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RP HPLC Purification of Small Molecules
Lou Cheng
Astrazeneca R&D Boston
2
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
3
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
4
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
5
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
6
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
8
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
9
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.
10
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
11
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
12
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
13
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)
14
AZ Example of Screening Sequence (2252-026)
15
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
16
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
17
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!)
18
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
19
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!
20
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,
21
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?
*
*
22
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
23
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
24
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
26
PrepLC Purification - MS-triggered Fraction Collection
27
PrepLC Purification - MS-triggered Fraction Collection
Recovery 80 %
28
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.
29
Acknowledgment
Members of Analytical Group: Tatyana, Camil, Nancy, Mark, Sharon, Milena, Ziling.
Randstad USA: Yushen Chang, Vincent Cianciaruso.
30
31
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.
32
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.
33
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.
34
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
36
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
37
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
38
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
39
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 %
40
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!
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