hplc & lc-mshplc & lc-ms techniques to improve speed, resolution and sensitivity without...

HPLC & LC-MSTechniques to Improve Speed, Resolution and Sensitivity

without Investing in Capital Equipment

2010 Innovation Seminar SeriesSupelco, Div. of Sigma-Aldrich

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

3

Chromatographer Needs Triangle

Sens

itivi

ty

Resolution

Speed

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

5

SurfaceChemistry & BondedPhases

Architecture

Size & Size Distribution

Composition

ParticleParameters

Levers: Particle Parameters

????

SurfaceChemistry & BondedPhases

????Architecture

????Size & Size Distribution

????Composition

RuggednessSelectivitySensitivitySpeedParticleParameters

Fundamentals InnovationNeeds

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

7

Resolution: Simple Equation

• Maximize: Differential migration ( tR)• Minimize: Band dispersion (w)

Rs =0.5 (wA + wB)

tR

Kinetic

Thermodynamic(chemistry)

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.

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

10

Flow

Typical HPLC packed bed

Well-ordered packed bed

Visual Depictions of HPLC Packed Bed

11

Depiction of Extra-Particle Volume

Non-uniform particlesHigh and variable extra-particle volume

Uniform particlesMinimize extra-particle volumeSmaller particles are better

12

Visualizing the van Deemter Relationship

H

u (linear velocity)

H = A + B/u + Cu

Flat (C)

Low (A)

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

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.

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

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

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

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

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

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

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

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

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

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

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)

26

SEM of Fused-Core Particles

X-section

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

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

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

• •

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)

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

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

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)

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

35

Hybrid Kromasil Eternity Particles

36

Selectivity and High pH NRs = kk+1

-14

• •

37

Particle Stability at High pH

• Comparison with conventional technology• Mobile phase: acetonitrile in 10 mM ammonium acetate, pH 10.5, 45 C

38

Summary

• Hybrid HPLC Particles: Kromasil® Eternity™

High pH operation

High pH operation--Composition

RuggednessSelectivitySensitivitySpeedParticleParameters

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

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

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)

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

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

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

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.

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

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

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

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

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

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

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