basics in fast hplc - sigma-aldrich: analytical, … © 2013 sigma-aldrich co. all rights reserved....
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sigma-aldrich.com/analytical
© 2013 Sigma-Aldrich Co. All rights reserved.
Basics in Fast HPLC
Dr. Frank Michel
© 2013 Sigma-Aldrich Co. All rights reserved.
What influences retention?
with
• tr = time of retention
• L = column length
• k’ = retention factor
• u = mobile phase velocity
2
tr = (k’+1)Lu
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Chromatographers Triangle
3
Speed
Sensitivity Efficiency
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4
Efficiency:
N = Lcol/H ���� H = Lcol/Nvan Deemter equation:
H = A + B/u + Cu
H
u
A = 2λλλλdP
From: Veronika R. Meyer,Practical High-Performance Liquid Chromatography
© 2013 Sigma-Aldrich Co. All rights reserved.
5
Efficiency:
N = Lcol/H ���� H = Lcol/Nvan Deemter equation:
H = A + B/u + Cu
H
u
A = 2λλλλdP
B = 2γγγγDm
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6
Efficiency:
N = Lcol/H ���� H = Lcol/Nvan Deemter equation:
H = A + B/u + Cu
H
u
A = 2λλλλdP
B = 2γγγγDm
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7
Efficiency:
N = Lcol/H ���� H = Lcol/Nvan Deemter equation:
H = A + B/u + Cu
H
u
A = 2λλλλdP
B = 2γγγγDm
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van Deemter and particle size
8Linear velocity of the mobile phase (mm/s)
HE
TP
(µm
)
1 2 3 4 5
16,000
35.00
30.00
25.00
20.00
15.00
10.00
5.00
Pre
ssure
(psi)
H = A + B/ u + Cu
HE
TP
(µm
)
1 2 3 4 5
16,000
35.00
30.00
25.00
20.00
15.00
10.00
5.00
Pre
ssure
(psi)
H = A + B/ u + Cu
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Methods to Increase the Speed of HPLC Separations
• Reduce column dimensions
• Reduce particle size
• Increase flow rate
• Increase temperature
• Change gradient profile
9
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Column Dimensions
10
0 10 20 30
Time (min)
1
2
3
4
5
6
0 10 20 30
1
23
4
5
6
0 10 20 30
1
2
3
4
5
6
0 10 20 30
12
34
5
6
15cm x 4.6mm ID
5cm x 4.6mm ID
2cm x 4.6mm ID
25cm x 4.6mm ID
Columns: Discovery™ C18, 5µm particles
Mobile Phase: CH3OH:H2O (45:55)Flow Rate: 1mL/min
Temp.: 20°CDet.: UV, 214nm
1. Barbital2. Phenobarbital3. Butabarbital4. Mephobarbital5. Pentabarbital6. Secobarbital
98-0348
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Column Dimensions
11
2cm x 4.6mm ID
25cm x 4.6mm ID
Columns: Discovery™ C18, 5µm
Mobile Phase: CH3OH:H2O (45:55)Flow Rate: 1mL/min
Temp.: 20°CDet.: UV, 214nm
1. Barbital2. Phenobarbital3. Butabarbital4. Mephobarbital5. Pentabarbital6. Secobarbital
0 10 20 30
Time (min)
1
2
3
4
5
6
1.0 2.0
1
2
34
5
6
98-0349
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Particle Size
12
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Particle size: Influence on efficiency and pressure
13
050
100150200250300350400
0 5 10 15 20
0
5,000
10,000
15,000
20,000
25,000
30,000
0 5 10 15 20
Particle (µm) psi bar N
1.8 5889 406 27,500
2.5 3089 213 20,000
3 2118 146 16,500
5 769 53 10,000
10 189 13 5,000
15 87 6 3,750
20 44 3 2,500
10 cm column, 3 mm/s linear velocity
dp (µm)
pdN
1∝
2
1
pdP ∝
Doubling the efficiency by halving the particle size results in a pressure increase by a factor of four.
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Flow Rate
14
2mL/min
3mL/min
4mL/min
1mL/min
Columns: Discovery™ C18 column,
5.0cm x 4.6mm, 5µm particlesMobile Phase: CH3OH:H2O (45:55)
Flow Rate: see figure
Temp.: 25°CDet.: UV, 214nm
1. Barbital2. Phenobarbital3. Butabarbital4. Mephobarbital5. Pentabarbital6. Secobarbital
1.0 2.0 3.0 4.0 5.0 6.0 7.0
Time (min)
1 2
3
4
5
1.0 2.0 3.0 4.0 5.0 6.0 7.0
1
2
1.0 2.0 3.0 4.0 5.0 6.0 7.0
1.0 2.0 3.0 4.0 5.0 6.0 7.0
98-0356
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Column Efficiency with Various Flow Rates
15
Flow Rate 1.0mL/min 2.0mL/min 3.0mL/min 4.0mL/min
N (Barbital) 1766 1460 1250 997
N (Phenobarbital) 2556 1821 1461 1307
N (Butabarbital) 3169 2215 1766 1506
N (Mephobarbital) 3524 2383 1924 1569
N (Pentobarbital) 3829 2494 2029 1704
N (Secobarbital) 3947 2584 2113 1742
98-0357
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Temperature Effect
16
1.0 2.0 3.0 4.0 5.0 6.0
1
2
3
40°°°°C
30°°°°C
20°°°°C
50°°°°C
Columns: Discovery™ RP-AmideC16 column,
5.0cm x 4.6mm, 5µm particlesMobile Phase: ACN:H2O (30:70)
Flow Rate: 2.0mL/minTemp.: see figure
Det.: UV, 254nm1. Clonazepam2. Chlorazepate3. Diazepam
1.0 2.0 3.0 4.0 5.0 6.0
1
2
3
1.0 2.0 3.0 4.0 5.0 6.0
Time (min)
1
2
3
1.0 2.0 3.0 4.0 5.0 6.0
1
2
3
98-0359
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Gradient Profile
17
10%/min gradient
16%/min gradient
20%/min gradient
6.67%/min gradient
Columns: Discovery™ RP-AmideC16 column5.0cm x 4.6mm, 5µm particles
Flow Rate: 1mL/min
Temp.: 30°CDet.: UV, 220nm
Elution: 10:90 ACN, 0.1%TFA: H2O, 0.1%TFA gradient to 90:10 ACN, 0.1%TFA:H2O,0.1%TFA
1. 1-hydroxy-7-azabenzotriazole2. 4-methoxybenzene sulfonamide3. Methyl-3-amino-2-thiophene-carboxylate4. 4-aminobenzophenone
1.0 2.0 3.0 4.0 5.0 6.0 7.0
Time (min)
1
23
4
1.0 2.0 3.0 4.0 5.0 6.0 7.0
1
2
3
4
5
1.0 2.0 3.0 4.0 5.0 6.0 7.0
1
2
3
4
1.0 2.0 3.0 4.0 5.0 6.0 7.0
1
2
3
4
98-0361
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Fast Separation
• Reduce column dimensions
• Reduce particle size
• Increase flow rate
• Increase temperature
• Change gradient profile
18
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Fast Separation
• Reduce column dimensions
• Reduce particle size ???• Increase flow rate
• Increase temperature
• Change gradient profile
19
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© 2013 Sigma-Aldrich Co. All rights reserved.
UHPLC performance on any HPLC system
Dr. Frank Michel
© 2013 Sigma-Aldrich Co. All rights reserved.
21
van Deemter curves for different particle sizes
HE
TP
(µ
m)
1 2 3 4 5
16,000
35.00
30.00
25.00
20.00
15.00
10.00
5.00
Pre
ssure
(p
si)
H = A + B/u + Cu
HE
TP
(µ
m)
1 2 3 4 5
16,000
35.00
30.00
25.00
20.00
15.00
10.00
5.00
Pre
ssure
(p
si)
H = A + B/u + Cu
Mobile phase velocity (mm/sec)
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22
050
100150200250300350400
0 5 10 15 20
0
5,000
10,000
15,000
20,000
25,000
30,000
0 5 10 15 20
Particle (µm) psi bar N
1.8 5889 406 27,500
2.5 3089 213 20,000
3 2118 146 16,500
5 769 53 10,000
10 189 13 5,000
15 87 6 3,750
20 44 3 2,500
10 cm column, 3 mm/s linear velocity
dp (µm)
pdN
1∝
2
1
pdP ∝
Doubling the efficiency by halving the particle size results in a pressure increase by a factor of four.
Particle size: Influence on efficiency and pressure
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23
PellicularPellicular particles do have onlya thin porous skin (low capacity)
History of HPLC particle design
Total Porous Current state-of-the-artin HPLC
Irregular Difficult to pack, clogging, not very robust
Fused-Core™ The NEW technologyFused-Core is a trademark of Advanced Materials Technology, Inc.
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24
• Innovative approach in HPLC by Dr. J. J. Kirkland in 2007
• Porous silica with high capacity on solid, non-porous core
• Highly pure silica
• 2.7 µm silica particle
• 1.7 µm solid core
• 0.5 µm porous SiO2 layer
• 90 Å pore size
• Very narrow particle size distribution
• C18, C8, HILIC (Si and OH5), RP-Amide, Phenyl-Hexyl, Peptide ES C18, PFP (F5)
0.5 µm
1.7 µm2.7 µm
Fused-Core technology
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25
• Innovative approach in HPLC by Dr. J. J. Kirkland in 2007
• Porous silica with high capacity on solid, non-porous core
• Highly pure silica
• 2.7 µm silica particle
• 1.7 µm solid core
• 0.5 µm porous SiO2 layer
• 90 Å pore size
• Very narrow particle size distribution
• C18, C8, HILIC (Si and OH5), RP-Amide, Phenyl-Hexyl, Peptide ES C18, PFP (F5)
0.5 µm
1.7 µm2.7 µm
Fused-Core technology
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26
Fused-Core provides higher efficiency
0.5 µm
1.7 µm2.7 µm
Diffusionpathway 1.5 µm
The shorter diffusion pathway facilitates the mass transfer (C term)!
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27
-500
0
500
1000
1500
2000
2500
3000
1 2 3 4 5 6 7
Particle Diameter, µm
Am
ou
nt
Fused-Core particle size distribution Average = 2.77 µm; standard deviation = 6% of mean
Particle size distribution of a typical commercial totally porous packingAverage = 3.78 µm; standard deviation = 19% of mean
Fused-Core provides higher efficiency
Narrow particle size distribution!
•Bed uniformity (better A term)•Larger frits (column lifetime)
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28
1 2 3 4 5
35.00
30.00
25.00
20.00
15.00
10.00
5.00
2 4 6 8 10 12
16,000
14,000
12,000
10,000
8,000
6,000
4,000
2,0002.7 µm Ascentis Express
H = A + B/u + Cu ∆∆∆∆P = 1000FηηηηLππππr2dp
2∆∆∆∆P =
3 µm
1.7 µm
2.7 µm FC
*50x4.6 mm columns, 55/45 ACN/water
1-2 mL/min
Linear velocity of the
mobile phase (mm/sec)
Comparison of pressure and efficiency
Linear velocity of the
mobile phase (mm/sec)
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29
Fused-Core technology – higher efficiency
Agilent 1200Ascentis Express C18, 15cm x 4.6mm, 2.7 µm1.0mL/min, 254nm, RT, 10uL inj.
4. Toluene N = 30,738
3. Benzene N = 31,696
2. Acetophenone N = 33,786
1. Uracil (marker for void time)
0 1 2 3 4 min
How much efficiency is possible on a normal HPLC system?
4
3
2
1
Pressure = 183 bar (2690 psi)
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300 2 4
Agilent 1100 HPLC systemColumn: 150 x 4.6 mm
Mobile phase: ACN/water
Flow rate: 1.5 mL/min
Injection: 2.0 µL
Detection: 220 nm
Usual C18, 3 µm72.5 % ACNN = 140,600 N/m, N = 21,090 N/col.Pressure = approx. 4,000 psi
Ascentis Express C18, 2.7 µm 65 % ACNN = 237,700 N/m, N = 35,655 N/col.Pressure = approx. 4,000 psi
Twice the efficiency compared to 3 µm at the same back pressure
1. p-Hydroxy ethylbenzene2. Napthalene3. p-Xylene4. Biphenyl
13
4
0 2 4
2
1
2
3
4
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31
0 2 4 6 8 10 12
Time (min)
020
40
60
80
100
120
140
mA
U
0 2 4 6 8 10 12
Time (min)
020
40
60
80
100
120
140
mA
U
Column:Column:Column:Column: 150 x 4.6 mm I.D.Mobile phase:Mobile phase:Mobile phase:Mobile phase:Ascentis Express C18 2.7 µm: 25:75, water: acetonitrileAscentis C18 3 µm: 30:70, water: acetonitrileFlow rateFlow rateFlow rateFlow rate: 1.0 mL/min.Temp.:Temp.:Temp.:Temp.: 30 °CDet.:Det.:Det.:Det.: UV at 365 nmInjection:Injection:Injection:Injection: 1 µLSample: Sample: Sample: Sample: 47285-U TO11/IP6A Carbonyl-DNPH Mix as indicated below in 40:60, water: acetonitrilePeak IDsPeak IDsPeak IDsPeak IDs1. Formaldehyde-2,4-DNPH (105 µg/mL)2. Acetaldehyde-2,4- DNPH (76.4 µg/mL)3. Acrolein-2,4- DNPH (63.2 µg/mL)4. Acetone-2,4- DNPH (61.5 µg/mL)5. Propionaldehyde-2,4- DNPH (61.5 µg/mL)6. Crotonaldehyde-2,4- DNPH (53.6 µg/mL)7. Butyraldehyde-2,4- DNPH (52.5 µg/mL)8. Benzaldehyde-2,4- DNPH (40.5 µg/mL)9. Isovaleraldehyde-2,4- DNPH (46.4 µg/mL)10. Valeraldehyde-2,4- DNPH (46.4 µg/mL)11. o-Tolualdehyde-2,4- DNPH (37.5 µg/mL)12. m-Tolualdehyde-2,4- DNPH (37.5 µg/mL)13. p-Tolualdehyde-2,4- DNPH (37.5 µg/mL)14. Hexaldehyde-2,4- DNPH (42 µg/mL)15. 2,5-Dimethylbenzaldehyde-2,4- DNPH (35 µg/mL)
Ascentis Express C18, 2.7 µmPeak 8N = 260,720 p/mN = 39,108 p/col
Ascentis C18, 3 µmPeak 8N = 146,587p/mN = 21,988p/col
TO11/IP6A Carbonyl DNPH Mix
8
8
Sensitivitygap
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32
Same efficiency compared to sub 2µm particles
1.0 2.0 3.0 4.0
Time (min)
12
3
4
5
Ascentis Express C18
0.3 mL/min45 % acetonitrile2130 psiN = 12,500
Sub-2 µm Column 2
0.3 mL/min51 % acetonitrile.7000 psiN = 12,170
1
2
3
4
5
1.0 2.0 3.0 4.0
Time (min)
Mobile Phase: water : acetonitrile; isoelutropic for β-Estradiol
Columns: 100 x 2.1 mmFlow: variableDet: 200 nm
Inj: 1µL
Elution order:1. Estriol
2. β-Estradiol
3. Contaminant
4. Estrone
5. Estrone degradant
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Fused-Core® Milestones - Pioneering the Particles
• 2007: First 2.7 µm particles to achieve efficiencies >250,000 N/m
• Ruggedness proven to hold up extremely well in use
• Efficiencies comparable to sub-2 µm particles
• Pressure drop (flow resistance) comparable to 3 µm particle columns
• Allows use of traditional 400 bar instruments
• 2012: First 5 µm particles to achieve efficiencies >150,000 N/m
• Operate at low pressures with unsurpassed ruggedness.
• Efficiencies exceed most 3 µm particles (150,000 N/m observed routinely at low pressure)
• Pressure drop of 5 µm particle columns
• Designed for traditional instruments & routine methods.
33
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Fused-Core Technology – 5 µm
34
© 2013 Sigma-Aldrich Co. All rights reserved.
Faster HPLC on any system
35
Fused-Core Particles
• More plates with less backpressure
• 2.7 µm replaces 1.7-3 µm
• 5 µm replaces 3-5µm
Fused-core
2.7 µm Fused Core
5 µm Fused Core
5.0 µm 3.0 µm 1.7 µm 2.5 µm
Higher Backpressure
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More Efficiency with Ascentis Express Columns
36
Fused-Core Outperforms Porous Particles
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50
flow (mL/min)
H
Fused Core 2.7um
Porous 3 um
Fused Core 5um
Porous 5 um
5µm porous
3µm porous
2.7µm Fused-CoreNmax = 37,500 (250K/m)
5µm Fused-CoreNmax = 25,000 (167K/m)
Column: C18 150 x 4.6mmMobile Phase: 60% AcetonitrileTemperature: 35ºC
Sample: 10µLToluene
N = L/H
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Separation of JWH-018, JWH-073 and their Primary and Secondary Metabolites using Ascentis Express C18, 2.7 um
1. JWH-073 4-Butanoic acid metabolite2. JWH-018 5-Pentanoic acid metabolite3. JWH-073 3-Hydroxybutyl metabolite4. JWH-018 4-Hydroxypentyl metabolite5. JWH-0736. JWH-018
0 2 4 6 8 10Time (min)
12
4
5
6
3
© 2013 Sigma-Aldrich Co. All rights reserved.
0 2 4 6 8 10Time (min)
0 2 4 6 8 10Time (min)
Comparison of 5 um and 2.7 um Ascentis Express C18 Columns
Particle Size: 2.7 µm
Backpressure: 2143 (start)
Particle Size: 5 µm
Backpressure: 1037 (start)
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Higher Speed and Resolution with Fused Core
42
Cl
N
N
N
NH
CH3
1. Oxazepam: FW = 2862. Alprazolam: FW = 3113. Clonazepam: FW = 3154. N-Desmethyldiazepam: FW = 2705. Diazepam: FW = 284
1 2 43 5
ClN
CH3
O
N
ClN
H
O
N
NO2N
H
O
N
Cl
ClNH
O
NOH
Mobile phase: 35:65 ACN:WaterDetection: 254 nmInjection: 10 µLTemperature: RT
Requirement on method: N (Peak 5) >20.000
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43
0 10 20 30 40
Conventional C1825 cm x 4.6 mm I.D., 5 µm1.0 mL/min., N= 22147Pressure: 128 bar (1880 psi)
0 2 4 6 8 10
Ascentis Express C1810 cm x 4.6 mm I.D., 2.7 µm1.0 mL/min., N = 22694Pressure: 167 bar (2450 psi)
*Agilent 1100 HPLC System
Requirement on method: N >20.000
Increasing speed on traditional HPLC systems*
0 2 4 6 8 10Time (min)
Ascentis Express C1810 cm x 4.6 mm I.D., 2.7 µm1.5 mL/min., N = 21297Pressure: 248 bar (3645 psi)
20 4 6
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44
0 10 20 30 40
Conventional C1825 cm x 4.6 mm I.D., 5 µm1.0 mL/min., N= 22147Pressure: 128 bar (1880 psi)
0 2 4 6 8 10
Ascentis Express C1810 cm x 4.6 mm I.D., 2.7 µm1.0 mL/min., N = 22694Pressure: 167 bar (2450 psi)
0 2 4 6Time (min)
*Agilent 1100 HPLC System
Requirement on method: N >20.000
0 2 4 6Time (min)
0 2 4 6Time (min)
0 2 4 6Time (min)
0 2 4 6Time (min)
0 2 4 6Time (min)
Increasing speed on traditional HPLC systems*
0 2 4 6 8 10Time (min)
Ascentis Express C1810 cm x 4.6 mm I.D., 2.7 µm1.5 mL/min., N = 21297Pressure: 248 bar (3645 psi)
20 4 6
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45
Ballistic gradientswith Ascentis Express
• Sample: plasma after protein preticipation
• Column: Ascentis Express
• Dimension: 50 x 2.1mm
• Gradient:
• A: 0.1% (aq) formic acid
• B: 0.1% formic acid in ACN
• Flow rate = 1.1 mL/min• Courtesy of: Dr. David Mallet, DMPK,
GSK, Stevenage, UK
T IC o f + M R M ( 4 p a i r s ) : f r o m S a m p l e 1 1 ( t e s t m i x 1 0 ) o f A s c e n t is t e s t m ix 2 2 0 4 0 8 . w if f ( T u r b o S p r a y )
0 . 1 0 . 2 0 . 3 0 . 4 0 . 5 0 . 6 0 . 7 0 . 8 0 . 9 1 . 0T i m e , m i n
0 . 0
1 . 0 e 5
2 . 0 e 5
3 . 0 e 5
4 . 0 e 5
5 . 0 e 5
6 . 0 e 5
7 . 0 e 5
8 . 0 e 5
9 . 0 e 5
1 . 0 e 6
1 . 1 e 6
1 . 2 e 6
1 . 3 e 6
1 . 4 e 6
1 . 5 e 6
1 . 6 e 6
1 . 7 e 6
1 . 8 e 6
1 . 9 e 6
2 . 0 e 6
2 . 1 e 60 . 7 2
0 . 7 70 . 5 7
0 . 3 4
18µL test mixInjection 9Pressure 434 bar
T IC o f + M R M ( 4 p a i r s ) : f r o m S a m p le 5 ( t e s t m i x ) o f A s c e n t is t e s t m ix 2 5 0 4 0 8 . w i f f ( T u r b o S p r a y )
0 . 1 0 . 2 0 . 3 0 . 4 0 . 5 0 . 6 0 . 7 0 . 8 0 . 9 1 . 0T i m e , m i n
0 . 0
1 . 0 e 5
2 . 0 e 5
3 . 0 e 5
4 . 0 e 5
5 . 0 e 5
6 . 0 e 5
7 . 0 e 5
8 . 0 e 5
9 . 0 e 5
1 . 0 e 6
1 . 1 e 6
1 . 2 e 6
1 . 3 e 6
0 . 6 7
0 . 7 30 . 5 4
0 .3 5
Injection 659Pressure 444 bar
T IC o f + M R M ( 4 p a i r s ) : f r o m S a m p l e 2 5 ( t e s t m i x ) o f A s c e n t i s t e s t m i x 2 5 0 4 0 8 . w i f f ( T u r b o S p r a y )
0 . 1 0 . 2 0 . 3 0 . 4 0 . 5 0 . 6 0 . 7 0 . 8 0 . 9 1 . 0T i m e , m i n
0 . 0
1 . 0 e 5
2 . 0 e 5
3 . 0 e 5
4 . 0 e 5
5 . 0 e 5
6 . 0 e 5
7 . 0 e 5
8 . 0 e 5
9 . 0 e 5
1 . 0 e 6
1 . 1 e 6
1 . 2 e 6
1 . 3 e 6
1 . 4 e 6
1 . 5 e 6
1 . 6 e 6
1 . 7 e 6
1 . 8 e 6
1 . 9 e 60 .6 4
0 . 7 3
0 . 5 3
0 . 3 4
Injection 1658Pressure 447 bar
T IC o f + M R M ( 4 p a i r s ) : f r o m S a m p l e 1 0 ( t e s t m i x 5 ) o f A s c e n t i s t e s t m ix 0 2 0 5 0 8 . w i f f ( T u r b o S p r a y )
0 . 1 0 . 2 0 . 3 0 . 4 0 . 5 0 . 6 0 . 7 0 . 8 0 . 9 1 . 0T i m e , m i n
0 . 0 0
5 . 0 0 e 4
1 . 0 0 e 5
1 . 5 0 e 5
2 . 0 0 e 5
2 . 5 0 e 5
3 . 0 0 e 5
3 . 5 0 e 5
4 . 0 0 e 5
4 . 5 0 e 5
5 . 0 0 e 5
5 . 5 0 e 5
6 . 0 0 e 5
6 . 5 0 e 5
7 . 0 0 e 5
7 . 5 0 e 5
8 . 0 0 e 5
8 . 5 0 e 5
9 . 0 0 e 5
9 . 5 0 e 5
1 . 0 0 e 6
0 . 5 3
0 . 7 2
0 .6 4
0 .3 5
Injection 3688Pressure 458 bar
Time (min) 0.00 1.00 1.05 1.30
% B 5 95 5 5
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46
Ruggedness of Ascentis Express
© 2013 Sigma-Aldrich Co. All rights reserved.
47
Summary
• Fused-Core particle provide a „kinetic advantage“
• Higher efficiency on any HPLC system
– 2,7 µm have 2x the efficiency compared to 3 µm particles
– 2,7 µm have 3x the efficiency compared to 5 µm particles
– 2,7 µm have half backpressure compared to sub-2 µm particles
– 5 µm have the efficiency of 3 µm particles at same backpressure as fully porous 5
µm particles
• Increase of the column length for increased efficiency
• Can be used on any HPLC system
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Summary
• Fused-Core particle provide a „kinetic advantage“
• Shorter analysis times maintaining the efficiency
– 2,7 µm have same efficiency like 3 µm columns (1/2 column length)
– 2,7 µm have same efficiency like 5 µm columns (1/3 column length)
– 2,7 µm have same speed and efficiency like sub-2 µm columns at half
backpressure => Increase of flow rate and speed
– 5 µm have same efficiency like 3 µm columns (1/2 column length), but with less
backpressure
• Ruggedness and durability as known from 5 µm columns