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RP HPLC Purification of Small Molecules

Lou Cheng

Astrazeneca R&D Boston

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HPLC Basics: Classification, k, α, N, Rs

Reversed-Phase HPLC – Isocratic and Gradient

Analytical Method Development – Screening & Optimization

Analytical Scale-up of Optimized HPLC Method

From Analytical Scale-up to PrepLC Purification

PrepLC Purification – Fraction Collection & Recovery

Summary

Outline

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HPLC Basics - Classification

Nonionic polarNonpolar Ionic

Water-insoluble Water-soluble

Increasing polarity

AdsorptionPartition

IonExchange

[ NP Partition]

[ RP Partition]

Size Exclusion

[Gel Permeation] [Gel Filtration]

Mo

lecu

lar

Wei

gh

t

102

103

104

105

106

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HPLC Basics - Classification

Nonionic polarNonpolar Ionic

Water-insoluble Water-soluble

Increasing polarity

AdsorptionPartition

IonExchange

[ NP Partition]

[ RP Partition]

Size Exclusion

[Gel Permeation] [Gel Filtration]

Mo

lecu

lar

Wei

gh

t

102

103

104

105

106

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Retention factor

tRi - t0k = t0

k2α =

k1

Selectivity

N = 162t

Wi

Efficiency

Rs = 0.25 (-1)N0.5 kk + 1

Resolution

tR1

tR2

t0

w1 w2

Minutes0 10

mV

olt

sHPLC Basics – k, α, N, Rs

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k: retention factorkw: retention factor by 100% water as mobile phase (φ = 0)S: a constant for a given sample compoundφ : organic fraction (volume) in binary mobile phase

log k = log kw – Sφ Eq. 1*

Kw: Hydrophobicity

Reversed-Phase HPLC – Isocratic

S: Sensitivity to change of mobile phase strength

*Ref: Practical HPLC Method Development, Lloyd Snyder, etc. Wiley, New York, 1997.

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F

O

N S

OO

NH

O

ID: 2146-197 (BCL2)

(MW = 426.5)

Log k = log kw – Sφ

N

N

O

O

NHNH

NH

O

Cl Cl (MW = 454.3)

Code: 1862-191-1 (CoaD) 0.30 0.35 0.40 0.45 0.50 0.55 0.60

-1.0

-0.5

0.0

0.5

1.0

Kw = 933, S = 4.53

0

3

6

9

12

15

0.30 0.35 0.40 0.45 0.50 0.55 0.60

-1.0

-0.5

0.0

0.5

1.0

kw = 2290, S = 7.85

log

k

0

3

6

9

12

15

Re

ten

tion

Fa

ctor (k)

Conditions: HPChem 10, XBridge C8, 4.6 × 50 mm, 10 mM NH4Form/AcN, 1.0 mL /min

Rule of Adjusting Isocratic Retention by φ: 5% Ξ 100 % k

Isocratic RP HPLC - AZ Example 1

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0.10 0.15 0.20 0.25 0.30 0.35 0.400.0

0.5

1.0

1.5

log

k

0

10

20

30

40

50

Retention F

actor (k)Kw = 28S = 3.35

Kw = 62, S = 3.67Kw = 72, S = 3.72

02154-137, 10 g (HE-TMK)

N N

ClCl

N N

ClO

N N

OCl

Mw = 163

Mw = 158

Mw = 158

+

log k = log kw – Sφ

Conditions: XBridge C18, 4.6 × 50 mm, 0.1% NH4OH/MeOH Flow Rate: 1.0 ml /min, room temperature.

The Kw and S of the two isomers are too close to be separable by RP gradient runs.

Isocratic RP HPLC - AZ Example 2

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Reversed-Phase HPLC – Gradient

*By assuming S = 4 for all small molecules, one gradient run is sufficient to resolve kw Two gradient runs can solve kw and S, by assuming log k/φ linear relationship

Automatic method development

Retention Prediction (Drylabs, Chromsword)

b = SVm/tGF log k0 = log kw - S0

tR = (t0/b) log [2.3k0b(ts/t0) + 1] + ts + tD Eq. 2*

T0: column dead volumn; b: gradient steepness; k0: k at the beginning of the gradient; ts: value of tR for a nonretained solute; tD: dwell time for gradient elution; : change of organic percentage in the mobile phase; S: system constant; Vm: column dead volumn; tG: gradient time; F: flow rate.

For Linear Solvent Strength (LSS) Gradient:

*Ref: Practical HPLC Method Development, Lloyd Snyder, etc. Wiley, New York, 1997.

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Retention Prediction of Gradient RP HPLC - AZ Examples

Sample

b tG tR, predicted tR, Exp

Error (%)(%/min) (%) (min.) (min.) (min.)

2146-197 (BCL2)

(Kw = 933, S = 4.53)

6.0 35-65 5 2.55 2.56 -0.3

12.0 35-95 5 2.28 2.35 -3.0

1862-191-1 (CoaD)

(Kw = 2290, S = 7.85)

9.0 50-95 5 3.00 2.82 +6.4

6.0 40-70 5 4.41 4.42 -0.2

Conditions: HPChem 10, XBridge C8, 4.6 × 50 mm, 10 mM NH4Form/AcN, 1.0 mL /min, room temperature. t0 = ts = 0.6 min, tD = 1.2 min.

tR = (t0/b) log [2.3k0b(ts/t0) + 1] + ts + tD

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Why Gradient?

Flexibility (b, , 0) to optimize separation with minimal effects on efficiency (N)

Samples with a wide k range, sometimes containing late-eluting interferences that

can either kill the column or carryover to subsequent runs

More precise, robust, and automatable

Dilute solutions of sample dissolved in a weak solvent

Gradient RP run is the best starting point for method development

Reversed-Phase HPLC – Gradient Over Isocratic

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B: ACNC: MeOHD: THF/H2O (9/1)

SolventsColumn Examples

1: XBridge C18

2: Gemini C6-Phenyl3: Atlantic dC18

4: YMC ODS AQ5: Synergi Hydro-RP6: YMC Carotenoid (C30)●●●●●●

162 conditions can be screened for one sample if necessary

RP HPLC Analytical Method Development – Screening

E: 0.1% TFAF: 0.1% Formic AcidG: 10 mM NH4Ac pH8H: 0.1% TEAJ: 0.1% NH4FormK: 0.1% NH4OHL: 10 mM NH4Ac/HOAc pH5M: 20 mM NH4AcN: 10 mM (NH4)2CO3

Valves: G1159A 6-ColSelector, G1160A, 12/13 SelvalvesDetectors: Agilent MSD and Sedex 75 ESDL Detector, Finnigan AQA mass spectrometer by closed contact Waters MicroMass Massspectrometer NP, chiral, SFC have similar settings

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Criteria for Evaluating and Optimizing HPLC Methods

General:• Low k, low tailing factor, high N

• High α, high Rs

Client-specific:

Clear communication with clients is a prerequisite to successful method development

●●●●●●

• MPS/library (universal applicability)

• Fraction collection for one component, multiple components, or all components?

• Purity/Recovery?

• pH stability of the desired components?

• Amount of the sample (high loading)

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AZ Example of Screening Sequence (2252-026)

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From Screening To PrepLC

Anal. Scale-up100 mg/ml, 4.6 × 100 mm XBridge C1830-80% MeOH, 10 min, 1 ml/min

Screening

2252-026 (QuinFF)0.8 mg/ml, XBridge C18 (4.6 × 50 mm)5-95% MeOH/10 mM HCOONH4, 5 minutes, 1.0 ml/min, 240 nm.

N

N

OH

Br

O

O

Tracked by MSD

*

*

*

Rs

1.8 Rs

2.2

Optimization

30-80% MeOH

Rs

3.6Rs

4.8

PrepLC19 × 100 mm XBridge C1830-80% MeOH, 10 min20 ml/min, 100 mg/ml

Baseline Resolution

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From Screening To PrepLC

Scale-up/loading100 mg/ml, 4.6 × 100 mm XBridge C1830-80% MeOH, 10 min, 1 ml/min

Screening

2252-026 (QuinFF)0.8 mg/ml, XBridge C18 (4.6 × 50 mm)5-95% MeOH/10 mM HCOONH4, 5 minutes, 1.0 ml/min, 240 nm.

N

N

OH

Br

O

O

Tracked by MSD

*

*

*

Rs

1.8 Rs

2.2

Optimization

30-80% MeOH

Rs

3.6Rs

4.8

PrepLC19 × 100 mm XBridge C1830-80% MeOH, 10 min20 ml/min, 100 mg/ml

Baseline Resolution

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Analytical Scale-up of Optimized HPLC Method

Goal: 1) Is the optimized analytical HPLC method good for preparative one? 2) If it is, what is the maximum loading for touching-band separation?

Optimization

EN00059-12Synergi Hydro-RP (4.6 × 50 mm)30-60% MeOH/TFA, 5 min, 1.0 ml/min, 240 nm, 0.6 mg/ml.

Product (MW =471.5)

Anal. Scale-up

Synergi Hydro-RP (4.6 × 100 mm)30-60% MeOH/TFA, 10 min, 1.0 ml/min, 240 nm, 12.5 ul, 160 mg/ml.

Product

This method is not practical for separation of 5 gram samples! (125 prep injections for 19 × 100mm column!)

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Scale-up of Optimized HPLC Method – Search for the Best

Optimization Anal. Scale-up

Gemini C6-Phenyl (4.6 × 50 mm)50-70% MeOH/NH4OH, 5 min, 1.0 ml/min, 240 nm, 0.6 mg/ml.

Gemini C6-Phenyl (4.6 × 100 mm)50-70% MeOH/NH4OH, 10 min, 1.0 ml/min, 240 nm, 12.5 ul, 160 mg/ml.

XBridge C18 (4.6 × 100 mm)30-60% CH3OH/NH4OH, 10 min, 1.0 ml/min, 240 nm, 12.5 ul, 160 mg/ml.

XBridge C18 (4.6 × 50 mm)30-60% CH3CN/NH4OH, 5 min, 1.0 ml/min, 240 nm,5ul inj, 0.6 mg/ml

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Gilson PrepLC via UV-triggered Fraction Collection

EN00059-12XBridge C18 (50 × 250 mm)30-60% CH3CN/NH4OH, 25 min, 100 ml/min, 240 nm, 6.0 ml, 160 mg/ml.Only 5 injections!

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Scale-up of Optimized HPLC Method – Theoretical Aspects

W

TR

W2 = W02 + Wth

2 Eq. 3

= 16 N-1 t02 (1 + 0.5 k)2 + 0.9 t0

2 k2 w ws-1

(column effect) (sample-weight effect)

Ws: column saturation capacity ( ≈ 0.4 surface area)

Overloading in reality may include mass overloading, and others. volume overloading,

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Optimized Analytical

2146-197 (BCL2)Xbridge C8 (4.6 × 50mm)20-50% CH3CN/NH4Ac (pH 8)5min, 1 ml/min, 254 nm, ~1 mg/ml5 μl injection

Loading of Analytical Scale-up - Theoretical vs. Practical

Scale Up2146-197 (BCL2)Xbridge C8, 4.6 × 100mm20-50% CH3CN/NH4Ac (pH 8)10 min, 1 ml/min, 254 nm, ~70 mg/mlInj Vol: 20 μl (Vth = 90 μl)

Rs3.5

Product

What is the maximum loading of touching-band separation?

*

*

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2146-197 (BCL2)Xbridge C8, 4.6 × 100mm20-50% CH3CN/NH4Ac (pH 8)10 min, 1 ml/min, 254 nm, ~70 mg/mlInj Vol: 5 μl, 10 μl, 15 μl, 20 μl

5 μl

10 μl

15 μl

20 μl

10 μl: Baseline separation

Analytical Scale-up of Optimized HPLC Method - Mass Loading Studies

15 μl: Touching-band separation 20 μl: No separation

*

*

*

Rt

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From Analytical Scale-up to PrepLC

F = (dprep/danal)2 x Lprep/Lanal

Constant:Constant:•Column chemistryColumn chemistry•Particle sizeParticle size•Sample concentration

To scale:To scale:•Flow rate•Injection volume

4.6 × 100 mm

19 × 100 mm

50 × 100 mm

VTB, 1 ml/min

17 VTB, 17 ml/min

118 VTB, 118 ml/min

× Fsemi

× Flarge

2146-197 (BCL2)XBridge C8, 20-50% CH3CN/NH4Ac (pH 8), 10 min, 20 ml/min, ~70 mg/ml, GilsonTM LC

Baseline Separation

200 (255) μl, 20 (17) ml/min, 19 × 100 mm15 μl, 1 ml/min, 4.6 × 100 mm

2146-197 (BCL2)XBridge C820-50% CH3CN/NH4Ac (pH 8) 10 min, 1.0 ml/min~70 mg/ml, Agilent 1100

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PrepLC Purification - UV-triggered Fraction Collection

XBridge C18 (50 × 250 mm)30-60% CH3CN/NH4OH, 25 min, 100 ml/min, 240 nm, 6.0 ml, 160 mg/ml.

2597 % Recovery (91 % without first fraction)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

0.0

5.0k

10.0k

15.0k

20.0k

25.0k

30.0k

Y = 6580 X - 65, r = 0.99994

Abs

orba

nce

@ 3

10 n

m

Concentration (mg/ml)

PrepLC Purification - Fraction Analysis & Recovery

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PrepLC Purification - MS-triggered Fraction Collection

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PrepLC Purification - MS-triggered Fraction Collection

Recovery 80 %

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Summary

Screening Optimization Anal. Scale-up Prep LC

Gradient RP analytical run is the best starting point for developing PrepLC method Screening, optimization, and scale-up are effective steps toward PrepLC method

development┐ The best analytical methods are not always the best PrepLC methods, and scale-up

experiments are imperative to validate the performance and loading of the analytical method under PrepLC conditions

UV-triggered fraction collection has high recovery and lower purity than MS-triggered fraction collection.

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Acknowledgment

Members of Analytical Group: Tatyana, Camil, Nancy, Mark, Sharon, Milena, Ziling.

Randstad USA: Yushen Chang, Vincent Cianciaruso.

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Analytical Method Screening - Results

XBridge C18 (4.6 × 50 mm)5-95% NH4OH/AcN, 5 min, 1.0 ml/min, 240 nm.

Gemini C6-Phenyl (4.6 × 50 mm)5-95% NH4OH/MeOH, 5 min, 1.0 ml/min, 240 nm.

Atlantis dC18 (4.6 × 50 mm)5-95% NH4Fm/MeOH, 5 min, 1.0 ml/min, 240 nm.

Synergi Hydro-RP (4.6 × 50 mm)5-95% TFA/MeOH, 5 min, 1.0 ml/min, 240 nm.

Luna C6-Phenyl (4.6 × 50 mm)5-95% AcONH4/MeOH, 5 min, 1.0 ml/min, 240 nm.

Curosil PFP (4.6 × 50 mm)5-95% HCOOH/CH3CN, 5 min, 1.0 ml/min, 240 nm.

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Analytical Method Optimization

XBridge C18 (4.6 × 50 mm)30-60% NH4OH/AcN, 5 min, 1.0 ml/min, 240 nm,5ul inj, 0.5 mg/ml

Gemini C6-Phenyl (4.6 × 50 mm)50-70% NH4OH/MeOH, 5 min, 1.0 ml/min, 240 nm.

Atlantis dC18 (4.6 × 50 mm)50-95% NH4Fm/MeOH, 5 min, 1.0 ml/min, 240 nm.

Synergi Hydro-RP (4.6 × 50 mm)30-60% TFA/MeOH, 5 min, 1.0 ml/min, 240 nm.

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Analytical Scale-up of Optimized Analytical Methods

XBridge C18 (4.6 × 100 mm)30-60% NH4OH/AcN, 10 min, 1.0 ml/min, 240 nm, 12.5 ul, 160 mg/ml.

Gemini C6-Phenyl (4.6 × 100 mm)50-70% NH4OH/MeOH, 10 min, 1.0 ml/min, 240 nm, 12.5 ul, 160 mg/ml.

Synergi Hydro-RP (4.6 × 100 mm)30-60% TFA/MeOH, 10 min, 1.0 ml/min, 240 nm, 12.5 ul, 160 mg/ml.

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EN00065-01-1cGemini C6-Phenyl (4.6X100mm)40-50% CH3CN/0.1% HCOOH14 min, 1 ml/min, 254 nm, ~30 mg/mlInj Vol: 3 μl, 6 μl, 12.5 μl, 25 μl

Analytical Scale-up of Optimized HPLC Method - Volume Loading

Rt

35

Effects of Buffer Concentration on Preparative Loadability

AG-166Gemini C6-Phenyl (4.6X100mm), 0-20% CH3CN, 20 min, 1 ml/min, 254 nm, 12 ul, ~100 mg/ml0% HCOOH 0.05% HCOOH0.1% HCOOH0.2% HCOOH

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Screening

1862-191-1 (CoaD)

1JB059551JB259551JB50955

Optimization

1JB606051JB505051JB40405

Scale-up/loading

4.6 × 100 mm XBridge C1840% AcN, 60% NH4Form, 30 min, 1 ml/min, 200 mg/ml

Purification

19 × 250 mm XBridge C1840 % AcN, 100 ml/min100 min, 1.0 ml (200mg/ml)

Difficult Purifications: Example 2

4.6 × 50 mm XBridge C1810mM NH4Form, 5min, 1 ml/min, 200 mg/mL

4.6 × 50 mm XBridge C1810mM NH4Form, 5min, 1 ml/min, 200 mg/mL

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Valves: G1159A 6-ColSelector, G1160A, 12/13 SelvalvesDetectors: Agilent MSD and Sedex 75 ESDL Detector Finnigan AQA mass spectrometer by closed contact

NP Chiral HPLCSolvent: HX, MeOH/EtOH(1/1), IPA, 0.1% DiethylamineDetectors: Advanced Laser Polarimeter, PDR_Chiral IncColumns: Chiralpak AD Chiralpak OD, Chiralpak AS, Chiralpak IA, Chiralpak IB, Chiralcel OD, Chiralcel OJ, Regis Pirkle covalent (S,S) whelk O2 10/100 FEC, Regis Pirkle covalent (S,S) whelk O1 5/100, Regis (S,S) ULMO 5/100,Regis (S,S) DACH DNB 5/100, Phenomenex Chirex ®-PGLY and DNB, Large 5cm X 50cm Prep Columns: Chiralpak AD Chiralpak OD, Chiralpak AS, Chiralcel OJ

NP Columns: Luna silica 10/100, YMC-PVA-sil 5/120, YMC-Pak Diol 5/60, YMC-Pak CN 5/120, Luna NH2 5/100, PrincetonSFC Pyridine 5/60

SFC Columns: Berger silica, Diol, CN, Pyridine, Chiralpak AD-H

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0.1% NH4OH 10mM NH4Form 0.1% FA 0.1% TFA MeOH k1 k2 k3 Rs(2,3)

Xbridge C18 95-60% 5-40% 7.76 9.4 9.8 1.6

80-60% 20-40% 4.75 6.9 7.51 1.8

60% 40% 1.43 2.27 2.56 1.15

70% 30% 2.73 4.74 5.545 1.78

75% 25% 3.8 6.81 7.92 2.07

80% 20% 5.59 10.39 12.16 2.29

85% 15% 8.76 16.83 19.75 2.35

90% 10% 15 29.57 34.66 2.3

75% 25% (Acn)

75% 25% 5.15 5.73 1.75

75% 25% 6.52 7.60 2.11

75% 25% 6.61 7.72 2.15

Atlantis T3 75% 25% 10.92 12.67 2.29

75% 25% 11.24 13.01 2.32

75% 25% 11.55 13.37 2.31

Atlantis D2 75% 25% 5.2 5.77 1.28

85% 15% 11.21 12.47 1.55

YMC ODS AQ 75% 25% 5.63 6.31 1.52

Xbridge C8 75% 25% 5.54 6.46 2.13

1.5 (no separation)

Performance

Column

Method Optimization Summary for 02154-137

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02154-137 Gradient Runs

Gradient and Isocratic Runs for 02154-137

5-40 % MeOH, Rs = 1.620-40 % MeOH, Rs = 1.8

XBridge C18, 4.6 × 50 mm, 0.1% NH4OH, 5 min, 1.0 ml /min.

02154-137 Isocratic Runs

40 % MeOH, Rs = 1.1520 % MeOH, Rs = 2.2910 % MeOH, Rs = 2.30

XBridge C18, 4.6 × 50 mm, 0.1% NH4OH, 1.0 ml /min.

20 % MeOH

10 %

40 %

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From Scale-up to Gilson Separation for 02154-137

2154-137 (HE-TMK)XBridge C18, 4.6 × 100mm20 % MeOH/0.1% NH4OH20 min, 1 ml/min, 254 nm, 100 mg/mlInj Vol: 8 μl, 12.5 μl, 25 μl Agilent HP 1100

2154-137 (HE-TMK)XBridge C18, 50 × 250mm20 % MeOH/0.1% NH4OH50 min, 100 ml/min, 254 nm, Inj Vol: 1.5 (1.6) ml,100 mg/ml)GilsonTM LS System

Touching-band loading still lowRetention still too large

NP HPLC in progress!