For the distributor in your country contact Phenomenex USA, International Department. U.S.A. 2320 W. 205th St. Torrance, CA 90501-1456 U.S.A. TEL: (310) 212-0555 FAX: (310) 328-7768 PUERTO RICO Oficina Correo 1559 Ave. Americo Miranda San Juan, Puerto Rico 00921U.S.A. TEL: (800) 541-HPLC FAX: (310) 328-7768 CANADA 2320 W. 205th St. Torrance, CA 90501-1456 U.S.A. TEL: (800) 543-3681 FAX: (310) 328-7768 UNITED KINGDOM Queens Avenue, Hurdsfield Ind. Est. Macclesfield, Cheshire SK10 2BN England TEL: 01625-501367 FAX: 01625-501796 GERMANY Zeppelinstr. 5 63741 Aschaffenburg Deutschland TEL: 06021-58830-0 FAX: 06021-58830-11 NEW ZEALAND P O Box 31-601 Milford Auckland TEL: 09-4780951 FAX: 09-4780952 AUSTRALIA P O Box 264 Pennant Hills NSW 1715 TEL: 1800 555 929 FAX: 1800 553 923 Chiral HPLC Separations: Advanced Tools and Method Development Techniques Chirex
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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.
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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
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$ 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
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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
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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
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.
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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
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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
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
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EnantioselectivityColumn Comparison
Optimized Separations of Metaproterenol
METAPROTERENOL(Bronchodilator)
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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
Column: 4.0mmID x 25cmMobile Phase:
Hexane/ethanol/trifluoroacetic acid(200:40:0.6)
Flow Rate: 1.0mL/minDetector: UV254nm
Column: 4.0mmID x 25cmMobile Phase:
Hexane/ethanol/trifluoroacetic acid(200:40:0.6)
Flow Rate: 1.0mL/minDetector: UV254nm
Column: 4.0mmID x 25cmMobile Phase:
Hexane/ethanol/trifluoroacetic acid(230:20:1)
Flow Rate: 1.0mL/minDetector: UV254nm
CHIREX 3017 � = 1.25 CHIREX 3019 � = 1.22
CHIREX 3018 � = 2.13 CHIREX 3020 � = 1.35
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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
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Chiral Stationary PhasesImprove Resolution by Increasing Column Length
ATROPINE (3-TROPANYL TROPATE)
Column: Chirex 3018Dimensions: 250 x 4.6mm (2x)Mobile Phase:
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
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
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)
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.