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1

Guidebook To Chiral HPLCCOLUMN SELECTION AND METHOD DEVELOPMENT TECHNIQUES

INTRODUCTION AND OBJECTIVES

Effective separations of enantiomers have become increasingly important to thepharmaceutical and agrochemical industry, as well as to biotechnology and mostother areas of natural products chemistry. The rapid introduction of optically activemedicinal drugs and pesticides, along with increasing government regulation,necessitates that rapid, sensitive and reliable stereochemical methods be de-vised for their analysis. Chromatography is by far the most powerful and sensitiveanalytical technique for resolving enantiomers, and chiral HPLC is probably themost versatile and important tool employed today.

This guidebook introduces the concepts of chirality and chiral separations byHPLC, and explores in detail the approach to column selection and method devel-opment. It summarizes the techniques used to develop and optimize chiral sepa-rations, and teaches by example and illustration. Data have been extracted from alarge number of applications developed by both Phenomenex and our customers.

A systematized approach to column selection is presented, and is highly depen-dent on the structure of your compound. Ultimately, however, column selectionbecomes an empirical process, based on what has already been shown to work,or by exploring column utility for related compounds, or more simply, by trial anderror. Once one or more columns are screened for their utility, optimization of theseparation on a given column is a more methodical and straightforward process.In this guidebook our intention is to suggest by illustration general separationstrategies you can use to successfully resolve and purify enantiomers directly andwithout difficulty. There are few hard and fast rules to follow. But in view of theapplicability and versatility of the chiral stationary phases described here, youshould have confidence that you are working with the most sophisticated toolsavailable today for chiral HPLC.

Phenomenex maintains a dedicated chiral separation services laboratory whichcan assist you in developing or improving your chiral separation. Please call on usfor help. We can assist you in column selection, method development, scale up orany other aspect of chiral chromatography. Phenomenex is the leader in advancedchiral separations.

2

Guidebook OutlineCHIRAL HPLC SEPARATIONS OF RACEMIC COMPOUNDS

� Chirality

� The Chirotechnology Revolution

� HPLC Separation Techniques

� Pirkle-Concept Separations

� Method Development Techniques

� Pharmaceutical Applications

3

Chirality and Molecular Structure

Causes of Molecular Asymmetry due to the arrangement of atoms in space

• Chiral Center C, N, P, S or B

• Chiral Axis Allene substitution

• Chiral Plane Helicenes,Natural polymers

• Atropisomerism Substituted Biaryls

• Molecular Strain Ortho-bridging ofBiaryls

4

Chiral Drugs and their Applications

as Single Isomers or Racemic Mixtures

� 40% of all man-made synthetic drugs are chiral

� 60% of all pharmaceuticals are chiral

� 45% of all chiral drugs are sold as racemates

Drugs1675

naturalsemisynthetic

475

synthetic1200

non-chiral6

chiral469

non-chiral720

chiral480

sold as single isomer461

sold as racemate8

sold as single isomer58

sold as racemate422

5

$ Millions

Antibiotic $26,752 $19,527 $20,064

Cardiovascular 36,580 17,530 20,046

Hormones/endocrinology 14,703 8,006 8,528

Oncology 11,558 6,803 7,513

Hematology 14,970 5,199 6,033

Antiviral 13,630 1,815 5,537

Central nervous system 36,069 4,065 5,231

Respiratory 29,088 1,201 2,327

Immunosuppressant 3,386 1,475 1,998

Anti-inflammatory/analgesic 18,309 895 1,483

Ophthalmic 6,432 675 1,087

Dermatology 14,789 545 843

Gastrointestinal 60,818 — 669

Benign prostate hyperplasia 8,026 450 626

Other 15,000 3,790 5,934

Total $310,110 $72,900 $87,919

Source: Technology Catalysts International

Total

drug sales

1997

Sales of

single-enantiomer

chiral drugs

1996 1997

Chiral Drug Sales Surge 21% Worldwide

to Almost $90 Billion

6

Stereospecific Activity

Enantiomers with different types of action. The analgesic action is incompatible with theantitussive, respiration depressant action. Both enantiomers are marketed, one as ananalgesic, the other as an antitussive (anti-cough) drug. Their chemical mirror-type

relationship is reflected in the names Darvon and Novrad [9].

2R.3S (+)dextropropoxypheneanalgesic

2S.3R ( - )levopropoxyphene

antitussive

S - ( + ) - K E T O P R O F E N( a n a l g e s i c , a n t i i n f l a m m a t o r y )

R - ( - ) - K E T O P R O F E N( s l o w s p e r i o d o n t a lb o n e l o s s )

DARVON NOVRAD

7

Enantiomers May Confer Benefits Over

Racemates for Therapeutic Uses

Properties of racemate Potential benefits of enantiomer

One enantiomer is exclusively active

The other enantiomer is toxic

Enantiomers have different pharmaco-kinetics

Enantiomers metabolized at differentrates in the same person

Enantiomers metabolized at differentrates in the population

One enantiomer prone to interactionwith key detoxification pathways

One enantiomer is agonist, the otherantagonist

Enantiomers vary in spectra ofpharmacological action and tissuespecificity

Source: Chiros

Reduced dose and load on metabolism

Increased latitude in dose and broaderuse of the drug

Better control of kinetics and dose

Wider latitude in setting the dose;reduction in variability of patients’responses

Reduction in variability of patients’responses; greater confidence insetting a single dose

Reduced interactions with othercommon drugs

Enhanced activity and reduction ofdose

Increased specificity and reduced sideeffects for one enantiomer; use ofother enantiomer for different indication

8

These drugs are Best Candidates

for Racemic Switches

CardiovascularAcebutolol Alprenolol

• Atenolol Betaxolol

Bisprolol Bopindolol

Bucumolol Bufetolol

Bufuralol Bunitrolol

Bupranolol Butofilolol

Carazolol Carvedilol

Curteolol Disopyramide

Dobutamine Indenolol

Mepindolol Metipranolol

• Metoprolol Nadolol

• Nicardipine • Oxpranolol

• Pindolol • Propanolol

Sotalol Toliprolol

• Verapamil Xibenolol

Central nervous systemDobutamine Fluoxetine

• Ketamine • Lorazepam

Meclizine Oxaprotiline

Phenylpropanolamine Thioridazine

Polycloramphetamine Tomoxetine

Toloxaton Viloxazin

Respiratory• Albuterol/salbutamol • Metaproteranol

• Terbutaline

A Racemic Switch is the redevelopment of a drug nowmarketed as a racemate to the pure enantiomer.

Anti-inflammatory and analgesicsCicloprofen Corticosteroids

Dihydroxythebaine Fenbuphen

• Fenoprofen • Flurbiprofen

• Ibuprofen • Indoprofen

• Ketoprofen Minoxiprofen

• Pirprofen Steroids

Suprofen Triamcinolone

Hormones, genitourinaryBenzyl glutamate Butoconazole

Calcitonin Estradiol

Fluorogesterone Gonadorelin

Ketodesogestrel/ Norgestrelestrogen

Progestin nitanol Testosterone

AnticancerCytarabine

Source: Technology Catalysts Interational

Antihistamine and cough/coldAstemizole Terfenadine

Antibiotics, antiinfectives,antivirals

Ciprofloxacin Norfloxacin

Ofloxacin

9

Study and Resolution of Optical Isomers

METHODS NOT INVOLVING SEPARATION� Polarimetry

� NMR Spectroscopy

� Isotope Dilution (Labeling)

� Calorimetry

� Enzymatic (asymmetric conversion/destruction)

METHODS BASED ON SEPARATION� Physical Sorting of Enantiomeric Crystals

� Selective Seeding of a Racemate Solution

� Crystallization in Optically Active Solvents

� Fractional Crystallization of Diastereomeric Salts

� Chromatography

� TLC

� GC

� LC (HPLC)

� SFC

� HPCE

10

Isomer Separation Flow Chart

ISOMERS

CONSTITUTIONAL STEREOISOMERS (CONFORMATIONAL)

GEOMETRIC ENANTIOMERS

RACEMATES OPTICALLY ACTIVE

SEPARATION OFDIASTEREOMERS

BY HPLC

NON-TRANSIENTCOMPLEX USING

CHIRAL DERIV.AGENTS (CDA)

TRANSIENT COMPLEX(WEAKLY ASSOCIATED)

CHIRAL MOBILEPHASE ADDITIVE

(CMPA)

ACHIRALCHROMATOGRAPHY

SILICA, ALUMINA, ETC.

CHIRAL STATIONARYPHASE (CSP)

CLASSIFICATIONTYPES I - V

11

Classification of

Chiral Stationary Phases (CSPs)

Type Description Chemistry Mechanism

I BRUSH Various chiral selectors Attractive interactions

(Pirkle) Ionic or covalent bonding Hydrogen Bonding

Charge transfer (�-� interaction)Dipole stacking

II HELICAL POLYMERS Cellulose derivatives Attractive interactives

Poly methacrylates Insertion complexes

Poly acrylamides

III CAVITY Cyclodextrins Inclusion complexes

IV LIGAND EXCHANGE Amino acid-metal complex Diastereomeric metal complex

V PROTEIN �-acid glycoprotein Hydrophobic interactionsBovine Serum Albumin Polar interactions

12

Pirkle-Concept CSPs

Mechanism of Interaction

Enantiorecognition involves hydrogen bonding, dipolar and charge transferinteractions and steric hindrance

Class-I CSPs and types of interactions that can be involved during the chiral recognition

process.

The three-point interaction model advanced by Dalgliesh

chiralselector

soluteenantiomers

achiralspacer

chiralmoiety

type of interactions:

dipole / dipole

hydrogen bonding

steric hindrance

silica

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13


CSP TYPE II. INSERTION COMPLEX

HELICAL POLYMERS (Natural CELLULOSES and

DERIVATIVES and VARIOUS SYNTHETIC POLYMERS)

Natural:

Synthetic:

Cellulose Derivatives:

Amylose

Chitosan Acetates

Dextran Esters

Carbamates

Polymethacrylates

Polyacrylamides

linear poly[1 - 4 - � - D -glucose]

(a) Use of chiral substituent:

chiral monomer

polymerizationcrosslinking

chiralpolymer network

(b) Use of chiral catalyst:

isotactic (stereoregular)polymer

Adopts a single helicalconformation

Y

C

14


CSP TYPE III. INCLUSION (CAVITY) COMPLEX

CYCLODEXTRINS and DERIVATIVES

Derivatives:

Acetates

Esters

Carbamates

The structure of �� and � cyclodextrin.

�-Cyclodextrin�-Cyclodextrin�-Cyclodextrin

Schematic showing the reversible inclusioncomplex formation typical of cyclodextrin bondedstationary phase.

15


CSP TYPE IV. LIGAND EXCHANGE

DIASTEREOMERIC METAL COMPLEX

Illustration of the principle of chiral ligand exchange used for chromatographic optical resolution.

Reversible formation of diastereomeric metal complexes is obtained in a reaction between a

chiral selector (left-hand side) and the respective enantiomers (right-hand side).

CSP TYPE V. PROTEIN

AGP, BSA, HSA, OVOMUCOID

16

Chiral HPLC Columnsfor

Enantiomeric Separations

The Chirex Advantage

� Pharmaceuticals

� Agrochemicals

� Derivatized and underivatized amino acids

� Biogenic amines, alcohols and amino alcohols

� Carboxylic acids, hydroxy acids and esters

� Wide variety of supports

� High enantioselectivity

� High efficiency, covalently bound columns

� Chemically stable and rigid supports

� Choice of elution order (opposite CSPs available)

� Packed columns from minibore to preparative

17

Chiral Stationary PhasesStationary phases in Chirex columns contain various chiral components. They are classified into

three types: amide, urea and ligand exchange types. In the amide type, asymmetric carbon atoms

are bonded directly with the CONH group, and in the urea type, asymmetric carbon atoms with the

NHCONH group, and they are chemically (covalently or ionically) bonded to silica gel. In the ligand

exchange type, the chiral components are coated hydrophobically on ODS.

CHIREX Chiral Component Mode* Application

Amide type

3001 (R)-phenylglycine NP

3002 (R)-phenylglycine (Ionic) NP

3005 (R)-I-naphthylglycine RP

3007 (R)-I-naphthylglycine (Ionic) NP

Urea type

3010 (S)-valine RP

3011 (S)-tert-leucine RP

3012 (S)-phenylglycine RP

(S)-valine,3013 (S)-I-(�-naphthyl)ethylamine NP

(S)-valine,3014 (R)-I-(�-naphthyl)ethylamine NP

(S)-proline,3017 (S)-I-(�-naphthyl)ethylamine NP

(S)-proline3018 (R)-I-(�-naphthyl)ethylamine NP

(S)-tert-leucine3019 (S)-I-(�-naphthyl)ethylamine NP

(S)-tert-leucine3020 (R)-I-(�-naphthyl)ethylamine NP

(S)-indoline-2-carboxylic acid,3021 (S)-I-(�-naphthyl)ethylamine NP

(S)-indoline-2-carboxylic acid,3022 (R)-I-(�-naphthyl)ethylamine NP

Ligand-exchange type

3126 (D)-penicillamine RP

esters, amides, carboxylic

acids, alcohols, ketones

carboxylic acids (including

Z-amino acids, BOC-amino

acids, benzoylamino acids)

amines, amides, amino

alcohols, alcohols, esters,

phosphoric amides

�-amino acids, �-hydroxy acids

3002 and 3007 are ionically bonded types.

*NP : Normal phase mode

RP : Reverse phase mode, typically consisting of ammonium acetate in methanol

Enantiomeric stationary phases are available except for 3021 and 3022, and on these phases the elution order of enantioners

are inverted.

18

Chirex Chiral Stationary Phases (CSPs)

3001 (R)-phenylglycine and 3,5-dinitrobenzoic acidamide linkage, electron acceptordesigned for the separation of• carboxylic acids, alcohols, esters• sulphoxides

3005 (R)-1-naphthylglycine and 3,5-dinitrobenzoic acidamide linkage, electron acceptordesigned for the separation of:• carboxylic acids, alcohols, esters• non-steroidal anti-inflammatory agents

3010 (S)-valine and 3,5-dinitroanilineurea linkage, electron acceptordesigned for the separation of:• carboxylic acids, amino acid derivatives• hydroxy acids• Dabsyl and Dansyl derivatives of amino acids

3011 (S)-tert-leucine and 3,5-dinitroanilineurea linkage, electron acceptordesigned for the separation of:• carboxylic acids, amino acid derivatives

3012 (R)-phenylglycine and 3,5-dinitroanilineurea linkage, electron acceptordesigned for the separation of:• carboxylic acids, amino acid derivatives

3014 (S)-valine and (R)-1-(�-naphthyl)ethylamineurea linkage, electron donordesigned for the separation of:• �-acceptor derivatives of amines, carboxylic acids and amino acids• the esters and amides of these acids• underivatized alcohols

3017 (S)-proline and (S)-1-(�-naphthyl)ethylamineurea linkage, electron donordesigned for the separation of:• amines, alcohols and amino acids• underivatized �-blockers• aromatic amines• pesticides

AMIDE TYPE

Phase Description Structure

UREA TYPE

R

19

Chirex Chiral Stationary Phases (CSPs)

3018 (S)-proline and (R)-1-(�-naphthyl)ethylamineurea linkage, electron donordesigned for the separation of:• amines, alcohols and amino acids• underivatized ß-blockers• aromatic amines• pesticides

3019 (S)-tert-leucine and (S)-1-(�-naphthyl)ethylamineurea linkage, electron donordesigned for the separation of:• esters, amino alcohols• underivatized ß-blockers• aromatic amines• cyano alcohols• pesticides

3020 (S)-tert-leucine and (R)-1-(�-naphthyl)ethylamineurea linkage, electron donordesigned for the separation of:• amines, amino alcohols, alcohols• underivatized �-blockers• aromatic amines• cyano alcohols• pesticides

3022 (S)-indoline-2-carboxylic acid and(R)-1-(�-naphthyl)ethylamineurea linkage, electron donordesigned for the separation of:• amines, amino alcohols, alcohols

3126 (D)-penicillamine ligand exchange,electron acceptordesigned for the separation of:• �-amino acids, their derivatives• �-hydroxy acids, amino alcohols

LIGAND EXCHANGE TYPE

UREA TYPE

Phase Description Structure

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20

Chiral Recognition Model

Propranolol and Phase 3014

21

Classification of Chiral Compounds

According to Functional Groups

22

23

Presence of Chemical Groupings in Chiral Solute

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Chirex Column Selection Guide

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24

25

Importance of Separation Factor �

Plots of plate numbers vs. �-values at different constant resolution values. Note thedrastic increase in plate numbers needed to maintain resolution at very low �-values

1.0 1.1 1.2 Separation Factor ��

To maintain the same level of resolutionseparations with low alpha valuesrequire very high efficiencies

2.0

3.0

4.0

5.0

Log Neff

Rs = 1.5

Rs = 1.0

26

EnantioselectivityColumn Comparison

Optimized Separations of Metaproterenol

METAPROTERENOL(Bronchodilator)

27

Chiral Stationary PhasesDifferences in Enantioselectivity

TOLPERISONE(Bronchodilator)

Column: 4.0mmID x 25cmMobile Phase:

Hexane/ethanol/trifluoroacetic acid(200:40:0.6)

Flow Rate: 1.0mL/minDetector: UV254nm








Hexane/ethanol/trifluoroacetic acid(230:20:1)


CHIREX 3017 � = 1.25 CHIREX 3019 � = 1.22

CHIREX 3018 � = 2.13 CHIREX 3020 � = 1.35

28

Chiral Stationary PhasesDifferences in Enantioselectivity

BENDROFLUMETHAZIDE(Diuretic)

Dimensions: 250 x 4.6mmMobile Phase:

Hex/DCE/EtOH-TFA(as indicated)

Flow Rate: 1.0mL/minDetector: UV @ 272nm

300155:35:10� = 1.11

302058:35:7� = 1.17

301458:35:7� = 1.14

301855:35:10� = 1.09

300558:35:7� = 1.07

301750:35:15� = P

29

Chiral Stationary PhasesImprove Resolution by Increasing Column Length

ATROPINE (3-TROPANYL TROPATE)

Column: Chirex 3018Dimensions: 250 x 4.6mm (2x)Mobile Phase:

Hexane/1,2-Dichloroethane/Methanol/Trifluoroacetic Acid(250:140:10:1)

Flow Rate: 1.0mL/minDetector: UV @ 254nm

30

Chiral Stationary PhasesImprove Resolution by Combining Different Column Selectivities

CYPERMETHRIN

Mobile Phase:Hexane/1,2-Dichloroethane/Ethanol (500:10:0.05)

Flow Rate: 1mL/minTemperature: AmbientDetector: UV @ 230nmSample: Racemic Cypermethrin

Chirex 3019 + 3020250 x 4.0mm (2X)

Chirex 3019250 x 4.0mm

31

Effect of Mobile Phase on the Separationof 2- (4-Chlorophenyl) -3-Methyl Butyric Acid

Mobile Phase: 0.1M Ammonium Acetate in:

Water/Methanol

(60:40)

Water/Acetonitrile

(60:40)

Water/Tetrahydrofuran

(60:40)

CONDITIONS:

Column: Chirex Phase 3012 (250 x 4.6mm)

Flow Rate: 1.0 mL/minDetector: UV @ 254nm

32

Comparison of Enantiomer Separations UsingDifferent Mobile Phase Components

Compounds stationary phase k1 � mobile phase

Ketoprofen Phase 3005 2.27 1.11 0.02M NH4AC 100% MeOH1.09 1.15 0.1M NH4AC 70% MeOH6.36 1.16 0.1M NH4AC 40% MeOH1.92 1.12 0.1M NH4AC 40% AN2.43 1.14 0.1M NH4AC 40% THF

19.07 1.19 0.1M NH4AC 20% THF12.82 1.03 Hexane/DCE/EtOH/AA

(190:9:1:1)

Naproxen Phase 3005 5.25 1.55 0.05M NH4AC 100% MeOH8.23 1.19 Hexane/DCE/EtOH/AA

(190:9:1:1)

Clidanac Phase 3011 6.85 1.56 0.01M NH4AC 100% MeOH4.01 1.19 Hexane/DCE/EtOH/AA

(490:9:1:1)

2-(4-Chlorophenyl)- Phase 3012 3.57 1.02 0.01M NH4AC 100% MeOH3-methyl butyric acid 4.85 1.04 0.1M NH4AC 40% MeOH

5.21 1.08 0.1M NH4AC 40% AN9.91 1.16 0.1M NH4AC 40% THF2.50 1.18 0.1M NH4AC 50% THF1.17 1.21 0.1M NH4AC 60% THF

A flow rate of 1.0 mL/min was used for the 250 x 4.6 mm ID column at room temperature. An injectionvolume of 1µL (5mg/mL) was typically used. k1, k2 = Capacity factors of first- and second-elutedisomer; � = separation factor (k

2/k

1).

NH4AC: ammonium acetate, MeOH: methanol, AN: acetonitrile, THF: tetrahydrofuran,DCE: 1,2-dichloroethane, EtOH: ethanol, AA: acetic acid

33

Normal vs. Reverse Phase on 3005

Hexane/1,2-dichloroethane/

ethanol/acetic acid

(190:9:1)

0.05M ammonium acetate

in methanol

CONDITIONS:



NAPROXEN(Antiinflammatory,Analgesic)

34

Enantiomer Separations of Propranolol byHPLC Effect of Mobile Phase

Column: Chirex Phase 3014 (250 x 4.0mm ID)

Mobile Phase: containing 0.25% trifluoroacetic acid

Flow Rate: 1.0mL/min

Detector: UV@254nm

TEST SYSTEM 1

TEST SYSTEM 2

Hexane/ethanol k1 �100 : 0 11.8 1.00

97.5 : 2.5 23.0 1.07

95 : 5 8.4 1.07

92.5 : 7.5 4.8 1.06

1,2-dichloroethane/ethanol k1 �100 : 0 0.8 1.00

99.5 : 0.5 5.5 1.00

97.5 : 2.5 4.6 1.07

95 : 5 1.6 1.08

35

Effect of Mobile Phase on Propranolol

Hexane

Hexane/ethanol

(95:5)


Mobile Phase: containing 0.25% trifluoroacetic acidFlow Rate: 1.0 mL/minDetector: UV @ 254nm

PROPRANOLOL(Antiarrhythmic,Antianginal)

36

37

Effect of Dichloroethane

73:20:7

58:35:7

43:50:7


Mobile Phase: Hex/DCE/EtOH-TFAFlow Rate: 0.7 mL/minDetector: UV @ 278nm

METAPROTERENOL(Bronchodilator)

MWD 1 A, Sig=278, 4 Ref=500, 80 of A:\HPCHEM\1\DATA\090192\TC01-42E.D

MWD 1 A, Sig=278, 4 Ref=500, 80 of A:\HPCHEM\1\DATA\090192\TC01-42C.D

MWD 1 A, Sig=278, 4 Ref=500, 80 of A:\HPCHEM\1\DATA\090192\TC01-42D.D

38

Effect of Solvent on Retention TimeMethoxamine Separation on Chirex 3014 250 x 4.6mm

39

Effect of Ethanol Concentration onCapacity and Separation Factors

40

Effect of Temperatureon the Separation of Lorazepam

Columntemp:

Column: Chirex Phase 3010Mobile Phase: hexane/dichloroethane/ethanol

(73:20:7,V/V/V)Flow Rate: (as indicated)Detector: UV @ 254nm

LORAZEPAM(Anxiolytic)

10°C 40°C

2.0 mL/min 1.0mL/min

Source: Journal of Liquid Chromatography, 16 (4), 879-891 (1993)

25

.06

82

8.0

17 2

4.3

33

26.9

41

Reversal of Elution Order

DINITROBENZENE RDERIVATIVE

S

NAPHTHYLENE RDERIVATIVE

S

UREIDES of �-METHYLBENZYLAMINE

CHIREX PHASE 3005

Heptane-Isopropanol (90:10)

42

Reversal in Elution OrderEnantiomeric Stationary Phases

LACTIC ACID

L L

D

D

Column temp: Room temperatureMobile Phase: 2mM copper (II) sulfate in

water/acetonitrile (95:5)Flow Rate: 1.0 mL/minDetector: UV @ 254nm

Chirex 3126(150 x 4.6mmID)

D-pennicillamine LEC L-penicillinamine LEC

43

Method Development

PROPRANOLOL(Antiarrhythmic,Antihypertensive)

238 292

Annotated Wavelengths:1 : Wavelength = 238 Result = 1.9124602 : Wavelength = 292 Result = 1.8724673 : Wavelength = 320 Result = 0.594666

44

Method DevelopmentPROPRANOLOL(Antiarrhythmic,Antihypertensive)

MWD 1 A, Sig=290, 4 Ref=500, 80 of A:\HPCHEM\1\DATA\1092\TC02_43A.D

55:35:10

60:35:5

76:20:4

Column: Chirex 3020(250 x 4.6mm)

Mobile Phase: Hex/DCE/EtOH-TFA (as indicated)Flow Rate: 1.0 mL/minDetector: UV @ 254nm

MWD 1 A, Sig=290, 4 Ref=500, 80 of A:\HPCHEM\1\DATA\1092\TC02_43B.D

MWD 1 A, Sig=290, 4 Ref=500, 80 of A:\HPCHEM\1\DATA\1092\TC02_43C.D

45

Method Development

MWD 1 A, Sig=250, 4 Ref=500, 80 of A:\HPCHEM\1\DATA\TC0893\TC0340C.D

BEPRIDIL(Antianginal)

55:35:10

60:35:5

62:53:3

Column: Chirex 3014Mobile Phase: Hex/DCE/EtOH-TFA (as indicated)Flow Rate: 1.0 mL/minDetector: UV @ 250nm

MWD 1 A, Sig=250, 4 Ref=500, 80 of A:\HPCHEM\1\DATA\TC0893\TC0340B.D

MWD 1 A, Sig=250, 4 Ref=500, 80 of A:\HPCHEM\1\DATA\TC0893\TC0340A.D

46

Method Development

MWD 1 A, Sig=280, 4 Ref=500, 80 of A:\HPCHEM\1\DATA\090192\TC01-34C.

VERAPAMIL(Antiaarrhythmic)

40:35:25

75:20:5

87:10:3


Mobile Phase: Hex/DCE/EtOH-TFA (as indicated)Flow Rate: 1.0 mL/minDetector: UV @ 280nm

MWD 1 A, Sig=280, 4 Ref=500, 80 of A:\HPCHEM\1\DATA\090192\TC01-34B.

MWD 1 A, Sig=280, 4 Ref=500, 80 of A:\HPCHEM\1\DATA\090192\TC01-34A.

47

Method DevelopmentMETHOCARBAMOL

(Skeletal Muscle Relaxant)

1. 40:35:25 2. 55:35:10

3. 75:20:5 4:77:20:3


Mobile Phase: Hex/DCE/EtOH-TFA (as indicated)


48

Method Development

MWD 1 A, Sig=248, 4 Ref=500, 80 of A:\HPCHEM\1\DATA\TC0893\TC0342C.D

CLENBUTEROL(Bronchodilator)

55:35:10

60:35:5

77:20:3




49

Method DevelopmentCHLOROQUINE(Antimalarial)

40:35:25

50:35:15




50

Method Development

WARFARIN(Anticoagulant)

Column: Chirex 3022Mobile Phase: Hex/DCE/EtOH-TFA

88:10:2Flow Rate: 1.0 mL/minDetector: UV @ 282nm

Area Percent Report

MWD A, Sig=282, 4 Ref=500,80

PK# Ret Time Area Height Type Width Area%

1 8.641 1480.076 123.785 BV 0.199 5.5719

2 9.003 1507.973 120.679 VV 0.198 5.6769

3 12.008 415.730 26.497 BV 0.249 1.5651

4 15.409 414.812 20.118 BB 0.323 1.5616

5 61.476 11175.077 204.599 BV 0.829 42.0696

6 63.504 11569.626 194.712 VB 0.893 43.5549

Total area = 26563

51

ReproducibilityOPTIMIZED SEPARATION OF N-BENZOYLVALINE

Column: Chirex 3012 (250 x 4.0mm)Mobile Phase: 10mM ammonium acetate in methanolFlow Rate: 1.0 mL/minDetector: UV @ 254nmInjection: 10µL of 3.4mg/2.0mL (17µg on-column)

MWD 1 A, Sig=254, 4 Ref=450, 80 of TC0506G.D

Retention Time (min)

52

EPINEPHRINE

CHIRAL IMPURITIESOR BREAKDOWN PRODUCTS?

Time �

TC02_21A.D: MWD A, Sig=282, 4 Ref=500, 80

Area Percent Report

Sample Name : EPINEPHRINE Injection Number :Run Time Bar Code : Sequence Line :Instrument Method : CRX_06.MAnalysis Method : CRX_06.MEPINEPHRINE 4.6MG/4ML IN HEX/EtOH 75:25 FLOW: 0.5mL/min 595psi MP:HEX/DCE/EtOH-TFA 55:35:10 EtOh-TFA 20:1 CRX 3022 MID-BORE 250 x 3.2 s/n 40644.MDW A, Sig=282, 4 Ref=500,80

PK# Ret Time Area Height Type Width Area%

1 8.641 1480.076 123.785 BV 0.199 5.5719

2 9.003 1507.973 120.679 VV 0.198 5.6769

3 12.008 415.730 26.497 BV 0.249 1.5651

4 15.409 414.812 20.118 BB 0.323 1.5616

5 61.476 11175.077 204.599 BV 0.829 42.0696

6 63.504 11569.626 194.712 VB 0.893 43.5549

Total area = 26563

53

Starter Columns for Method Development

STARTER COLUMNColumn Length: 50mmFlow Rate: 0.2 mL/min

STANDARD ANALYTICALColumn Length: 250mmFlow Rate: 1.0 mL/min

Separation conditions were the samefor both columns.

54

Midbore Comparison

STANDARD ANALYTICALColumn ID: 4.6mmFlow Rate: 1.0 mL/min

MIDBORE DIMENSIONSColumn ID: 3.2mmFlow Rate: 0.5 mL/min

Separation conditions were the samefor both columns.

55

Column Hardware ComparisonLABETOLOL(Antihypertensive)

Column: Chirex 3022(dimensions as indicated)

Mobile Phase: Hex/DCE/EtOH-TFA (55:35:10)

Flow Rate: as indicatedDetector: UV @ 308nm

MWD 1 A, Sig=308, 4 Ref=500, 80 of A:\HPCHEM\1\DATA\1092\TC02_26A.D

50 x 4.6mm“Starter”0.2mL/min

250 x 3.2mmMidbore ID0.5mL/min

250 x 4.6mmStandard Analytical1.0 mL/min

56

Preparative Separations

Mobile Phase: 2% acetic acid in methanol/ 1.2-dichloroethane (70:30)

Flow Rate: 1.0mL/minDetector: UV @ 254nmSample amount: 5µg

Mobile Phase: 2% acetic acid in methanol/ 1.2-dichloroethane (70:30)

Flow Rate: 150mL/minDetector: UV @ 300nmSample amount: 2.3µg

57

Preparative Separations

��

Operating conditions for preparative scale chromatography according to the analyticalvalue of selectivity �

resolution

speed capacity

Vel2 = Vel1 (R2/R1)2

Load2 = Load 1(L2/L1)(R2/R1)2

Inner Amount of Approximatediameter stationary maximum load

Column type (mm) Use phase (g) (mg)

Analytical 1-5 Isolation of pure samples for 0.2-3 0.2-36-11 MS or IR (0.001-0.1mg)

Analytical to 6-11 Isolation of pure samples for 3-25 3-25semi-preparative NMR or elemental analysis

(0.1-25mg)

Semipreparative 10-30 Small scale synthesis (0.1-1g) 25-100 20-1000to preparative

Preparative 20-100 Large scale synthesis (1-100g) 100-104 100-104

Industrial 100-1000 Industrial scale synthesis 103-105 103-105

SelectivityOperatingconditions

1.05< �<1.25 1.25< �<2 �>2

Behavior linear (analytical) intermediate non-linear(mass overload)

dp (µm) 5-7 µm 7, 10, and 15-40 µm 15-40µm and >40 µm

Loading rate g/g of CSP 10-4-10-3 g >10-3 g >10-2 g

Comments possibility of recycling semipreparative scale “flash chromatography”

58

Separation Utility of 7 Chirex CSPs

3001 3005 3014 3017 3018 3020 3022 ALL

S 3 4 24 3 7 29 29 46P 0 1 11 3 5 6 13 10T 31 24 66 26 28 64 65 70

%S 10 17 36 12 25 45 45 60%S + P 10 21 53 23 43 55 65 80

S = Separated enantiomeric pair (baseline or valley height between the pair is lessthan 25% of the height of peak one).

P = Partial separation of the enantiomeric pair (valley height is greater than 25% ofthe height of peak one).

T = Total number of compounds tested.

59

Chirex Pharmaceutical Applications

Phase: 3020 Phase: 3014 Phase: 3020CHLORTHALIDONE DIPERODON DOXYLAMINE(Diuretic; Antihypertensive) (Local (Antihistaminic)

Anesthetic)

�� = 1.08 � = 1.20 � = 1.07

Phase: 3020 Phase: 3020 Phase: 3020LABETOLOL METHOCARBAMOL METHOXYVERAPAMIL(Antihypertensive) (Skeletal Muscle (Antiarrhythmic)

Relaxant)

�� = 1.09 � = 1.15

Phase: 3014 Phase: 3014 Phase: 3022NICOTINE PRILOCAINE SYNEPHRINE(Anti-Smoking) (Local (Vasopressor)

Anesthetic)

�� = 1.10 � = 1.13 � = 1.12

60

Chiral Separations of Important

Clinical and Pharmaceutical Compounds

Phase: 3020 Phase: 3014 Phase: 3020CHLORTHALIDONE DIPERODON DOXYLAMINE(Diuretic; Antihypertensive) (Local (Antihistaminic)

Anesthetic)

� = 1.08 � = 1.20 � = 1.07

Phase: 3020 Phase: 3020 Phase: 3020LABETOLOL METHOCARBAMOL METHOXYVERAPAMIL(Antihypertensive) (Skeletal Muscle (Antiarrhythmic)

Relaxant)

�� = 1.09 � = 1.15

Phase: 3014 Phase: 3014 Phase: 3022NICOTINE PRILOCAINE SYNEPHRINE(Anti-Smoking) (Local (Vasopressor)

Anesthetic)

�� = 1.10 � = 1.13 � = 1.12

61

Pharmaceuticals

SALBUTAMOL(Bronchodilator)

Column: Chirex 3022Size: 250 x 4.6mm IDMobile Phase: Hexane/

1,2-Dichloroethane/Methanol/Trifluoroacetic Acid(240:140:20:1)


NICARDIPINE(Antianginal)

Column: Chirex 3018Size: 250 x 4.0mm IDMobile Phase: Hexane/

1,2-Dichloroethane/Methanol/Trifluoroacetic Acid(240:140:10:1)


� = 1.33

� = 1.06

62

Pharmaceuticals

NAPROXEN(Antiinflammatory)

Column: Chirex 3014Size: 250 x 4.0mm IDMobile Phase: Hexane/Ethanol/

Trifluoroacetic Acid(200:40:0:6)


CHLORPHENIRAMINE(Antihistaminic)

Column: Chirex 3005Size: 250 x 4.0mm IDMobile Phase: 0.03 Ammonium Acetate

in MethanolFlow Rate: 1.0 mL/minDetector: UV @ 254nm

� = 1.24

� = 1.35

63

NSAIDs

KETOPROFEN



FLURBIPROFEN



� = 1.09

� = 1.10

64

Amino Acids

LEUCINE



Z-LEUCINE

Column: Chirex 3126Size: 150 x 4.6mm IDMobile Phase: 2mM Copper (II) Sulfate

in Water/Methanol (85:15)Flow Rate: 1.0 mL/minDetector: UV @ 254nm

� = 1.17

� = 1.56

65

Conclusions

• CHIREX column line has WIDE UTILITY for the separation of enantiomers

• WIDE VARIETY of CSPs to choose from

• ENANTIOMER SEPARATION is often possible on TWO or MORE COLUMNS

• 50mm “STARTER” COLUMNS permit RAPID and ECONOMICAL METHODDEVELOPMENT

• MIDBORE column dimensions SAVE SOLVENT and INCREASE SENSITIVITY

66

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�� ;�� !� ;�� ;�� ;��& �� ;��&�� ;��&�� ;��&�*�� ;��#�� ! ��;��#�� "�� ;��#�� ;�� ;�� ;�� ;��!��&��+��,��

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NOTE: A dash ("—") under separation factorindicates the compound has more than onechiral center. Separation involves more thanone enantiomeric pair

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68

Chiral HPLC Method Development Kits

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The broad range of Pirkle-concept (Type I,or brush type) columns carefully chosen forthis Kit will enable the chiral chromatogra-pher to quickly and easily survey stationaryphase utility and separation conditions for awide variety of enantiomeric compounds.Used with normal phase solvent systems,these latest generation chiral columns arehighly efficient tools particularly well-suitedfor enantiomeric separations of pharmaceu-ticals and agrochemicals.

This kit contains five columns, 50 x 4.6mm:

Column 1: Chirex 3001; 3,5-dinitrobenzoicacid derivative of (R)-phenylglycineColumn 2: Chirex 3005; 3,5-dinitrobenzoicacid derivative of (R)-1

naphthylphenylglycineColumn 3: Chirex 3010; 3,5-dinitroaniline

derivative of (S)-valineColumn 4: Chirex 3014; (R)-1-(�-naphthyl)

ethylamine derivative of(S)-valineColumn 5: Chirex 3020; (R)-1-(�-naphthyl)

ethylamine derivative of(S)-tert- leucine

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This Kit contains a collection of chiral col-umns for method development of enantio-meric �-amino acids, �-hydroxy acids, andaromatic amines, alcohols and aminoalcohols. The kit includes four Pirkle-con-cept columns and one ligand-exchange typecolumn. The Pirkle-concept columns arechosen for their high utility/versatility in sepa-rating derivatized amino acids, whereas theligand-exchange column confers excellentenantioselectivity for underivatized �-aminoacids, and �-hydroxy acids.

This kit contains five columns, 50 x 4.6mm:

Column 1: Chirex 3010; 3,5-dinitroanilinederivative of (S)-valine

Column 2: Chirex 3011; 3,5-dinitroanilinederivative of (S)-tert-

leucineColumn 3: Chirex 3012; 3,5-dinitroaniline

derivative of (R)phenylglycineColumn 4: Chirex 3014; (R)-1-(�-naphthyl)

ethylamine derivativeof (S)-valineColumn 5: Chirex 3126; Ligand-Exchange type basedon (N,S) dioctyl-(D)-penicil-lamine complexed withcopper(II)

Phenomenex offers two very economical Chiral method development column kits. Each kitis composed of five short-length columns which maintain resolving power while offering themost flexible approach to column selection and general method development.

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Chiral on Guide