Faster HPLC by innovative HPLC Particle Technologies in ... · PDF fileFaster HPLC by...

Faster HPLC by innovative HPLC Particle Technologies in Pharmaceutical Analysis

•Dr. Frank Michel•[email protected]

2

Efficiency:N = Lcol/H H = Lcol/Nvan Deemter equation: H = A + B/u + Cu

H

u

A = 2dP

B = 2Dm

3

van Deemter curves for different particle sizes

HE

TP (

m)

1 2 3 4 5

16,000

35.00

30.00

25.00

20.00

15.00

10.00

5.00

HE

TP (

m)

1 2 3 4 5

16,000

35.00

30.00

25.00

20.00

15.00

10.00

5.00

Mobile phase velocity (mm/sec)

4

050

100150200250300350400

0 5 10 15 20

05,000

10,00015,00020,00025,00030,000

0 5 10 15 20

Particle (µm) psi bar N 1.8 5889 406 27,5002.5 3089 213 20,0003 2118 146 16,5005 769 53 10,00010 189 13 5,00015 87 6 3,75020 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

5

PellicularPellicular particles do have only

a 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.

6

•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

7

•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

8

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)!

9

-500

0

500

1000

1500

2000

2500

3000

1 2 3 4 5 6 7

Particle Diameter, µm

Am

ount

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)

10

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 =1000FLr2dp

2P =

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)

11

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

21

Pressure = 183 bar (2690 psi)

120 2 4

Agilent 1100 HPLC systemColumn: 150 x 4.6 mmMobile phase: ACN/waterFlow rate: 1.5 mL/minInjection: 2.0 LDetection: 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

34

13

0 2 4 6 8 10 12Time (min)

020

4060

8010

012

014

0m

AU

0 2 4 6 8 10 12Time (min)

020

4060

8010

012

014

0m

AU

Column: 150 x 4.6 mm I.D.Mobile phase:

Ascentis Express C18 2.7 µm: 25:75, water: acetonitrileAscentis C18 3 µm: 30:70, water: acetonitrile

Flow rate: 1.0 mL/min.Temp.: 30 °CDet.: UV at 365 nmInjection: 1 µLSample: 47285-U TO11/IP6A Carbonyl-DNPH Mix as indicated

below in 40:60, water: acetonitrile

Peak IDs

1. 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

14

Agilent 120055cm x 4.6mm (>100,000 N in 14 min)60% Acetonitrile1.1mL/min, 254nm, 50°C, 10uL

Toluene N = 104,235

Benzene N = 106,096

Acetophenone N = 117,475

Pressure = 473 bar (7,000 psi)

Ascentis Express separates …

0 2 4 6 8 10 12 14Time (min)

Toluene N = 105,657

Benzene N = 106,260

D6 Benzene N = 105,235



0 2 4 6 8 10 12 14Time (min)

D6-Benzene

Benzene

deuterated isomers

4 columns

15

Agilent 120055cm x 4.6mm (>100,000 N in 14 min)60% Acetonitrile1.1mL/min, 254nm, 50°C, 10uL

Toluene N = 104,235

Benzene N = 106,096



Ascentis Express separates …

0 2 4 6 8 10 12 14Time (min)

Toluene N = 105,657

Benzene N = 106,260

D6 Benzene N = 105,235



0 2 4 6 8 10 12 14Time (min)

D6-Benzene

Benzene

deuterated isomers

4 columns

10

10

DB

B

10

10

DB

B

Clindamycin

16

R1 R2 R3

Clindamycin C3H7 H Cl

Lincomycin C3H7 OH H

Clindamycin B C2H5 H Cl

7-epi-Clindamycin C3H7 Cl H

Lincomycin B C2H5 OH H

7-epi-Lincomycin C3H7 H OH

N

CH3

R1

HN

O

CH3

R3R2

H

OOH

S

OH

OH

CH3

From Presentation “Development of European Pharmacopoeia HPLC Methods for the Analysis of some Drugs and Comparison of Fused Core Column with other Columns” (2008)Prof. Dr. J. P. Surmann, Institute for Pharmacy, Free University Berlin

Determination of Clindamycin-HCl

17

-0,01

0,04

0 2 4 6 8 10 12 14Retention Time [min]

Abs

orba

nce

[AU

]

Ascentis Express C18

Conventional column: Spherisorb ODS

From Presentation “Development of European Pharmacopoeia HPLC Methods for the Analysis of some Drugs and Comparison of Fused Core Column with other Columns” (2008)Prof. Dr. J. P. Surmann, Institute for Pharmacy, Free University Berlin

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

Fused-Core Technology – 5 µm

19

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 µmBackpressure: 2143 (start)

Particle Size: 5 µmBackpressure: 1037 (start)

Higher Speed and Resolution with Fused Core

21

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

22

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

HPLC in Pharmacopoeias (EP, USP)

•Working acc. to monographs in the Pharmacopoeias?

23

EU Pharmacopoeia, Chapter 2.2.46., Chromatographic Separation Techn.

24

Challenges in QC, in-process control and stability testing of Herbal Medicinal Products

Guideline on Quality of Herbal Medicinal Products/Traditional Herbal Medicinal Products (http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003370.pdf). Guideline on declaration of herbal substances and herbal preparations in herbal medicinal products/traditional herbal medicinal products (http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003272.pdf).

Constituent with known therapeutic activityConstituents which are generally accepted to contribute substantially to the therapeutic activityActive MarkerGenerally accepted to contribute to the therapeutic activity but are not responsible for the full therapeutic effectAnalytical MarkerCharacteristic constituents that serve sole analytical purposes

25

O

AU

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

Minutes0,00 10,00 20,00 30,00 40,00 50,00 60,00 70,00 80,00 90,00


Osthol

0 20 40 60 80 90Time [min]

Angelica root extract

Stat. Phase:Reprosil-Pur ODS-3, 150 x 4,6 mm, 3 µmMob. Phase: Wasser/TFA/ACN/MeOH,GradientFlow rate:1,0 ml/minTemperature:45 °CDetection:323 nmHPLC system:Waters Alliance 2695

* Presented at Annual Meeting DPhG 2009 (German Pharmacist Society)

26

Ost

hol -

65,

920

AU

0,00

0,05

0,10

0,15

0,20

0,25

0,30

0,35

0,40

0,45

0,50

0,55

0,60

0,65

0,70

Minutes0,00 10,00 20,00 30,00 40,00 50,00 60,00 70,00 80,00 90,00


Herbal Medicinal Product Acontaining Angelica root extract

0 20 40 60 80 90Time [min]

Stat. Phase:Reprosil-Pur ODS-3, 150 x 4,6 mm, 3 µmMob. Phase: Wasser/TFA/ACN/MeOH,GradientFlow rate:1,0 ml/minTemperature:45 °CDetection:323 nmHPLC system:Waters Alliance 2695

* Presented at Annual Meeting DPhG 2009(German Pharmacist Society)

27

Improvements in analysis of Herbal Medicinal Products by Fused CoreTM technology

silib

in a

- 29

,120

silib

in b

- 30

,812

AU

0,000

0,010

0,020

0,030

0,040

0,050

0,060

0,070

0,080

0,00 5,00 10,00 15,00 20,00 25,00 30,00

Herbal MedicinalProduct B

Stat. Phase:Ascentis Express C18,100 x 2.1 mm (2.7 µm)Mob. Phase: Water/TFA/ACN/MeOH,GradientFlow rate:0,4 ml/minTemp.: Det.:30 °C 287 nmHPLC system:Waters Acquity UPLC

0 10 20 30Time [min]

• Further Reference on application of AscentisExpress in Pharmaceutical Analysis:

• D. Steinmann, M. Ganzera, J Pharm Biomed Analysis, 55/4 (2011) 744-757

• E.R. Badman et al., J. Chromatogr. B 878 (2010) 2307–2313

• A. Abrahim et al., Journal of Pharmaceutical andBiomedical Analysis 51 (2010) 131–137

* Presented at Annual Meeting DPhG 2009(German Pharmacist Society)

Conventional C18 Column (250 x 4.6 mm)

•Separation of alkaloids from Canadian bloodroot (Sanguinaria canadensis)

N

O

O

OO

CH3+

N

OCH3

OCH3

OO

CH3+

From: Klaus Reif, Phytolab, Presentation „HPLC Method development and validation in analysis of HPMs“ (2009)

Ascentis Express C18 (150 x 4.6 mm; 2.7 µm)

•Separation of alkaloids from Canadian bloodroot(Sanguinaria canadensis)

N

O

O

OO

CH3+

N

OCH3

OCH3

OO

CH3+


Separation of ionic compounds in HILIC(Hydrophilic Interaction Liquid Chromatography)

•Stachydrin in motherwort (Leonurus cardiaca) extracts

Ascentis Express HILIC (150 x 2.1 mm; 2.7 µm) in combination withELSDN

CH3H 3C

C O O H+


31

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 le 1 1 ( t e s t m i x 1 0 ) o f A s c e n t i s t e s t m ix 2 2 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 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 l e 5 ( t e s t m i x ) o f A s c e n t is 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 60 . 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 le 2 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

1 . 4 e 6

1 . 5 e 6

1 . 6 e 6

1 . 7 e 6

1 . 8 e 6

1 . 9 e 6 0 .6 4

0 . 7 3

0 . 5 3

0 . 3 4


T IC o f + M R M ( 4 p a i r s ) : f r o m S a m p le 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 i x 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 60 . 5 3

0 . 7 2

0 .6 4

0 .3 5


Time (min) 0.00 1.00 1.05 1.30% B 5 95 5 5

32

Ruggedness of Ascentis Express

33

Lead Discovery at Boehringer-Ingelheim

•Requirements for analytical investigations in Lead Discovery• In-time support of project groups!!!• Through-put of up to 2,000 compounds per week• Handling of the samples in micro titer plates (384 wells)• Usually no requirements regarding sensitivity• Sample composition

–Maximum chromatographic resolution• Maximum security in determining the molecular weight of

the target compound–Exact mass determination–Isotopic fingerprint

Courtesy of: Markus Christ, Boehringer-Ingelheim, GER

34

•Selection of HPLC columns of different manufacturers:• 1.) Ascentis Express C18, 2.1 x 30 mm, 2.7 µm (Supelco)• 2.) Zorbax SB-C18, Rapid Resolution HT, 2.1 x 50 mm,

1.8 µm (Agilent)• 3.) Luna 2.5 µm C18(2)-HST (Phenomenex)• 4.) UltraHT Pro C18, 2.0 x 50 mm, 2.0 µm (YMC)• 5.) XBridge C18 2.5 µm, 2.1 x 50 mm (Waters)• 6.) Nucleodur C18 Gravity, 1.8 µm, 2.0 x 50 mm

(Macherey-Nagel)

Columns tested for suitability


Info6

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Info6 Does your presentation require photos, charts, tables etc.? A number of predefined slide layouts are available in a layout template file, which you will find on your computer under My Documents\Layout_Template.ppt (Eigene Dateien\Layout_Template.ppt).Copy the slides that are useful for you from Layout_Template.ppt into your presentation and fill them with your content.Info; 24.09.2004

35

1

Flow rate = 2.5

ml/min

T = 60°C

2

Flow rate = 1.8

ml/min

T = 90°C

3Flow rate = 1.9

ml/min

T = 60°C

4

Flow rate = 1.4

ml/min

T = 50°C

5

Flow rate = 1.7

ml/min

T = 60°C

6

Flow rate = 1.2

ml/min

T = 60°C

Columns tested for suitability


Info7

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36

# Inj15

11116

Ascentis Express, 2.1 x 50 mm, 2.7 µmRapid resolution, 1 µl Inj.volume2.5 ml/min @60°C: 520 bar (> 4,500 psi)

Abso

rban

ce, m

AU

1000

2000

3000

6000

0.24 0.26 0.28 0.30 0.32 0.34Time, min

0.3 s

Ruggedness of Ascentis Express


37

Ultrahigh speed HPLC …

DAD

MS

9 Seconds !!!Courtesy of: Markus Christ, Boehringer-Ingelheim, GER

Info8

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38

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

39

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

40

•C18/Peptide ES C18•C8•F5•RP-Amide•Phenyl-Hexyl•ES-Cyano•OH5•HILIC (Si)

Thank you!

Faster HPLC by innovative HPLC Particle Technologies in ... · PDF fileFaster HPLC by...

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