High performanceliquid chromatography

Jared Benedict, Ph.D.

AkzoNobel/Eka Chemicals Separation Products, 281 Fields LaneBrewster, NY 10509

Internet: http://www.kromasil.com

Tel: 845-276-8220 Fax: 845-277-1406 Email: jared.benedict@akzonobel.com

Robust Particles for HPLC & Scale-up for Peptide Purification

2

Outline of the presentation

Introduction of Kromasil Eternity

Product characteristics

Applications & case study using Kromasil Eternity Analytical HPLC Preparative HPLC

Description of preparative HPLC

R&D peptide purification case study

Conclusion

3

Controlling and utilizing pH

Controlling and utilizing pH as a method development tool will greatly enhance your chromatography

pH strongly influences the efficiency of separation of ionizable compounds

Ideally, mobile phase pH should be at least 1 pH unit away from the analyte’s pKa

The ability to choose the optimal pH from a wide range pH 1 – 12 is very beneficial

4

Kromasil Eternity platform

Eternity platform

Particle sizes: 2.5 µm & 5 µm

Surface Area (porous): 330 m2/g

Pore Size: 100 Å

5

Kromasil Eternity C18 & PhenylHexyl

Eternity platform

Ligand: C18

Endcapping: Proprietary

pH Range: 1 - 12

USP: L1

Eternity C18

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Product characteristics:Ligand: C18

Particle sizes: 2.5 µm & 5 µm

Carbon Load: 14 %

Endcapping: Proprietary

pH Range: 1 - 12

Surface Area: 330 m2/g

Pore Size: 100 Å

USP: L1

7

Long term high pH stability – a comparison

8

Importance of pH control

The retention time of acidic and basic compounds depends upon the pH value.

0.1 pH unit can render a shift in retention time by 10%

0

5

10

15

20

25

30

35

0 2 4 6 8 10 12

pH

k' (a

cid)

0

5

10

15

20

25

30

0 2 4 6 8 10 12

pH

k' (b

ase)

± 1 pH unit retention changes by a factor of 5 - 6

Acidic analyte Basic analyte

!

9

Influence of the pH on the packing material

pH

SiO-SiOH

2 3 4 5 6 7 8 9

C4 C4 C4 C4C4 C4

C1 C1 C1 C1OH OHOH

Ser at pH 2 - 5

O OCH

NH

HO

H

pH < pKA (Si-OH)

O-

Lys at 6 < pH < 10

CHO

NH

+H3N

pKA (AA) > pH > pKA (Si-OH)

10

pH variation to control selectivity

11

Scale-up or Scale-down

12

Increase efficiency – Save time

N = 194 000 / mRs1 = 5.8Rs2 = 25ΔP = 383 bar

Stationary phase: Kromasil Eternity-2.5-C18Column size: 4.6 x 100 mmFlow rate: 1.6 ml/min

Stationary phase: Kromasil Eternity-5-C18Column size: 4.6 x 250 mmFlow rate: 1.0 ml/min

N = 77 000 / mRs1 = 6.9Rs2 = 26ΔP = 125 bar

Save time without

resolution loss by decreasing particle size

START

Mobile phase: ACN/H20/formic acid (25/75/.01)

1. Uracil2. suplathiazole3. sulphamerazin4. sulphamethoxazole

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Stationary phase: Kromasil Eternity-5-C18Column size: 4.6 x 250 mmFlow rate: 1.0 ml/min

N = 77 000 / mRs1 = 6.9Rs2 = 26ΔP = 125 bar

Stationary phase: Kromasil Eternity-2.5-C18Column size: 4.6 x 50 mmFlow rate: 2.7 ml/min

N = 123 000 / mRs1 = 2.9Rs2 = 13ΔP = 366 bar

Increase efficiency – Save time

Run at 1/14 of original analysis

time

START END

Mobile phase: ACN/H20/formic acid (25/75/.01)

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Reduce solvent consumption – An exampleN = 194 000 / mRs1 = 5.8Rs2 = 25ΔP = 383 bar

Stationary phase: Kromasil Eternity-2.5-C18Column size: 4.6 x 100 mmFlow rate: 1.6 ml/min

Stationary phase: Kromasil Eternity-5-C18Column size: 4.6 x 250 mmFlow rate: 1.0 ml/min

N = 77 000 / mRs1 = 6.9Rs2 = 26ΔP = 125 bar

Save solvent without

resolution loss

Particle size Col. Dim. Flow rate Cycle time (µm) (mm x mm) (ml/min) (min)

(ml) (%)5 4,6 x 250 1,0 12 12 100

2,5 4,6 x 100 1,6 3 4,8 402,5 3,0 x 100 0,7 3 2,1 182,5 2,1 x 100 0,33 3 1 8

Mobile phasevolume/ cycle

START

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Applications at high pH

Separation of some classical histamine antagonists

Test conditions:

Stationary Phase: Kromasil Eternity-5-C18Column size: 4.6 x 250 mmMobile Phase: methanol/water, 50 mM triethyl-

amine acetate buffer, pH 11 (70/30) Flow rate: 1.4 ml/minTemperature: 25°CDetection: UV 254 nm

1 = Maleic acid 2 = Doxylamine 3 = Chlorpheniramine 4 = Diphenhydramine 5 = Triprolidine

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" – The long-lasting phase whenalternative selectivity is required"

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Eternity PhenylHexyl provides alternate selectivity

18

pH variation – the perfect tool for method development

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Separations of aromatics

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Kromasil Eternity – Analytical scale

Easy to scale up to 5µm

2.5 µm particle size

UHPLC and HPLC (> 200 000 pl/m)

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Utilizing pH to increase loading in semi-preparative chromatography

Need to purify milligram to gram amounts of compound for further research

Huge difference in the loadability of ionized vs. un-ionized species The loadability difference can be as much as 50-fold

Un-ionized compounds can be loaded at a much higher concentration thus there is a need for robust stationary phase at all pH’s

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Case study: Exploring pH effect on the loadability of basic compounds

Goal: Purify at least 500 mg of 1-(2-chlorophenyl)piperazine on a 21.2 X 150mm Kromasil Eternity column

The pKa of 1-(2-chlorophenyl) piperazine is around 9.7

Since Kromasil Eternity provides the ability to develop methods using pH 1 to 12, the effect of pH on loadability of this basic compound is explored

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Prep application at low and high pH [1(3)]

0.0

0.2

0.4

0.6

0.8

1.0

0 10 20 30 40

% MeCN

Log

k'

● pH 2.5 ● pH 11

0

0't

ttk R

k’: retention factor tR: retention timet0: retention time of non-retained compound

Relationship of 1-phenylpiperazine retention with mobile phase strength and pH

1-(2-chlorophenyl)piperazine

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Prep application at low and high pH [2(3)]

Chromatographic conditionsSample: crude tryptamine and 1-(2-chlorophenyl)piperazineInjection: 2.5 mg (0.5 ml of 5 mg/ml sample solution)Column: Kromasil Eternity 5-C18 4.6 x 150 mmMobile phase: Acetonitrile/0.1 M potassium phosphate pH 2.5 or pH 11.0Flow rate: 1 ml/minDetection: UV @ 300 nm and 280 nm

Development of preparative separation method for a basic crude compound at acidic and basic pH

0 2 4 6 8 10 12 14

Time [min]

0 2 4 6 8 10 12 14

Time [min]

1-(2-chlorophenyl)piperazine

10% MeCN 25% MeCN

pH 2.5 pH 11.0

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Prep application at low and high pH [3(3)]

0 2 4 6 8 10 12 14

Time [min]

50mg200mg500mg

Scale up of column dimension followed by 10 times load increase without reaching touching bands

Chromatographic conditionsSample: crude 1-(2-chlorophenyl)piperazineInjection: 10 ml of 5, 20, and 50 mg/ml sample,sample dissolved in acetonitrile/0.1 M potassium phosphate pH 13 30/70Column: Kromasil Eternity 5-C18 21.2 x 150 mmMobile phase: Acetonitrile/0.1 M potassium phosphate pH 11 30/70 v/vFlow rate: 21 ml/minDetection: UV @ 280 nm

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Purification of crude amitriptyline

Column: Kromasil Eternity 5 μm C18 (21.2 x 50 mm),Mobile phase: 25 mM NH4HCO3 pH 10.5/ACN,Gradient: 45-80% ACN/10 min. Flow: 21 mL/min, 25°C, 254 nm

Purity assessment of fraction pool: Column: Kromasil Eternity 2.5 μm C18 (4.6 x 50 mm), Mobile phase: 10 mM NH4HCO3 pH 10.5/MeCN (45/55) Flow rate: 1.7 mL/min, 25°C, 254 nm

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Product assortment1,2,3

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Kromasil Eternity – The Benefits

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Analytical vs. preparative HPLC

Analytical HPLC:

Objective:Identification and/or quantificationof compounds

Output:Information

Compound destination:waste

Preparative HPLC:

Objective:Purification and isolation ofcompounds

Output:purified compound

Compound destination:Fraction collection

Column dimension is irrelevant, it is the SCOPE of the separation determines whether it is analytical or prep HPLC

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HPLC vs. LPLC

HPLC

P

time

LPLC

time

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Chromatographic modes

MW (Da)

Polarity

102 106105104103

RP-bonded

IEC

RP-HIC

GPC

GF

CSP

NP

org. soluble water soluble

H P L C

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Working areas of preparative HPLC

Compound amount Working area

µg - Isolation of e.g. enzymes for research purpose

mg - Biological and biochemical testing- Structure elucidation and characterization of

side products, metabolites, natural products

g - Reference compounds (standards)- Compounds for toxicological screenings

kg - tons - Industrial scale, APIs, drugs

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Column typesFixed- bed, pre-packed Self-packers DAC-columns

2.1 – 50 mm ID 25 – 100 mm ID 50 – 1000 mm ID

Semi-prepCombi-ChemR&D

DevelopmentPilot PlantProduction

DevelopmentPilot Plant

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Example of a 1m I.D. column

35

Linear vs. Non-linear chromatographycS

cM

Ana

lytic

al

HPL

C

Prep

arat

ive

HPL

C

Typical injection: 0.005 mg/g Typical injection: 10 mg/gX 2500

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Volume – vs. concentration overload

t

zinj

a) Analytical Injection

t

Vinj

c) Concentration Overload Concentration overload can lead to displacement effects…….

t

Vinj VinjVinj

b) Volume Overload

Volume overload should beavoided

Not detector signal saturation

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Compression

Analytical injection

Preparative injection(real profiles)

98% purity75% yield

Preparative injection(individual profiles)

98% purity60% yield

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Tag-along

Analytical injection

Preparative injection(individual profiles)

Preparative injection(real profiles)

98% purity90% yield

98% purity60% yield

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Case Study

Peptide Purification– R&D

(Example of Reversed Phase Application

Development)

40

Peptide Purification R&D

Characteristics of R&D Application:

lost crude

labor

solvent

packing

system

Cost Distribution:

Only very small amount of material to work with

Free hands no restrictions concerning e.g. mobile phase

Short time frame

41

Peptide Purification R&D – Case Study

Objectives:

9 amino acid peptide has to be purified from ca 75% to 98% purity

The peptide is stable below pH 9

1 g of peptide has to be purified per injection

Recovery > 30%

42

Peptide Purification R&D

Strategies:

1. Compare different pH values αmax

2. Compare different buffer systems αmax, preferences

3. Compare different stationary phases αmax

4. Overload fraction analysis purity / recovery calculations

5. Calculation of productivity required column dimension

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Peptide Purification R&D

50 mM potassium phosphate / MeCN28 – 31 % MeCN / 30 minKR100-5-C18, 4,6 x 250 mm1 mL/min230 nm

1. Comparing pH value

Better selectivity at higher pH

pH 3 pH 7

Small peptide (< 10 amino acids) no need for gradient elution

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Peptide Purification R&D

2. Try TRIS as buffer

3. Run under isocratic conditions

4. Compare C18 with C4

C18 C4

0.1M Tris pH 8 / MeCN 73/27 (v/v)1 mL/min230 nm

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Peptide Purification R&D

5. Overloaded injection fraction analysis purity / recovery assessement

0.1M Tris pH 8 / MeCN 73/27 (v/v)KR100-10-C4 (4.6 x 250 mm)10 mg injection1 mL/min230 nm

0

1000

2000

3000

4000

5000

6000

1 2 3 4 5 6 7 8 9Frac#

Area unit

l

Purity : 98.4%Recovery: 69.4%

Product

Tail peakFront peak

Reconstructed elution profile

OK

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Peptide Purification R&D6. Productivity calculation required column dimension

minj.: 10 mgmpacking: 2.36 grelative load: 4.24 mg/ginitial purity: 75%recovery: 69%

cycle time: 45 min (incl. washing and re-equilibration)

productivity: 5.65 mgcrude. h-1 . gpacking

-1

2.92 mgproduct. h-1 . gpacking

-1

Scale-up factor: 100

mpacking : 236 g 414 ml column volume I.D. column = 2.3 cm

Required column dimension: 30 x 250 mm

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Conclusions

Reversed phase HPLC is a powerful, versatile tool for peptide purification Adequate for difficult separations Fast separation high productivity Concentrated product fraction cost effective work-up

Kromasil Eternity 2.5µm particle size for efficient analytical chromatography Broad pH range (1-12) provides maximum method development

flexibility 5µm particle size provides robust choice for small scale purification C18 & PhenylHexyl phases

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Thank you for your attention!

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