Sunshell Seminar

Schematic Diagram of Core Shell silica

Particle diameter: 2.6 m, Core diameter: 1.6 m, Thickness of porous silica: 0.5 mPore volume: 0.30mL/g, Specific surface area: 150 m2/g, Pore diameter: 9 nmThe ratio of porous silica volume: 77%

0.5 m

2.6 m Core

Porous silica

0.5 m

Core

Porous silica

2.6 m

1.6 m

Relationship between t0/N2 （ impedance time) and theoretical plate

3

13,162,278 316,228 31,623 3,162

1,000,000 100,000 10,000

100

10

1000

N

t 0/N

2 (nS

)1.4 m

2 m3 m5 m

t0=1 st0=10 st0=100 s

t0=1000 s

t0=10000 s

40 MPa

Core Shell 2.6 m

Figure 1. Kinetic plot analysis at 40 MPa. The curves for particulate columns were obtained by assumingη=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 1.4, 2.0, 3.0 , 5.0 μm, core shell 2.6 m.

Increase of plate numberNeed low HETP

HETP≒2.2 x Dp (Dp: Particle diameter)

Particle diameter 5 m 3 m 2 m 2.6 m

HETP 11.0 m 6.6 m 4.4 m 4.4 m

The smaller particle diameter, the lower HETP. ?

Comparison between core shell and totally porous particles

2.6 m

2.0 m

2.0 mCa. 2.0 m

sample sample

Diffusion area orDiffusion height

1.6 m

Core shell Totally porous

HETP is related to diffusion height, not particle diameter.

Comparison of Plate Height Plots

Column: C18, 50 x 4.6 mm Mobile phase: Acetonitrile/water=(60/40)Temperature: 25 oC Sample : Naphthalene

Comparison of Reduced Plate Height Plots

Column dimension: 50 x 2.1 mm Mobile phase: Acetonitrile/water=(70/30)Temperature: 25 oC

Thermo

Agilent Zorbax

Waters Acquity

D10:1.75mD50: 2.01mD90: 2.31 mD90/D10=1.32

D10:1.67 mD50: 2.09 mD90: 2.65 mD90/D10=1.59

m)0.5 1 2

D10:2.46mD50: 2.63mD90: 2.82 mD90/D10=1.15

Comparison of Particle Size Distribution

Company F 2 m

Sunniest 2 m

SunShell 2.6 mJapanese silica manufacturerFuji SIlicia

Comparison of Back Pressure for High Throughput Columns

Column dimension: 50 x 2.1 mm Mobile phase: Acetonitrile/water=(70/30)Temperature: 25 oC

Thermo

Agilent Zorbax

Waters Acquity

Phenomenex Kinetec

Comparison of Performance by Plate/Pressure

Plates Pressure(MPa) Plate/pressureSunniest C18 –HT 2.0 m 9,900 16.7 593Brand A C18 1.9 m 7,660 16.3 470Brand B C18 1.8 m 10,100 19.6 515Brand C C18 1.7 m 11,140 32.0 348SunShell C18 2.6 m 9,600 9.7 990

Sunniest C18 –HT 2.0 m

Brand A C18 1.9 m

Brand B C18 1.8 m

Brand C C18 1.7 m

SunShell C18 2.6 m

Column: 50 x 2.1 mm C18, Mobile phase: Acetonitrile/water=(70/30), Temperature: 25 oC

Thermo

Agilent Zorbax

Waters Acquity

Comparison of retention between fully porous silica C18 and core shell silica C18s

Fully porous silica C18Sunniest C18 　 5 mm

Core shell silica C18SunShell C18 2.6 mm

Core shell silica C18

Kinetex C18 2.6 mmSpecific surface area 340 m2/g 150 m2/g Effective 200 m2/g

Packing weight 1.5 g per column 2.7 g per column (180%)

Retention time ( ｔ R)

Retention factor (k)

Retention time ( ｔ R)


Retention time ( ｔR)


1 = Uracil 1.70 0 1.35 0 1.36 02 = Caffeine 1.90 0.12 1.47 0.09 1.49 0.103 = Phenol 2.17 0.28 1.65 0.22 1.61 0.184 = Butylbenzene 13.35 6.85 10.01 6.41 6.19 3.555 = o-Terphenyl 19.19 10.29 14.24 9.55 8.15 4.996 = Amylbenzene 19.96 10.74 15.09 10.18 8.75 5.437 = Triphenylene 24.35 13.32 20.33 14.06 9.44 5.94

Mobile phase: Methanol/water(75:25)Temperature: 40oCColumn dimension: 150 x 4.6 mmFlow rate: 1.0 mL/min　　

0 5 10 15 20 25 30Retention time/min

Sunniest C18 　 5 m1

2 34

56

7

SunShell C18 2.6 m12 3

4

5

6

7

Column: SunShell C18, 2.6 m 100 x 2.1 mmMobile phase: CH3CN/H2O=60/40 Flow rate: 0.3 mL/minPressure: 12.5 MPaTemperature: 25 ºC

Sample: 1 = Uracil 2 = Toluene　　　　　　 3 = Acenaphthene 4 = BUtylbenzene


Chromatogram of standard samples of SunShell C18

1

23 4

Plates(3)=38,000

Retention time/min

Plates(3)=21,30021

3

4

Comparison of standard samples

0 2 4 6 8 10 12 14 16 18 20 22 24

Retention time/min

Column: Kinetex C18, 2.6 m 150 x 4.6 mm Ascentis Express C18, 2.7 m 150 x 4.6 mm SunShell C18, 2.6 m 150 x 4.6 mmMobile phase: CH3OH/H2O=75/25 Flow rate: 1.0 mL/minTemperature: 40 ºC Sample: 1 = Uracil, 2 = Caffeine, 3 = Phenol, 4 = Butylbenzene 5 = o-Terphenyl, 6 = Amylbenzene, 7 = Triphenylene

Kinetex C18

Ascentis Express C18

SunShell C18 Hydrogen bond (Caffeine/Phenol)

Hydrophobicity(Amylbenzene/Butylbenzene)

Steric selectivity(Triphenylene/o-Terphenyl)

Kinetex C18 0.48 1.54 1.20

Ascentis Express C18 0.44 1.60 1.31

SunShell C18 0.40 1.59 1.47

1

1

1

2

2

2

7

3

3

3

4

4

4

5

5

5

6

6

6

7

7

Comparison of pyridine

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Retention time/min

Column: Kinetex C18, 2.6 m 150 x 4.6 mm Ascentis Express C18, 2.7 m 150 x 4.6 mm SunShell C18, 2.6 m 150 x 4.6 mm Mobile phase: CH3OH/H2O=30/70Flow rate: 1.0 mL/minTemperature: 40 ºC Detection: UV@250nmSample: 1 = Uracil　　　　　 2 = Pyridine 3 = Phenol

1

1

2

3

2

3

1

2 3

Kinetex C18


SunShell C18

Comparison of oxine, metal chelating compound

0 1 2 3 4 5 6 7 8 9 10

Retention time/min

Column: Kinetex C18, 2.6 m 150 x 4.6 mm Ascentis Express C18, 2.7 m 150 x 4.6 mm SunShell C18, 2.6 m 150 x 4.6 mm Mobile phase: CH3CN/20mM H3PO4=10/90Flow rate: 1.0 mL/minTemperature: 40 ºC Detection: UV@250nmSample: 1 = 8-Quinolinol (Oxine)　　　　　 2 = Caffeine

21

12

21

Kinetex C18


SunShell C18

Comparison of formic acid

0 1 2 3 4 5 6

Retention time/min

1

1 2

21

2

3

3

3

Column: Kinetex C18, 2.6 m 150 x 4.6 mm Ascentis Express C18, 2.7 m 150 x 4.6 mm SunShell C18, 2.6 m 150 x 4.6 mmMobile phase: CH3CN/0.1% H3PO4=2/98Flow rate: 1.0 mL/minTemperature: 40 ºC Detection: UV@210nmSample: 1 = Formic acid　　　　　 2 = Acetic acid 3 = Propionic Acid

Kinetex C18


SunShell C18

Comparison of basic compounds I

18

1

1

1

2

2

2

3

3

3

4

4

4

A

B

C

N(4) =11,000TF(4) =1.03

N(4) =9,000TF(4) =1.06

N(4) =12,500TF(4) =1.04

Brand　E　C18

4

4

4Brand　E　C18

Brand　E　C18

Column:　Sunniest　C18, 5 m 150　x　　4.6　mm　　　　　　　　　　　　　　　　Re:　Brand　E　C18,　5 m 150　x　　4.6　mmMobile　phase:　　A)　CH3OH/20mM　Phosphate　buffer　pH7.5　=　80/20　B)　CH3OH/20mM　Phosphate　buffer　pH6.0　=　80/20　C)　CH3CN/20mM　Phosphate　buffer　pH7.0　=　60/40　Flow　rate:　1.0　mL/minTemperature:　40　ºC　for　A,　C　and　D,　22　ºC　for　B　Sample:　1　=　Uracil,　　　　　　　　　　　　　　　　　2　=　Propranolol,　

　　　　　　　　　　　　　　　　3　=　Nortriptyline,　

　　　　　　　　　　　　　　　　4　=　Amitriptyline,　

TF: USP tailing factor

O

OH

NH CH3

CH3

N

CH3

CH3

NHCH3

N(4)=10,000TF(4)=1.50

N(4)=5,800TF(4)=2.86

N(4)=4,300TF(4)=5.24

Column　size:　150　x　4.6　mm　for　A,　D　and　E　　　　　　　　　　　　　　　　　　　　　　　100　x　2.1　mm　for　B　and　C　　Mobile　phase:　　CH3CN/20mM　Phosphate　buffer　pH7.0=60/40Flow　rate:　1.0　mL/min　for　A,　D,　and　E　　　　　　　　　　　　　　　　　　0.4　mL/min　for　B　and　CTemperature:　40　ºCSample:　1　=　Uracil　　　　　　　　　　　　　　　　2　=　Propranolol　　　　　　　　　　　　　　　

　　　　　　 3　=　Nortriptyline

　　　　　　　　　　　　　　　　4　=　Amitriptyline

　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　

Comparison of basic compounds II

O

OH

NH CH3

CH3

NHCH3

N

CH3

CH3

TF: USP tailing factor

TF(4)=1.04

TF(4)=1.28

TF(4)=5.24

TF(4)=1.18

Sunniest　C18　(HMODTS),　5　m

Brand　E　C18,　5　m1

1

1

2

1

2

2

2

３

3

3

3

4

4

4

Sunniest　C18-HT(HMODTS),　2　m

SunShell　C18(HMODTS),　2.6　m　core　shell　typecore　shell　type

4

1 23 4

TF(4)=2.24

Brand　D　C18,　2.6　m　(Kinetex)　core　shell　type　core　shell　type　

A

B

C

D

E

4.4%

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

0 1 2 3 4 5 6 7 8 9 10Retention time/min

Loading capacity of amitriptyline I

Theoretical plate was calculated by 5σ method using peak width at 4.4% of peak height.

Kinetex C18

Acsentis Express C18

SunShell C18

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

100 times

Loading capacity of amitriptyline II

0 1 2 3 4 5 6 7 8 9 10Retention time/min

SunShell C18

30 times

Mobile phase: Acetonitrile/10mM acetate ammonium 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

Kinetex C18

Acsentis Express C18

4 5 min 6

Loading capacity of amitriptyline III

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.

USP Tailing factor

3.6

2.9

2.4

1.8

1.3

7 times

Mobile phase: Acetonitrile/0.1% formic acid=(30:70)Column dimension: 150 x 4.6 mm, Flow rate: 1.0 mL/min, Temp.: 40oC

Stability under acidic pH condition

Durable test conditionColumn size: 50 x 2.1 mmMobile phase: CH3CN/1.0% TFA, pH1=10/90 Flow rate: 0.4 mL/minTemperature: 80 ºC

Measurement conditionColumn size: 50 x 2.1 mmMobile phase: CH3CN/H2O=60/40Flow rate: 0.4 mL/minTemperature: 40 ºC Sample: 1 = Uracil 2 = Butylbenzene

Kinetex

Ascentis Express

Stability under basic pH conditionDurable test conditionColumn Size: 50 x 2.1 mmMobile phase: CH3OH/20mM Sodium borate/10mM NaOH=30/21/49 (pH10) Flow rate: 0.4 mL/minTemperature: 50 ºC

Measurement conditionColumn Size: 50 x 2.1 mmMobile phase: CH3OH/H2O=70/30Flow rate: 0.4 mL/minTemperature: 40 ºC Sample: 1 = Butylbenzene

Kinetex

Ascentis Express

SunSell C18

Brand L C18

＋ Q1: 5.997 min to 7.999 min of Sample

Column size: 50 x 2.1 mmMobile phase: A) 0.1% acetic acid B) CH3CNGradient: Time: 0min 1min 5min 7min %B: 5% 5% 100% 100%Flow rate: 0.4 mL/minTemperature: 40 ºC MS ： ABI API-4000Ionization: Turboionspray (cation)Measurement mode: Q1 Scan m/z 100-1000

Bleeding test using LC/MS

TIC of ＋ Q1

YMC Pro C18

0 2 4 6 8 10 Sample: 1 = Uracil 2 = Toluene　　 3 = Acenaphthene 4 = BUtylbenzene


Comparison between HPLC and UHPLC

1

23 4

Plates(3)=38,000

Retention time/min

26

UHPLC

1 2

3

4

Plates(3)=31,000

Retention time/min

HPLC

80% performance to compare with UHPLC

Still 1.5 times more separation than that of a 3 um column

Column: SunShell C18, 2.6 m 100 x 4.6 mm 150 x 4.6 mmMobile phase: CH3CN/H2O=70/30 Flow rate: 1.0 mL/minPressure: 9.5 MPa,13.5 MPaTemperature: 25 ºC

Separation of standard samples using HPLC

1 2

3

4Plates(3)=20,000

Retention time/min

27

HPLC

12

3

4

Plates(3)=31,000

Retention time/min

HPLC

Sample: 1 = Uracil 2 = Toluene　　　　　　 3 = Acenaphthene 4 = BUtylbenzene

The same efficiency as 5 m, 250 x 4.6 mm

Saving 60% for analytical time and consumption of solvent

Examples of transfer (isocratic separation)

28

SunShell C18, 2.6 m 100 x 4.6 mm



0 2 4 6 8 10 12 14 16 18 20 22 24Retention time/min

HPLC

UHPLC

1/3 of analysis time

1

1

1

1

1

2

2

2

2

2

3

3

3

3

3

4

4

4

4

4

5

5

5

5

5

ACE C18, 5 m 250 x 4.6 mm

1.0 mL/min

1.8 mL/min

N(4)=20,287

N(4)=22,424

N(4)=19,313

N(4)=24,124

N(4)=20,287

Analysisi time decreases to 1/6 to compare with a 5m column sized 250x4.6mm.

Column: ACE C18, 5 m 250 x 4.6 mm SunShell C18, 2.6 m 100 x 4.6 mmMobile phase:CH3CN/20mM Phosphoric acid = 45/55Flow rate: 1.0 mL/min, 1.8 mL/min at the lowest chromatogramTemperature: 25 ºCPressure: 9.5 MPa for Brand F C18 5 m 13.4 MPa for SunShell C18 2.6 mDetection: UV@230 nm

Sample: 1 = Benzydamine 2 = Ketoprofen 3 = Naproxen 4 = Indomethacin 5 = Ibuprofen

HPLC: Hitachi LaChrom ELITE 　　　　　 (using 0.25 mm i.d. tubing) UHPLC: Jasco X-LC



0 2 4 6 8 10 12 14 16 18 20 22 24Retention time/min

HPLC

UHPLC

1/2 of Analysis time

1

1

1

1

2

2

2

2

3

3

3

3

4

4

4

4

5

5

5

5

Sunniest C18, 5 m 150 x 4.6 mm

SunShell C18, 2.6 m 75 x 4.6 mm

7 8 9

6

6

6

7

7

8 9

7

8 9

8 9

8 9

76

Column: Sunniest C18, 5 m 150 x 4.6 mm SunShell C18, 2.6 m 75 x 4.6 mmMobile phase: A) 0.1% Phosphoric acid B) CH3CNGradient program for Brand G C18

for SunShell C18

Flow rate: 1.0 mL/min, Temperature: 25 ºCDetection: UV@250 nmSample: Oolong tea1 = Gallocatechin, 2 = Epigallocatechin,3 = Catechin, 4 = Caffeine, 5 = Epicatechin, 6 = Epigallocatechin gallate, 7 = Gallocatechin gallate, 8 = Epicatechin gallate, 9 = Catechin gallate

HPLC: Hitachi LaChrom ELITE 　　　　　 (using 0.25 mm i.d. tubing)UHPLC: Jasco X-LC

Examples of transfer (gradient separation)

Time 0 min 15 min 20 min %B 2% 25% 25%

Time 0 min 7.5 min 10 min %B 2% 25% 25%

SunShell C18, C8, PFP

30

Chracteristics of SunShell Core shell silica Bonded phase

Particle size

(m)

Pore diameter (nm)

Specific surface area (m2/g)

Carbon content (%)

Bonded phase Maximum operating pressure

Available pH range

SunShell C18 2.6 9 150 7 C18 60 MPa or 8,570 psi 1.5 - 10

SunShell C8 2.6 9 150 4.5 C8 60 MPa or 8,570 psi 1.5 - 9

SunShell PFP 2.6 9 150 4.5 Penta-fluorophenyl

60 MPa or 8,570 psi 2 - 8

SunShell C8 and PFPSeparation of amitriptyline using C8 Separation of xanthines Separation of isomers of xylene

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Sunshell Seminar