sunshell seminar
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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 factor (k)
Retention time ( tR)
Retention factor (k)
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
Column: SunShell C18, 2.6 m 150 x 4.6 mmMobile phase: CH3CN/H2O=70/30 Flow rate: 1.0 mL/minPressure: 11.5 MPaTemperature: 25 ºC
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
Ascentis Express 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
Ascentis Express 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
Ascentis Express 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
Column: SunShell C18, 2.6 m 150 x 4.6 mmMobile phase: CH3CN/H2O=70/30 Flow rate: 1.0 mL/minPressure: 13.5 MPaTemperature: 25 ºC
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 1 2 3 4 5 6Retention time/min
0 1 2 3 4 5 6Retention time/min
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 1 2 3 4 5 6Retention time/min
0 1 2 3 4 5 6Retention time/min
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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