (0.2 μm)
SunShell C8-30 2.6 μm (0.5 μm)
0
5
10
Retention time/min
15
20
Surface area works well for separation.
25
86
Separation of Proteins Comparison of thickness of porous layer
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 5 or 35 min
%B 20%
65%
Flow rate: 0.5 mL/min , Temperature: 60 or 80 oC, Detection: [email protected] nm,
In case of fast separation, 0.2 μm of
0.5
Sample:1 = Cytochrome C, 2 = Lysozyme, 3 = BSA, 4 = Myoglobin, 5 = Ovalbumin
porous layer showed better separation
W =0.87 sec
2
W0.5=0.93 sec
1
3
than 0.5 μm of porous layer.
5
4
80 oC and 5 min gradient
Prototype 3.4 μm (0.2 μm)
SunShell C8-30 2.6 μm (0.5 μm)
W0.5=4.8 sec
80
oC
and 35 min gradient
There is no difference between 0.2 μm and
0.5 μm of porous layer at 80 oC
W0.5=4.2 sec
Prototype 3.4 μm (0.2 μm)
SunShell C8-30 2.6 μm (0.5 μm)
0
5
10
Retention time/min
15
20
Surface area works well for separation.
25
87
Which is better?
SunShell particle
Particle size: 2.6 μm
Thickness of porous layer: 0.5 μm
Specific surface area: 40 m2/g
Prototype particle (sales from 2015 )
Particle size: 3.4 μm
Thickness of porous layer: 0.2 μm
Specific surface area: 15 m2/g
It 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 of efficiency 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 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.
88