HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing...
Transcript of HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing...
![Page 1: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/1.jpg)
HPLC & LC-MSTechniques to Improve Speed, Resolution and Sensitivity
without Investing in Capital Equipment
2010 Innovation Seminar SeriesSupelco, Div. of Sigma-Aldrich
![Page 2: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/2.jpg)
2
Overview of Presentation
•Chromatographer needs triangle• What in your work has driven the need for us to innovate?
•Levers• What particle parameters do we have to pull to fulfill needs?• Why do these parameters work?
•Results• Two specific HPLC innovations
![Page 3: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/3.jpg)
3
Chromatographer Needs Triangle
Sens
itivi
ty
Resolution
Speed
![Page 4: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/4.jpg)
4
Levers: Particle Parameters
• Physical Attributes:1. Particle Size & Size Distribution2. Architecture
– Unique structures or morphology
• Chemical Attributes:3. Composition
– What the particle is made out of4. Particle surface and bonded phases
}Kinetic
}Thermodynamic
![Page 5: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/5.jpg)
5
SurfaceChemistry & BondedPhases
Architecture
Size & Size Distribution
Composition
ParticleParameters
Levers: Particle Parameters
????
SurfaceChemistry & BondedPhases
????Architecture
????Size & Size Distribution
????Composition
RuggednessSelectivitySensitivitySpeedParticleParameters
Fundamentals InnovationNeeds
![Page 6: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/6.jpg)
6
Resolution: Competing Processes
• Result of two competing processes in the column• Differential migration ( tR)• Band dispersion (w)
Inject A and B
A B A B
tBtA
tR
wBwAtr L while w L1/2
![Page 7: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/7.jpg)
7
Resolution: Simple Equation
• Maximize: Differential migration ( tR)• Minimize: Band dispersion (w)
Rs =0.5 (wA + wB)
tR
Kinetic
Thermodynamic(chemistry)
![Page 8: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/8.jpg)
8
Resolution and Column Efficiency
• But how to get at a physical means to improve resolution?
NRs = kk+1
-14
• •
N = L/H
H = A + B/u + Cu N 1/Hsmall H values are good.
![Page 9: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/9.jpg)
9
van Deemter Equation Terms
Small H values are good, so we need to minimize the A, B & C terms.
H = A + B/u + Cu
Outside the particle
Inside the particle
•C = Mass transfer• Particle size• Particle porosity• Stationary phase thickness
•A = Eddy (axial) diffusion• Particle shape, size, distribution• Bed uniformity
Flow
•B = Longitudinal diffusion• Flow rate and mobile phase
Flow
![Page 10: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/10.jpg)
10
Flow
Typical HPLC packed bed
Well-ordered packed bed
Visual Depictions of HPLC Packed Bed
![Page 11: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/11.jpg)
11
Depiction of Extra-Particle Volume
Non-uniform particlesHigh and variable extra-particle volume
Uniform particlesMinimize extra-particle volumeSmaller particles are better
![Page 12: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/12.jpg)
12
Visualizing the van Deemter Relationship
H
u (linear velocity)
H = A + B/u + Cu
Flat (C)
Low (A)
![Page 13: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/13.jpg)
13
Levers: Particle Parameters
• Physical Attributes:1. Particle Size & Size Distribution2. Architecture
– Unique structures or morphology
• Chemical Attributes:3. Composition
– What the particle is made out of4. Particle surface and bonded phases
}Kinetic
}Thermodynamic
![Page 14: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/14.jpg)
14
Resolution Equation & Selectivity
NRs = kk+1
-14
• •
k = (tR – t0)/t0
= k2/k1
1.00 1.05 1.10 1.15 1.20 1.250.0
0.5
1.0
1.5
2.0
2.5
3.0
0 5K 10K 15K 20K 25K
0 5 10 15 20 25
N
k
Nk
Res
olut
ion
(R)
Yun Mao, PhD Dissertation, University of Minnesota, 2001.
![Page 15: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/15.jpg)
15
Influencing Selectivity
•How do we influence selectivity ( )?• Bonded phase (surface interactions)• Mobile phase
Silica
1 2
1
2
3
1 2
C18
1
2, 3
Change bonded phase, change
![Page 16: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/16.jpg)
16
Influencing Selectivity
•How do we influence selectivity ( )?• Bonded phase• Mobile phase
– Influence ionization state of analyte– Influence solvent polarity (solvation)
pH
acidic conditions basic conditions
AH
A-
reve
rsed
pha
se re
tent
ion
(hyd
roph
obic
ity)
BH+
B
A- + H3O+ < > AH + H2OBH+ + OH- < > B + H2O
>pH 8 silica unstable
![Page 17: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/17.jpg)
17
Resolution Levers: Particle Parameters
• Physical Attributes:1. Particle Size & Size Distribution2. Architecture
– Unique structures or morphology
• Chemical Attributes:3. Composition
– What the particle is made out of4. Particle surface and bonded phases
Rs =0.5 (wA + wB)
tR
}Minimize band dispersion
} Increase selectivity
![Page 18: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/18.jpg)
18
Summary – What Particle “Levers” We Can Pull
• Small particles• Low particle size distribution• Well-packed bed• Minimal resistance to mass transfer
Fused-Core™ technology (Ascentis® Express, established 2007)Hybrid HPLC particles (Kromasil® Eternity)Both use existing HPLC instrumentation (no capital investment)
• Choices in bonded phases• Ability to operate at pH extremes
H = A + B/u + Cu
van
Dee
mte
r
NRs = kk+1
-14
• •Rs
![Page 19: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/19.jpg)
19
monoliths,CEC
Particle Innovation Chronology
Particle Size and Architecturespeed, efficiency
Particle Composition
silica polymers oxides Hybrids
expand pH range
Kromasil®Eternity™
Ascentis®
Express
Fused-Core
Permeability wall
5 μm 3 μm <2 μm
![Page 20: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/20.jpg)
20
Fused-Core Ascentis Express Particles
• Comparison of architecture of Fused-Core™ particles vs. porous 3 μm particles
H = A + B/u + CuH = A + B/u + Cu
![Page 21: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/21.jpg)
21
u (mm/sec.)
Virtually no performance loss at higher flow rates
Architecture: Speed Implications
•Data provided by Prof. Luigi Mondello, U. Messina, Messina, Italy
H = A + B/u + CuH = A + B/u + CuVan Deemter curve at 35 C
column USHW001402
0.00
2.00
4.00
6.00
8.00
10.00
12.00
0.00 1.00 2.00 3.00 4.00 5.00 6.00
H (μ
m)
2.7 μm Fused-Core
![Page 22: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/22.jpg)
22 Mobile Phase Velocity (mm/sec)
2 4 6 8 10 12
16,000
14,000
12,000
10,000
8,000
6,000
4,000
2,000
Pres
sure
(psi
)
3 μm
1.7 μm
2.7 μm Fused-Core
~ 1 mL/min.
Architecture: Speed Implications
• Pressure rises faster than efficiency and resolution as particle size is reduced
• P 1/dp2
• N 1/dp
• Fused-Core Ascentis Express particles permit high speed operation at conventional pressures.
P 1/dp2P 1/dp
2
![Page 23: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/23.jpg)
23
Size & Architecture: Speed Implications• Scaling methods to fast LC on traditional HPLC systems
1.0 2.0 3.0 4.0Time (min)
0.2 0.4 0.6 0.8 1.0Time (min)
At least 4-fold increase in throughput with 3-fold solvent-saving due to short column length.
1.5 mL total run volume
N~11,000; Rs~ 1.8; 885 psi
0.5 mL total run volume
N~11,000; Rs~1.8; 1750 psi
5 μm C18, 15 cm
Fused-Core Ascentis Express, 5 cm
![Page 24: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/24.jpg)
24
0 10 20 30Time (sec)
Size & Architecture: Speed Implications (cont’d)
• Virtually no loss of resolution at 4X normal flow rate: benefit of flat van Deemter
0 2 4 6 8 10 12 14 16 18Time (sec)
9-fold increase in throughput comparedto the original method on 5μm particle.
15-fold increase in throughput compared to the original method (not possible with 5 or 3 μm due to loss of N).
30 sec.; 3700 psi (1.2 mL/min.)Fused-Core Ascentis Express, 5 cm
18 sec.; 6400 psi (2 mL/min.)
Fused-Core Ascentis Express, 5 cm
![Page 25: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/25.jpg)
25 Particle Diameter, μm
Ascentis Express: 2.7 μm +/- 6%
Typical porous silica particle: Average +/- 19%
Tight Particle Size Distribution of Fused-Core (A-term Implications)
-500
0
500
1000
1500
2000
2500
3000
1 2 3 4 5 6 7
Num
ber
H = A + B/u + CuH = A + B/u + Cu
• Bed uniformity (A-term)• Frits (lifetime)
![Page 26: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/26.jpg)
26
SEM of Fused-Core Particles
X-section
![Page 27: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/27.jpg)
27* D.N. Mallett (GSK), C. Ramírez-Molina, J. Pharm. Biomed. Anal., 2009, 49(1), 100-107.
Ruggedness: Ascentis Express Chosen for High Speed LC-MS DMPK Studies*
The authors conclude: “The partially porous stationary phase material has demonstrated equivalent resolving power to sub-2μm materials under the ballistic gradient chromatography conditions employed, and shown to exhibit excellent resilience and performance over the analysis of thousands of protein precipitated plasma extracts, suggesting that this type of column is a valuable tool for pharmaceutical bioanalysts.”
• Express columns chosen by GSK group for ruggedness and performance
![Page 28: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/28.jpg)
28
Sensitivity Demonstration• Ascentis Express Peptide ES-C18 vs. 5 μm C18 (expanded
scale)
15.0 16.0 17.0Time (min)
010
020
0
15.0 16.0 17.0 18.0Time (min)
010
020
0
Ascentis Express Peptide ES-C18~20 peaks
C18, 5μm~10 peaks
0 10 20 30 40Time (min)
010
020
00 10 20 30 40
Time (min)
010
020
0
N 1/w2N 1/w2
Area = ½ h wArea = ½ h w
![Page 29: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/29.jpg)
29
Selectivity Choices in Fused-Core Particles
•C18•C8•Phenyl-hexyl•RP-Amide•HILIC•Peptide C18
AscentisExpressC18
1
23 4
5
0 2 4 6Time (min)
AscentisExpressRP-Amide
12 3
4
5
AscentisExpressPhenyl-Hexyl
1
2 3
4
5
NRs = kk+1
-14
• •
![Page 30: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/30.jpg)
30
0 2 4 6
Resolution Advantages of Fused-Core
Conventional C18, 3 μm
0 2 4 6
Ascentis Express C18 (2.7 μm)
Peak ID1. Uracil2. Nordoxepin (50 mg/L)3. Desipramine (50 mg/L)4. Nortiptyline (50 mg/L)5. Doxepin (50 mg/L)6. Norclomipramine (50 mg/L)7. Imipramine (50 mg/L)8. Amitriptyline (50 mg/L)9. Clomipriamine (50 mg/L)
![Page 31: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/31.jpg)
31
UHPLC Users: Want more resolving power
Ascentis Express C1875 cm (five 15 cm cols.)0.6 mL/min.903 bar (13,500 psi)k(toluene) = 5.3
0 10 20 30Time (min)
020
4060
mA
U 2
50 n
m
1
23 4
0 2 4 6 8 10 12 14Time (min)
020
40
mA
U 2
50 n
m
sub-2μ C1830 cm (two 15 cm cols.)0.3 mL/min.780 bar (11,300 psi)k(toluene) = 5.3
3 mm I.D. columns, water:acetonitrile 56:44, 60 C, peaks: uracil, acetophenone, benzene, toluene (deuterated analogs)
At the system pressure limit, Ascentis Express provides more resolvingpower because you can use longer columns.
G004353
![Page 32: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/32.jpg)
32
Summary
• Fused-Core™ Particles: Ascentis® Express
-Choices in bonded phases--
SurfaceChemistry & BondedPhases
--Fused (solid)
core =Low C-term
Fused (solid) core =
Low C-term (flat van Deemter)
Architecture
2.7 μm, uses 2 μm frits-Low A-term2.7 μm = low
pressureSize & Size Distribution
RuggednessSelectivitySensitivitySpeedParticleParameters
-Choices in bonded phases--
SurfaceChemistry & BondedPhases
--Fused (solid)
core =Low C-term
Fused (solid) core =
Low C-term (flat van Deemter)
Architecture
2.7 μm, uses 2 μm frits-Low A-term2.7 μm = low
pressureSize & Size Distribution
RuggednessSelectivitySensitivitySpeedParticleParameters
-Choices in bonded phases--
SurfaceChemistry & BondedPhases
--Fused (solid)
core =Low C-term
Fused (solid) core =
Low C-term (flat van Deemter)
Architecture
2.7 μm, uses 2 μm frits-Low A-term2.7 μm = low
pressureSize & Size Distribution
RuggednessSelectivitySensitivitySpeedParticleParameters
-Choices in bonded phases--
SurfaceChemistry & BondedPhases
--Fused (solid)
core =Low C-term
Fused (solid) core =
Low C-term (flat van Deemter)
Architecture
2.7 μm, uses 2 μm frits-Low A-term2.7 μm = low
pressureSize & Size Distribution
RuggednessSelectivitySensitivitySpeedParticleParameters
![Page 33: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/33.jpg)
33
Fused-Core Features & Benefits Summary
• Features:1. Unique particle architecture2. ~ 3 μm particle size3. Tight particle size distribution
• Benefits:1. Efficiency equal to sub-2 μm particles2. Even higher efficiency possible than sub-2 μm particles by using longer
columns3. Maintains efficiency at high flow rates4. One-half the pressure of sub-2 μm particles5. Uses conventional HPLC instruments (e.g. what you currently have)
![Page 34: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/34.jpg)
34
Ascentis Express Application Areas
• Process Chemistry• Pharmaceutical/Med. Chem.• Food & Beverage• Natural Products/Agriculture• Forensic• Bioanalytical (DMPK, clinical)• Biochromatography• Environmental• Others…
Using UHPLC:• want more resolving power• want more speed• want more rugged columns• want to transfer methods onto conventional systems (e.g. QA/QC)
Using conventional columns:• want more speed• want more resolving power • basically want UHPLC performance on conventional systems
![Page 35: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/35.jpg)
35
Hybrid Kromasil Eternity Particles
![Page 36: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/36.jpg)
36
Selectivity and High pH NRs = kk+1
-14
• •
![Page 37: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/37.jpg)
37
Particle Stability at High pH
• Comparison with conventional technology• Mobile phase: acetonitrile in 10 mM ammonium acetate, pH 10.5, 45 C
![Page 38: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/38.jpg)
38
Summary
• Hybrid HPLC Particles: Kromasil® Eternity™
High pH operation
High pH operation--Composition
RuggednessSelectivitySensitivitySpeedParticleParameters
![Page 39: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/39.jpg)
39
Summary: Etiology of Two Particle Innovations
•Customer needs drive innovations•Leverage the fundamental equations:
• van Deemter: • Resolution: NRs = k
k+1-1
4• •
NRs = kk+1
-14
• •
H = A + B/u + Cu
•Fused-Core™ Ascentis® Express• High speed• High efficiency• Conventional and UHPLC systems
•Kromasil® Eternity™• Hybrid HPLC technology• Durable particles for high pH separations
![Page 40: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/40.jpg)
40
Other Supelco/Fluka HPLC and MS Innovations
• Chromatography:• Chiral – Unique CSPs for HPLC, GC, LC-MS, SFC, Prep (Astec line)• Ionic liquids for GC – extremely polar selectivity• Fast GC phases and techniques• Carbonaceous adsorbents (Carboxen®)
• LC-MS and MALDI-MS:• Phospholipid removal (HybridSPE-PPT®)• Chiral LC-MS (Astec CHIROBIOTIC®)• High-purity MS solvents and additives (CHROMASOLV®)• Ionic liquids for MALDI and ESI• Packed pipette tips for proteomics
• High performance fittings and accessories
![Page 41: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/41.jpg)
41
Acknowledgements/Collaborators
• Dr. Jack Kirkland, AMT, Inc.• Prof. Luigi Mondello, U. Messina, Messina, Italy• Dr. Richard Henry, Consultant• Jared Benedict, Ph.D., Eka Nobel• Supelco and Fluka R&D Teams
Fused-Core is a trademark of Advanced Materials Technologies, Inc. (AMT)
![Page 42: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/42.jpg)
42
When to Choose Fused-Core Ascentis Express
• Process Chemistry• Pharmaceutical/Med. Chem.• Food & Beverage• Natural Products/Agriculture• Forensic• Bioanalytical (DMPK, clinical)• Biochromatography• Environmental• Others…
Using UHPLC:• want more resolving power• want more speed• want more rugged columns• want to transfer methods onto conventional systems (e.g. QA/QC, prep)
Using conventional columns:• want more resolving power• want more speed• want more sensitivity• want UHPLC performance on conventional systems
Using either UHPLC or HPLC:• want different selectivity• want solvent savings
![Page 43: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/43.jpg)
43
UHPLC Users: Want more resolving power
1.0 2.0 3.0 4.0 5.0 6.0Time (min)
1.0 2.0 3.0 4.0Time (min)
1400 psi
1650 psi
Ascentis Express C18, 10 cm
Sub-2 μm C18, 5 cm
2.1 mm I.D. columnswater:acetonitrile 55:45 (top) or
54:46 (lower)0.2 mL/min.ambient temperatureUV at 200 nm1 μL inj.peaks: estradiol, ß-estradiol,
impurity, estrone, estrone degradant
Ascentis Express provides twice the resolving power as sub-2 μm particlesat equal pressures.
G003973
G003974
![Page 44: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/44.jpg)
44
UHPLC Users: Want more speed
3000 psi
Ascentis Express C18, 10 cm0.4 mL/min.
3000 psi
Sub-2 μm C18, 10 cm0.2 mL/min.
2.1 mm I.D. columnswater:acetonitrile 49:51 (top) or
55:45 (lower)ambient temperatureUV at 200 nm1 μL inj.peaks: estradiol, ß-estradiol,
impurity, estrone, estrone degradant
Ascentis Express provides twice the speed as sub-2 μm particlesat equal pressures.
G003975
G003976
![Page 45: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/45.jpg)
45D.N. Mallett (GSK), C. Ramírez-Molina, J. Pharm. Biomed. Anal., 2009, 49(1), 100-107.
UHPLC Users: Want more rugged columns
5 cm x 2.1 mm I.D. columns; 0.1% formic acid in water:acetonitrile, gradient of 5 – 95% acetonitrile in 1 min.; MS/MS detection; 1.1 mL/min.; 40 C or 65 C; peaks: bromo-guanosine, labetalol, reserpine, SB243213A (GSK)
Sub-2 μm C18~700 bar (>10,000 psi)65 C
Ascentis Express C18~447 bar (6500 psi)40 C
• Ascentis Express has the efficiency and resolution of sub-2 μm particles, but with much lower pressure drop and is much more resistant to plugging. With the repeated injections of plasma extracts shown here, the authors observed increasing and excessive pressure and retention variation on the sub-2 μm column.
![Page 46: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/46.jpg)
46
UHPLC Users: Want to transfer methods onto conventional systems (e.g. QA/QC, prep)
Ascentis Express C18, 10 cmPressure: 167 bar (2450 psi)N (peak 5): 22,700
4.6 mm I.D. columns; water:acetonitrile 65:35; 1 mL/min.; ambient temperature; UV at 254 nm; peaks: oxazepam, alprazolam, cloazepam, N-desmethyldiazepam, diazepam
Methods on Ascentis Express are easily scalable to conventional HPLC systems, and vice versa.
G004040
5 μm C18, 25 cmPressure: 128 bar (1880 psi)N (peak 5): 22,150
G004039
40302010
![Page 47: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/47.jpg)
47
Conventional HPLC Users: Want more resolving power
15.0 16.0 17.0Time (min)
010
020
0
15.0 16.0 17.0 18.0Time (min)
010
020
0
Ascentis Express Peptide ES-C18~20 peaks
C18, 5μm~10 peaks
0 10 20 30 40Time (min)
010
020
0
0 10 20 30 40Time (min)
010
020
0
Higher efficiency means higher peak capacity and more information from Ascentis Express methods than from conventional HPLC columns.
5 cm x 2.1 mm I.D. columns; 0.1% formate in water:acetonitrile gradient; 1 mL/min.; 35 C; ESI(+)-TOF; sample: HAS tryptic digest
![Page 48: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/48.jpg)
48
Conventional HPLC Users: Want more speed
3 mm I.D. columnswater:acetonitrile 31:6935 Cpeaks: uracil, acetophenone,
benzene, toluene, naphthalene
At least eight-times the speed on conventional HPLC systems.
Ascentis Express C18, 5 cm1.2 mL/min.24 second run
5 μm C18, 15 cm0.4 mL/min.3.8 minute run
G004158
G004157
![Page 49: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/49.jpg)
49
Conventional HPLC Users: Want UHPLC performance (speed) on conventional systems
1.0 2.0 3.0 4.0Time (min)
0.2 0.4 0.6 0.8 1.0Time (min)
At least 4-fold increase in throughput with 3-fold solvent-saving due to short column length.
0.4 mL/min.4 min. run time1.5 mL total run volumeN~11,000; Rs~ 1.8; 885 psi
0.6 mL/min.1 min. run time0.5 mL total run volumeN~11,000; Rs~1.8; 1750 psi
5 μm C18, 15 cm
Ascentis Express, 5 cm
5 cm x 3 mm I.D. columns; water:acetonitrile 20:80 (top) or 31:69 (bottom); 0.4 mL/min. (top) or 0.6 mL/min. (bottom); 35 C; uracil, acetophenone, benzene, toluene, naphthalene
![Page 50: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/50.jpg)
500 10 20 30
Time (sec)
Conventional HPLC Users: Want UHPLC performance (speed) on conventional systems
•Virtually no loss of resolution at 4X normal flow rate: benefit of flat van Deemter.
•9-fold increase in throughput compared to the original method on 5 μm particle.
1.2 mL/min.30 sec. run time3700 psi
1.0 2.0 3.0 4.0Time (min)
0.4 mL/min.4 min. run time885 psi
5 μm C18, 15 cm
Ascentis Express, 5 cm
![Page 51: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/51.jpg)
51
Conventional HPLC Users: Want better sensitivity
Higher efficiency provides better sensitivity on Ascentis Express vs. 3 μm or 5 μm totally porous particles.
10 cm x 4.6 mm I.D. columns; water:acetonitrile 65:35; 1.8 mL/min.; 35 C; UV at 254 nm; 5 μL inj.; peaks: uracil, acetophenone, benzene, toluene
G004330
![Page 52: HPLC & LC-MSHPLC & LC-MS Techniques to Improve Speed, Resolution and Sensitivity without Investing in Capital Equipment 2010 Innovation Seminar Series Supelco, Div. of Sigma-Aldrich](https://reader036.fdocuments.us/reader036/viewer/2022071609/61476da0afbe1968d37a0de3/html5/thumbnails/52.jpg)
52
UHPLC or HPLC Users: Want different selectivity
•Variety of phase chemistries of Ascentis Express:• C18• C8• Phenyl-hexyl• Amide• HILIC• Peptide C18
AscentisExpressC18
1
23 4
5
0 2 4 6Time (min)
AscentisExpressRP-Amide
12 3
4
5
AscentisExpressPhenyl-Hexyl
1
2 3
4
5