basics in fast hplc - sigma-aldrich: analytical, … © 2013 sigma-aldrich co. all rights reserved....

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© 2013 Sigma-Aldrich Co. All rights reserved.

Basics in Fast HPLC

Dr. Frank Michel

[email protected]


What influences retention?

with

• tr = time of retention

• L = column length

• k’ = retention factor

• u = mobile phase velocity

2

tr = (k’+1)Lu


Chromatographers Triangle

3

Speed

Sensitivity Efficiency


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


5

Efficiency:


H = A + B/u + Cu

H

u

A = 2λλλλdP

B = 2γγγγDm


6

Efficiency:


H = A + B/u + Cu

H

u

A = 2λλλλdP

B = 2γγγγDm


7

Efficiency:


H = A + B/u + Cu

H

u

A = 2λλλλdP

B = 2γγγγDm


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


Methods to Increase the Speed of HPLC Separations

• Reduce column dimensions

• Reduce particle size

• Increase flow rate

• Increase temperature

• Change gradient profile

9


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


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



0 10 20 30

Time (min)

1

2

3

4

5

6

1.0 2.0

1

2

34

5

6

98-0349


Particle Size

12


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.


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



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


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


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


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


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


Fast Separation


• Reduce particle size

• Increase flow rate



18


Fast Separation


• Reduce particle size ???• Increase flow rate



19

sigma-aldrich.com/analytical


UHPLC performance on any HPLC system

Dr. Frank Michel

[email protected]


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)


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


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.


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


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


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)!


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)


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)


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)


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


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


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


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


Fused-Core Technology – 5 µm

34


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


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


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


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)


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


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)


20 4 6


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


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)


20 4 6


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


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


Time (min) 0.00 1.00 1.05 1.30

% B 5 95 5 5


46

Ruggedness of Ascentis Express


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


48

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

basics in fast hplc - sigma-aldrich: analytical, … © 2013 sigma-aldrich co. all rights reserved....

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