Chiral separations by hplc

“Chiral Separations by HPLC”
Anand Khatavkar
DAICEL CHIRAL TECHNOLOGIES (INDIA) Pvt Ltd
Email : anand@chiral.daicel.com
Mobile : 09373313040
Seminar on Chromatographic Techniques in Pharma [API]
7th October 2015

• What is Chirality
• Importance of Chirality
• Chiral Chromatography
• Types of Chiral Stationary Phases (CSP’s)
• Method development on Polysaccharide CSP’s
• API Applications
Overview

What's a “Chiral” Molecule?
Non-super imposable mirror image isomers
Chirality due to assymetric centre:
.
c

Lamp
Slit
standard light plane-polarized light
Sample/Cell
Analyzer
Observe
plane-polarized light
(twisted)
Levorotatory: Rotates in a counter-
clockwise direction. (-), （ｌ）
Dextrorotatory: Rotates in a
clockwise direction. (+), （ｄ）
Any compound that rotates plane polarized light in a clockwise or
counterclockwise direction.
Optical Activity

Importance of Chirality
• In an achiral environment, all physical properties of
enantiomers are identical, except for optical activity
• Enantiomers often behave differently in animals and plants
because they are chiral. Enzymes & proteins discriminate
between enantiomers.

Compound R-enantiomer S-enantiomer
Vomiting
(Dexamisole)
Antihelmenthic
(Levamisole)
Anti-
inflamatory
No activity
5 times less active
than its antipode
Anticoagulant
Thalidomide
N
O
O
N
H
O
O
Sedative Teratogenic
N
SN
Tetramisole
CH3
COOH
O
Ketoprofen
Activity differences between enantiomers
O O
OH
OWarfarin

Drug Discovery Today, vol. 9, No. 3, 105-111 (2005)
Distribution of Worldwide-Approved Drugs

Chiral Chromatography from
Analytical to Production Scale

CHIRAL CHROMATOGRAPHY
Fastest and more economical way to:
 Analyse chiral compounds
 Isolate individual enantiomers
 Obtain the two enantiomers for
clinical/toxicological studies
 Scale-up from bench- to pilot-scale
Early Stages of Development
Highly competitive way to:
 Isolate single enantiomers (final
drugs or intermediates) at
production-scale
Full Development / Production
5 kg to 100 tons
Until Industrial Scale Production
mg to 10 Kg

Types of CSP
Introduction to Chiral Stationary Phases

Types of CSPs and Their Loading Capacities
Drug discovery today, volume 10, Number 8, April 2005
Type CSPs Loading capacity
(mg solute / g CSP)
I Pirkle type (Brush type) 1-50
II Polysaccharide derivatives 5-150
III Macrocyclic type
Cyclodextrins 0.1-5
Glycopeptides 0.1-5
Chiral Crown ether 0.1-5
IV Ligand exchange 0.1-1
V Protein type 0.1-0.2

Chiral Recognition Mechanism
Y. Okamoto, E. Yashima, Angew. Chem. Intl. Ed. Engl. 37 (1998) 1020-1043.
•Attractive interactions
Hydrogen bonding
pi-pi interactions
dipole -dipole
•Inclusion complexes

1999 2001 2003 2008
Polysaccharide
80％
5%
3%
12%
87％
4%
2% 7%
86％
2%
1% 11%
92％
4%
2% 2%
1995-2003： Tetrahedron Asymmetry
2008 : J. Am. Chem. Soc.
Which type of CSP？
Pirkle
Protein
Others

Coated Chiral Stationary Phases

Coated Polysaccharide derived CSP’s

Normal phase conditions:
 Alkane/2-propanol
 Alkane/Ethanol
Polar mode:
 Acetonitrile
 Ethanol
 Methanol
 Other alcohols
Reversed Phase Conditions (for RH-versions):
 Water/alcohol or acetonitrile
 Phosphate buffer (pH 2-8)/alcohol or acetonitrile
 KPF6 pH 2/acetonitrile
 Bicarbonate buffer (pH 9)/alcohol or acetonitrile
Mobile Phases for Coated CSPs’

These will
irreversibly destroy
the Coated CSP
Coated CSPs Are Not Stable With All Solvents
NEVER USE
(even as a sample solvent)
 Chloroform
 Methylene chloride
 Ethyl acetate
 Acetone
 THF
 DMF
 DMSO
 Dioxane
 MtBE
Mobile Phases for Coated CSPs
TO AVOID ABSOLUTELY

1. Flush all the HPLC unit with a compatible solvent – preferably 2-propanol.
2. Flush the entire unit with the column storage mobile phase.
Before connecting the column to the system:
Pump Column to detector connection
ALL inlet solvent lines Pump to
Injector
connection
Injection Loop
Injection Wash
Solvent
Injector to column connection
Detector
Before Use

Immobilized
Chiral Stationary Phases

Traditionally Prepared by Coating
the Polysaccharide Derivative onto
the Silica Support
Immobilised Polysaccharide derived CSP’s
Coated Polysaccharide - Derived
Chiral Stationary Phases (CSPs)
 Highly Selective
 Broad Application Domain
 High Loading Capacity
LIMITED CHOICE OF SOLVENTS
Immobilisation of the Chiral Polymer on the silica support
 Makes the CSP resistant to a broader range of solvents
 Enlarges the application domain of polysaccharide-derived CSPs, when the appropriate
immobilisation process is applied

Immobilised Polysaccharide derived CSPs

ADVANTAGES
New Generation Chiral Stationary Phases
•High Sucess Rate and Broad Application Domain
•Highly Durable and can be Regenerated
•Compatible with All Miscible Solvents
• Rugged Phases Hence Carefree Operation
• High Preparative Potential
•Compatibility with RP, NP and Polar mode

+
Coated
polymer
SiO2SiO2
THF
EA
CHCl3
CH2Cl2
MtBE
ACT
Dioxane
…
Non-standardsolvents
IMMOBILISATION
SiO2
SiO2
SiO2
SiO2
SiO2
SiO2
SiO2
SiO2
SiO2
SiO2
SiO2
SiO2
SiO2
SiO2
SiO2
Immobilised
polymer
Non-standard
solvents
Any solvents
 Compatible with All Solvents
 New Selectivity Profile
 Robustness
 Extended Durability
Coated & Immobilised Polysaccharide derived CSPs
Solvent Compatibility

Conventional
NP Mode
CHIRALPAK IA
CHIRALPAK IB
CHIRALPAK IC
CHIRALPAK ID
CHIRALPAK IE
CHIRALPAK IF
Polar Mode
Non Standard
NP Mode
RP Mode
New Generation Chiral Columns - Applications
Chromatographic Modes

NP Mode Polar Mode /
RP Mode
Switching the Chromatographic Modes
NP Mode
Transition Solvent: Ethanol / IPA (50/50)
Polar Mode

RP Mode NP Mode /
Polar Mode
Step 1: Wash the column with EtOH @0.5mL/min for 30 min
Step 2: Wash the column with DMF or THF @0.5mL/min for 2 hrs
Step 3: Wash the column with EtOH @0.5mL/min for 30 min
Switching the Chromatographic Modes

Method Development –
Immobilized Phases

Screening Approach- i CHIRAL-6
Normal Phase Mode:
 n-Hexane/ EtOH (80/20)
 n-Hexane/ 2-PrOH (80/20)
 MtBE /EtOH (95/5)
 n-Hexane /DCM/ EtOH (49/49/2)
Polar Mode:
 Ethanol (100%)
 Methanol (100%)
 Acetonitrile (100%)
Reverse Phase mode:
 H2O / ACN (50/50)
 Formic acid (pH:2.0) / ACN (50/50)
 Bicarbonate buffer (pH:9.0) / ACN
(50/50)

0 2 4 6 8 10 12
50
100
150
200
250
300
= 2.81
MtBE/EtOH
95:5
0 10 20
0.00
0.01
0.02
0.03
0.04
0.05 n-hexane/CH2Cl2
75:25
= 2.36
Method Development on CHIRALPAK® IA
Applications with Different Solvents
CH3
N
N
O
CH3
Methaqualone
= 1.96
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
0
0.1
0.2
0.3
toluene/n-hexane/EtOH
70:25:5
0 2 4 6 8 10 12 14
0.0
0.2
0.4
0.6
0.8
1.0
= 1.79
n-hexane/CHCl3
50:50
0 10 20
0.00
0.01
0.02
0.03
0.04
= 1.70
n-hexane/methyl acetate
80:20
0 2 4 6 8 10 12 14
0
50
100
150
200
250
= 1.65
n-hexane/2-PrOH
80:20
0 10 20
0.0
0.1
0.2
= 1.63
n-hexane/THF
85:15
= 1.33
0 2 4 6 8 10 12 14
0.0
0.2
0.4
0.6
0.8
1.0
n-hexane/acetone
85:15

Method Development on CHIRALPAK® IA
Effect of Solvent Modifiers
Lorazepam
N
N
H
Cl
O
OH
Cl
MtBE/EtOH
95:5
 = 1.33
12.1
15.0
MtBE 100%
 = 1.32
27.8 35.7
The addition of 5% of alcohol
significantly improves peak shape and
reduces retention times
MtBE/MeOH
95:5
 = 1.29
9.7
11.7
min
UV 230

Prilocaine
Immobilised CSPs -
Complementary with Each Other

CHIRALPAK® IC
Operating under Various Chromatographic Modes
THF 100%
1.0ml/min
min0 2 4 6 8 10 12 14 16
N
N
O
O
Praziquantel
min0 2 4 6 8 10 12 14 16
O
NH
O
OH
O
OH
n-hexane / EtOH / TFA (85:15/0.1 v/v/v)
1.0ml/min
CBZ-DL-Serine
min0 2 4 6 8 10 12 14
N
N
S
Tetramisole
ACN / DEA (100:0.1 v/v)
1.0ml/min
Polar mode
ACN / H2O (50:50 v/v)
0.5ml/min
OH
OH
min0 2.5 5 7.5 10 12.5 15 17.5
1,1'-Bi(2-Naphthol)
RP mode

N
H
N
O
O O
O
9.7
21.1
UV 270nm
in DMF
Mobile phase: MeOH 100% 1.0ml/min. 25°C
9.7
21.1
UV 270nm
in Dioxane
UV 294nm
9.6
20.8
in Pyridine
9.6
20.9
UV 270nm
in DMSO
Diverse Injection Media on CHIRALPAK® IA

Mobile Phases Additives
Amines, Acids, Salts
Analyte Additive Form of Analyte
Acid TFA, CH3COOH Neutral
Amine
DEA, Butylamine,
Ethanolamine, Ethylenediamine
Neutral
TFA Charged
Amino Acid /
Amphoteric
Compounds
TFA
Amine Charged, Acid
Neutral

0
10
20
30
mV
0 2 4 6 8 10
Minutes
5.41
5.93
Hexane / 2-Propanol / Diethylamine
(90:10:0.1)
N
H
Cl
N
Chlorpheniramine
Hexane / 2-Propanol (90:10)
0.0
0.4
0.8
1.2
1.6
2.0
mV
0 2 4 6 8 10 12 14
Minutes
6.98
Column: CHIRALPAK® AD-H
Temp.: 25°C
Flow rate: 1.0 ml/min.
Detection: UV 254 nm
Effects-1

6.44
9.15
0 2 4 6 8 10 12 14
Retention Time (min)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Intensity(AU)
n-heptane/EtOH/AcOH
(60:40:0.5)
O
OH
O
O
Warfarin
5.62
8.15
0 2 4 6 8 10 12 14
Retention Time (min)
0.0
0.1
0.2
0.3
0.4
0.5
Intensity(AU)
n-heptane/EtOH/TFA
(60:40:0.5)
Column: CHIRALCEL® OD
Temp.: 25°C
Flow rate: 0.7 ml/min.
Detection: UV 254nm
Effects-2

CHIRALPAK®AD (250 x 4.6 mm, 10µm)
Acetonitrile 0.1% DEA
6.72
8.37
0 2 4 6 8 10 12 14
RetentionTime(min)
0.00
0.01
0.02
0.03
0.04
0.05
0.06
Absorbance(AU)
Acetonitrile 0.1% Butylamine
R R'
NH2
xO
Primary amine
Effects-3

Additive Memory Effect
• Sometimes a new column is found not to reproduce a separation
developed on an existing one.
• The problem is often blamed on the new column when in fact it is
due to the previous use and “Memory’ of the old one.
A column, once used with acidic or basic solvent additives, often is
found to exhibit a modified selectivity when re-tested under neutral
conditions.
This is due to a “memory Effect” as the additive remains adsorbed on
the CSP surface long after it is removed from the mobile phase.

Illustration of additive memory effect
NH2
1-(1-Naphthyl)ethylamine

Column Regeneration - Immobilized
Phases

Mobile Phase History & Column Performance
Regeneration in Practice
PURPOSE
Recover the specific supra molecular structure
Ensure the consistency of the column performance
PROCEDURES
CHIRALPAK® IA, IB, IC, ID, IE & IF
 Flush with EtOH at 0.5 ml/min for 30 min
 Flush with DMF 100% at 0.3 ml/min for 2 h
 Flush with EtOH at 0.5 ml/min for 30 min

Ezetimibe
tR1: 10.7 tR2:
12.6
Rs: 3.0
CHIRALPAK IC
n-Hexane/IPA/TFA (85/15/0.1, v/v/v)
Flow rate:1.0 ml/min ; UV at 240 nm;
Col Temp: 35 °C
tR1: 7.6
tR2: 9.3
Rs: 4.0
n-Hexane/EtOH/DEA (50/50/0.3, v/v/v)
Flow rate: 1.0 ml/min ; UV at 225 nm;
Col Temp: 25 °C
Darifenacin
iCHIRAL-6 Applications: NP mode

Tirofiban
CHIRALPAK IC
Mobile phase: n-Hexane/ MeOH/IPA/EDA/TFA (30/30/40/0.3/0.3, v/v/v/v/v)
Flow rate: 1.0 ml/min ; Detection: UV at 230 nm; Temp: 25 °C
Sample diluent: n-Hexane/ MeOH/IPA/TFA (30/30/40/0.3, v/v/v/v)
tR1: 6.7
tR2: 8.7
Rs: 3.5

CHIRALPAK IE
Fexofenadine-Meta isomer separation
n-Hexane/EtOH/DEA/TFA 65/35/0.1/0.1
Col Temp: 25 °C
Alvimopan
n-Hexane: EtOH: TFA (75:25:0.1)
Col Temp: 40 °C
m in2 4 6 8 10
m AU
0
200
400
600
800
DAD1 A, Sig=220,4 Ref=off (123280912278.D)
6.064-Meta-Isomer
7.346-Fexofenadine

Montelukast sodium
CHIRALPAK IA
Mobile phase: n-Hexane/EtOH/Dioxane/TFA/DEA (70/20/10/0.3/0.05, v/v/v/v/v)
tR1: 7.0
tR2: 8.0
Rs: 3.1
iCHIRAL-6 Applications: non-std NP mode

Fulvestrant
CHIRALPAK IA
Mobile phase: MtBE:EtOH (70/30, v/v)
tR1: 5.4
tR2: 6.9
Rs: 4.2
Note: Diastereomeric separation

CHIRALPAK IE
Sitagliptin
MtBE: ACN: MeOH: DEA (80:10:10:0.1)
Col Temp: 25 °C
min2 4 6 8 10 12
mAU
0
50
100
150
200
250
DAD1A,Sig=270,4Ref=off(122750812124.D)
NH2 F
F
F
N N
N
N
FF
F
O
min2 4 6 8 10 12 14 16 18
mAU
0
50
100
150
200
250
VWD1A,Wavelength=215nm(121030612009.D)
Mitiglinide
MtBE: ACN: TFA (80:20:0.3)
Col Temp: 25 °C

Mirabegron
Mobile phase: n-Hexane / MtBE/ MeOH/DEA (30/30/40/0.1, v/v/v/v)
CHIRALPAK IF-3
tR1: 8.2
tR2: 10.0
Rs: 3.1

Pramipexole
Mobile phase: MeOH/DEA (100/0.1, v/v)
CHIRALPAK IA
tR1: 3.7
tR2: 4.4
Rs: 3.5
iCHIRAL-6 Applications: Polar Organic mode

Propafenone
tR1: 4.4
tR2: 5.6
Rs: 5.5
CHIRALPAK IA
Mobile phase: MeOH/DEA (100/0.1, v/v)

Emtricitabine
Mobile phase: Ethanol/IPA (50/50, v/v)
Flow rate: 0.5 ml/min ; Detection: UV at 280nm; Temp: 25 °C
CHIRALPAK IC
tR1: 8.1
tR2: 9.2
Rs: 3.1

Dexrazoxane
Mobile phase: MeOH/ACN/DEA (60/40/0.1, v/v/v)
CHIRALPAK ID
tR1: 3.7
tR2: 5.0
Rs: 6.5

Lamivudine
CHIRALPAK IC
Mobile phase: Ethanol/IPA /DEA (90/10/0.1, v/v/v)
tR1: 8.0
tR2: 9.1
Rs: 3.1

Bicalutamide
CHIRALPAK IA
Mobile phase: 10 mM HCOONH4/ACN (60/40, v/v)
tR1: 6.8
tR2: 7.4
Rs: 2.0
iCHIRAL-6 Applications: RP mode

STIRIPENTOL DOLUTEGRAVIR
Conditions:
CHIRALPAK IF-3 (4.6x250mm, 3µm);
10mM KPF6 in (water:MTBE:ACN 40/05/55, v/v/v);
Flow Rate: 1.0mL/min; Col Temperature: 25°C;
Detection: 260nm (UV)
tR1: 12.28,
tR2: 13.85;
Rs:3.96
Conditions:
CHIRALPAK IF-3 (4.6x150mm, 3µm) ;
10mM Ammonium acetate: ACN (30/70, v/v);
Flow Rate: 1.0mL/min; Col Temperature: 25°C;
Detection: 268nm (UV)
CHIRALPAK IF-3
tR1: 3.77,
tR2: 4.52;
Rs:3.57

Entecavir
CHIRALPAK IE-3 (150 x 4.6) mm
Mobile phase: 5 mM Aq Amm bicarbonate / EtOH (5/95, v/v)
tR1: 5.7
tR2: 6.6
Rs: 2.6
CHIRALPAK IE-3

Sertraline
CHIRALPAK IA-3
n-Hexane/EtOH/MeOH/DEA (98/01/01/0.1, v/v/v)
ColTemp: 25 °C
iCHIRAL-6 Applications– 4 isomers analysis
Pitavastatin
n-Hexane/EtOH/HCOOH (90/10/0.1, v/v/v)
Flow rate: 1.5 ml/min; UV at 245 nm;
Col Temp: 25 °C
Diluent 1: MeOH;
Diluent 2: n-Hexane / EtOH (90/10)

Darunavir
CHIRALPAK IE
n-Hexane/EtOH/DEA (70/30/0.1)
Col Temp: 25 °C
Aliskiren
MtBE:ACN:1.4-Dioxan:EDA (80:20:01:0.05)
Flow Rate: 1.0 mL/min; UV: 280 nm;
Column Temp: 40ºC
CHIRALPAK IE-3
CHIRALPAK IE/IE-3

Rosuvastatin
CHIRALPAK IB-3
n-Hexane/EtOH/IPA/TFA (90/05/05/0.3)
Col Temp: 25 °C
Diluent 1: MeOH; Diluent 2: n-Hexane / EtOH (90/10)
Solifenacin
CHIRALPAK IC
n-Hexane:IPA/EtOH:DEA (60/25/15/0.1)
Flow Rate: 1.0 mL/min; UV: 220 nm;
Column Temp: 30ºC

CHIRALPAK IE-3
n-Hexane/EtOH/THF/DEA (80/10/10/0.1)
Col Temp: 25 °C
Diluent 1: MeOH; Diluent 2: n-Hexane / EtOH (90/10)
Lurasidone
iCHIRAL-6 Applications– Multiple isomers analysis

Daclatasvir: Gradient elution
Column : Chiralpak ID-3, 250x4.6mm (3mm)
Mobile Phase A : ACN/DEA 100/0.1 v/v
Mobile Phase B : MeOH / DEA 100/0.1 v/v
Flow Rate : 1.0 mL/min
T/%B : 0/20, 10/80, 15/80, 15.1/20, 22/20
Injection Volume: 10 mL
Detection : 315 nm
Column Temp : 400 C
Concentration : 0.5 mg / mL (refer sample preparation)
Diluent : ACN/MeOH/DEA 50/50/0.1 v/v/v

Warfarin5micron3micron
Mobile phase: H2O: ACN:HCOOH (5/95/0.1, v/v/v)
Flow Rate: 1.0 mL/min, Column Temp: 40°C
3 3.5
LC-MS Compatible methods - 3 µm Chiral Columns

Licarbazepine
CHIRALPAK IC-3 (150 x 4.6)mm
Mobile phase: 5 mM Aq. CH3COONH4 / MeOH (10/90, v/v)
tR1: 3.5
tR2: 4.1
Rs: 2.6
LC-MS Compatible methods
3 µm Chiral Columns

Esomeprazole
CHIRALPAK IC-3 (150 x 4.6)mm
Mobile phase: 5 mM NH4HCO3 in H2O / MeOH (10/90, v/v)
tR1: 4.3
tR2: 5.4
Rs: 3.6
LC-MS Compatible methods
3 µm Chiral Columns

Conclusions
 Chiral HPLC is a versatile technique for determining the enantiomeric
purity of APIs
 Polysaccharide derived CSPs have very high success rate
 Immobilised CSPs + few mobile phases have simplified the chiral
HPLC method development process.
 Immobilised CSPs provide carefree column operation
 Immobilised chiral columns are suitable for the analysis of complex
Chiral APIs while including process impurities

Chiral separations by hplc

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