1 1 1 2 LCGC NORTH AMERICA VOLUME 25 NUMBER 11 NOVHHBEfi2[»7 www. chromatogr9phyonline.com

A Strategy for Developing HPLCMethods for Chiral Drugs

Molecules that relate to each other where an object and its mirrorimage are not superimposable are called chiral {from Greek word cheiro,meaning "hand"); that is, they are like a pair of hands. These moleculesalso are called enantiomers. Major differences in biological activity havebeen observed in chiral molecules. The difference in spatialarrangements of atoms in a molecule (that is, its stereochemistry) caninfluence its pharmacological, metabolic, or toxicologic activity. This iswhy regulatory requirements in the pharmaceutical industry demanddetailed investigations of chiral molecules. Before initiating methoddevelopment, it is important to develop a basic understanding ofstereochemistry (1-5). Basic information on stereochemistry is providedin this article to help readers develop better understanding of theseparation mechanisms that come into play in various separationmethods used for chiral compounds. This knowiedge can allow readersto select a desirable chiral separation method, based upon themolecular structure of the chiral compound of interest. Logical reasonsfor the selection process are discussed later in this article.

Satinder (Sut) Ahuja

Ahuja Consulting, Calabash, NC

Please direct correspondence to SutAhuja at sutahuja@atmc,net

T he most difficult problem for aninvestigator in chiral separations isto determine where to start. Cosr

considerations, availabiliry of equipment,and know-how play important roles inthe selection process (1,3-5). The scien-tific literature suggests that chromato-graphic methods are generally favoredover nonchromatographic methods. Ofthe various chromatographic methods(such as thin-layer chromatography, gaschromarography [GC], high perform-ance liquid chromarography [HPLC],supercritical fluid chromatography[SFC], or capillary elecrrochromatogra-phy), HPLC methods are likely to befound most useful (exceptions can befound in references 1 and 3-5). The nextstep is to select an appropriate column,based upon various considerations (seeshort discussion in the following sec-tion). The primary concern for any inves-tigator in this area, notwithstanding thehigh cost of the columns, is which col-umn will work best in a particular situa-

tion. This article attempts to answerthese questions, and it provides examplesthat will help readers make intelligentdecisions in this complex field.

Determine the Chirality of theMoleculeFirst the investigator must take a closelook at the molecule that is to be resolvedand then answer the following question:Is there a stereogenic center? The simplestexample of this is an asymmetric carbonwith four different substituents. As simpleas it might sound, this process appearscumbersome when you are looking atmany carbon atoms in a molecular struc-ture. The simplest way to counter thisproblem is to number all carbon atoms inthe structure and look at each of them inturn to see if they are asymmetric or not.

If no asymmetric carbon is found,look at the plane of symmetry of thewhole molecule and other atoms, suchas suifur and nitrogen, which also canconfer chirality.

1 1 1 4 LCGCNORTHAMERICA VOtUME JS NUMBER n NOVEMBER 2007

Normal phase Reversed phase

Derivat izat ion

necessary?Analyte ligates

t o Cu(ll}?

pi of acid-base

close t o C*? .

5trong cat ion/

anion nearC*?

Type 1

CSPType 4 Type 5

CSP CSP

Hydrophobic g roup

f i ts cyclodextrin?

Cyclodextrin

CSPOther inclusion

CSP

Figure 1: Flow diagram to select an appropriate CSP (1).

If one asymmetric carbon or scere-ogenic center is found, the investigatorcan expecr two enantiomers. For twosrereogenic centers, the number of enan-tiomers is four. It should be clear thatthis nLimber grows rapidly as the numberof asymmetric centers increases; the 2"rule applies, except for fused rings, where« IS the number of asymmerric centers.

Stereoisomerism: Molecules that arcisomeric but have a different spatialarrangement are called stereoisomers.Symmetry classifies stereoisomers aseither enantiomers, as defmed previotisly,or diastereomers. Sterecisomerisni canresult from a variety of sources besides thesingle chiral carbon (stereogenic or chiralcenter) mentioned previoLisly— that is, achiral atom that is a tetrahedral atomwith four different substituenrs. Detaileddiscussion on these topics can be found inseveral books and review articles (1-8); ashort summary is provided here. It is notnecessary for a molecule to have a chiralcarbon to exist in enantiomcric forms.

but it is necessary that the molecule, as awhole, be chiral. There are two simplemolecular sources of chirality; moleculeshaving a stereogenic center and thosehaving a stereogenic axis. Stereoiso-merism is possible in molecules that haveone or more centers of chirality, helicity,planar—axial—torsional chirality, or copo-logical asymmetry.

The amounts of energy necessary toconvert given stereoisomers into theirisomeric forms can be used for their clas-sification, Scereoisomers with low-energybarriers to this conversion are termedconformational isomers, whereas high-energy-barrier conversions are describedas configurational isomers. Diastere-omers differ in energy content and, thus,in every physical and chemicai property;however, the differences can be sominute as to be nearly indistinguishable.

Stereochemistry and biologicalactivity: The importance of determiningthe stereo isomeric composition of chemi-cal compounds, especially those of phar-

maceutical importance, has been well rec-ognized for some time now (9-12). How-ever, a fairly large number are still used inracemic or diastereomeric forms. It isimportant to remember that some enan-tiomers might exhibit potentially differ-ent pharmacologic activities, and thepatient might be taking a useless or evenundesirable enantiomer when ingesting aracemic mixture. To ensure the safety andefFea of currendy used and newly devel-oping drugs, it is necessary to isolate andexamine both enantiomers separately.Furthermore, it is necessary, in at leastthree situations, to measure and controlthe stereochemical composition of drugs.Each situation presents a specific techni-cal problem during manufacture, whereproblems of preparative scale separationsmight be involved; quality control (orregulatory analysis), where analyticalquestions of purity and stability predom-inate; and metabolic and pharmacologicstudies of plasma disposition and drugefficacy, where ultratrace methods can berequired (1).

Accurate assessment of the isomericpurity of substances is critical since iso-meric impurities can have unwanted tox-icologic, pharmacologic, or orher effects.Such impurities can be carried throughthe synthesis, preferentially react at oneor more steps, and yield an undesirablelevel of another impurity. Frequently,one isomer of a series can produce adesired effect, while another can be inac-tive or even produce some undesiredeffect. Some examples of activity differ-ences are given in Table I. Large differ-ences in activity between stereoisomerspoint out the need to accurately assessisomcric purity of pharmact'uticals.

Regulatory requirements: the Foodand Drug Administration (FDA) of theUnited States issued a set of initial guide-lines in 1987 on the submission of newdrug applications, where the questionsrelating to stereochemistry wereapproached directly in the guidelines onthe manufacture of drug substances(13,14). The finalized guidelines requirea full description of the methods used inthe manufacture of the drug, includingtesting to demonstrate its identity,strength, quality, and puricj'. Therefore,the submissions to the FDA should showthe applicant's knowledge of the molecu-lar structure of the drug substance. For

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(«,/?) Whelk-OICSP (5,S) Whelk-0 1 C5P

(S)

More stable adsorbate Less stable adsorbate

Figure 2: Whelk-0 1 CSP.

.̂ ^r-̂ °v ,0 \^_-r~--.\,^'

HO

HO HOHO

HO HO

Circle 49

Figure 3: Cellulose, a linear polymer.

chiral compounds, this includes identifi-cation of all chirai centers. The enan-tiomer ratio, although 50:50 by the defi-nition for a racemate, should be definedfor any other admixture of srereoisomers.The proof of structure should considerstereochemistry and provide appropriatedescriptions of the molecular structure.An enantiomeric form is considered animpurity, and therefore, it is desirable roexplore potential in vivo differencesbetween these forms.

Separation MethodsApproximarely 40 years ago, systematicresearch was initiated for the design ofchiral stationary phases functioning toseparate enantiomers by GC. This led tomolecular design and preparation of chi-

ral phase systems for LC. Lately, theseefforts have been directed toward findingnew types of chiral stationary and mobilephases on the basis of the stereochemicalviewpoint. Many factors can be responsi-ble for the extent of interactions ofstereoisomeric molecules in any environ-ment such as dipole-dipole interactions,electrostatic forces, hydrogen bonding,hydrophobic bonding, inductive effects,ion-dipole interactions, ligand forma-tion, panition coefficient differences, pA"differences, resonance Interactions-stabi-lization, solubilities, steric interference(size, orientation, and spacing of groups),structural rigidiry-con format ion al flexi-bility, temperature, and van der Waalsforces. The nature and effects of some ofthese factors can influence the chro-

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Table I: Activities of some chirai compounds

Amphetamine

Propoxyphene

Epinephrine

Synephrine

Propanolol

Warfarin

Ascorbic acid

d-lsomer is a potent CNS stimulant; l-isomer has little effect

u-l is antitussive; r«-d is analgesic

l-isomer is 10 times more active as a vasoconstrictor than d-isomer

l-isomer has 60 times the pressor activity of d-isomer

S-(-) isomer has only p-adrenergic blocking activity

S-{-) isomer is 5 times more potent anticoagulant than «-( + ) isomer

d-isomer is anti-ascorbutic; l-isomer is not active

matogniphy of stereoisomers; therefore.they must be carefully reviewed beforedeveloping a separation method { see ftir-rhcr discussion below).

It is well recognized now that HPLCmethods offer distinct advantages overclassic techniques in the separation andanalysis of stereoisomers, especially forenantiomers that are generally muchmore difficult to separate. These meth-ods show promise for moderate-scaleseparations of synthetic intermediates aswell as for final products (15-19). For

large-scale separations and in considera-tion of the cost of plant-scaJe resolutionprocesses, the separation methods offersubstantial increases in efficiency overrecrystallization techniques.

There are basically two approaches to[he separation of an cnantiomer pair byHPLC. In the indirect approach usedrarely, the enantiomers can be convertedinto covalem, diastereomeric compoundsby a reaction with a chiral reagent, andthese diastereomers typically are sepa-rated on a routine, achiral stationary

Progrumide Separation onEpitomize CSPl Chiral

; Tram-

! Oxide

HPLC<:olufnn20 x 2S0 mm

k'l 0.46

1 6 0

"[K-l/k-21 3,5N /m 5'2S5

1.33 Atropine Separation onEpitomize CSPl Chirai

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Circle 51

phase. In the direct approach which isoften called chiral HPLC, the enan-tiomers or their derivatives are passedthrough a column containing a chiralstationary phase (CSP). Chiral HPLCand SFC are Ideally suited for large-scalepreparation ot optical isomers.

Separation of ChiralCompounds by HPLCAs mentioned previously, the chromato-graphic separation of enantioniers can beachieved by various methods; however, itis generally desirable to use some kind ofchiraJ discriminator or selector (20,21).Two different types of selectors can be dis-tinguished: a chirai additive in the mobilephase or a chiral stationary phase. Ofthese approaches, chiral stationary phasesare more commonly used for separationsof enantiomers, and they are discussed atsome length in the following sections.

Mechanism for chiral separations: Todevelop an optimuni method, it isimportant to understand the mechanismof chirai separation. Our understandingof chiral separations with some of thesystems is quite good, while it remainspoor for protein and cellulose stationaryphases. The separation basis with variouschiral stationary phases is discussedbelow in their respective group; somegeneral comments are included here. Anumber of chirai recognition modelshave been proposed to account for opti-cal resolutions by HPLC; these are oftenbased upon the three-point interactionrule advanced by Daigiiesh (22) in 1952.He arrived at this conciu.sion from paperchromatographic studies of certain aro-matic amino acids. He assumed that thehydroxyi groups of the celluiose werehydrogen-bonded to the amino carboxylgroups of the amino acid. A third inter-action was caused, according to theseviews, by the aromatic ring substituents.It ied to the postulation that three simul-taneously operating interactions betweenan enantiomer and the stationary phaseare needed for chiral discrimination.However, this is not always necessary assteric discrimination also could resultfrom steric interactions.

Chiral separations aiso are possiblethrough reversible diastereomeric associa-tion between an enantiomeric solute and achiraJ environment that is introduced intothe column. Because chromatographic res-

1 1 2 0 LCGC NOFfTH AMERICA VOLUME 25 NUMBER n NOVEMBER 2007 www.chromatog raphyo nline.com

Functional group

Yes

1. Chiralcel OB, OD, AD, OJ, MA, WH2. ChiralcelOB, WF, WH3. Chiralcel AD, OB. OD.OJ4. Chiralcel OD. OJ, OC5. Chiralcel OC,OD,OJ6. Chiralcel AD, OD,OJ. OB

7. ChiralcelOB, OJ,OD8. Chiraicel OB, OJ, OD, OT9. Chiralcel OB, OJ, OD, OC, OT, WH10. Chiralcel OD.OJn.ChiralcelOC, OD, AD

Figure 4: Guide to selection of columns, based on functionalities of the solute (1).

olutions are possible under a \^riety of con-ditions, it might be concluded that the nec-essary difference in association can beobtained by many types of molecular inter-actions. The association, which may beexpressed quantitatively as an equilibriumconstant, will be a function of the magni-tudes of the binding as well as the repulsiveinteractions involved. The latter are usuallysteric, although dipoie-^dipole repulsionsalso could occur, whereas various kinds ofbinding interactions can operate. Theseinclude hydrogen bonding, electrostatic

and dipole-dipoie attractions,charge-transfer interaction, and hydropho-bic interaaion (in aqueous systems).

CSPs, where steric fit is of primaryimportance, inciude those based uponinclusion phenomena, such as cyclodex-trin and crown ether phases. It is possibleto construct chiral cavities for the prefer-ential inclusion of only one enantiomer.Moiecuiar imprinting techniques arevery interesting in this respect (1,23).The idea is to create rigid chirai cavitiesin a polymer network in such a way that

only one of two enantiomers wili find theenvironment acceptabie.

A Primer for Selecting aSuitable CSPAs mentioned earlier, it is important tostudy the solute to determine what kindof interactions it can bring about. Thisinformation is very heipful in the selec-tion of the desired CSP. The chromato-graphic analysis of entantiomeric com-pounds can be based upon whether thenormai-phase or reversed-phase mode isdesirabie (1). However, the preferredapproach is to select a CSP based uponvarious considerations discussed in thefoiiowing. A conventional classificationof common chirai stationary phases isprovided in Table IL The table also pro-vides a quick review of various interac-tions on CSPs. Alternatively, the chro-matographic separations of chiraicompounds can be viewed in terms ofnormal or reversed phase; the termsused more commonly by the chro-matographers. A flow diagram (Figure1) is provided to help readers seiect anappropriate CSP.

Brush-type cotumns (Type 1): TheCSP in brush-type or Pirkle coiumns iscomposed of various seiectors capabie ofionic or covaient bonding. These CSPsare generally composed of an opticallypure amino acid bonded to 7-amino-propyl-silanized silica gei. An amide orurea linking of a IT-electron group to theasymmetric center of amino acid pro-vides for TT-electron interactions and onepoint of chiral recognition. It has beenproposed that at least three points ofinteraction are necessary between thechiral molecule and the CSP. One ofthese interactions must be stereochemi-caily dependent. When three points ofinteraction occur, a transient diastere-omeric complex is formed between thesolute and the CSP. The TT acids, 17 bases,hydrogen bond donors and acceptors,amide dipoles, and other functionalgroups linked to a chiral selector of theCSP provide the required three-pointinteraction and confer overall selectivityto the column. Other points that provideattractive or repulsive interactions alsocontribute to enantioselectivity. Thecompounds that can be separated withthese CSPs should• Form hydrogen bonds

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Table II: Conventional classification of chiral stationary phases

Description

Brush-type(donor-acceptors)

Polysaccha rides

Inclusion CSPs, e.g.,cyclodextrins

Examples

DNB-glycine,DNB leucine,

naphthylalanine

Chirakel-OA, -08, -OF,-OJ, etc.

Normal phase(polar modifier)

Normal phase(polar modifier)

Cyclobond 1-3. Chiralpak-

Ligand exchangers

Proteins

Proline, OH-proline

Albumin, glycoprotein

phase, for exam-ple, aqueous acetonitrile,

aqueous methanol

Reversed phase, for exam-ple, aqueous buffers

pie, aqueous buffers

• Have TT-TT interactions• Have dipoie stacking• Have other attractive interactions

If the sample of interest does not con-tain the neceSxSary sites of recognition,the sites have to be added by formingappropriate derivatives — a process nottoo popular with separation scientists.Enantiomeric compounds that can beseparated on these columns are as fol-lows: amines, amino acids, carboxylicacids, esters, ethers, hydroxy acids,ketones, and lactones.

Two types of columns can be used. Forthe older brush-type columns, derivaciza-cion was frequently required. This wascumbersome, and it complicated separa-tions unnecessarily. Columns such as a-Burke columns and Whelk-O 1 that obvi-ate rlie need for derivarization have evolved.

Underivatized separations: An a-Burke II column (Regis Technologies,Morton Grove, Illinois) is useful for res-olution of metoprolol and related com-pounds. A mobile phase composed of85:10:5 methylene chloride-ethanol-methanol containing 10 mM ammo-nium acetate is used at a flow rate of 1mL/min for a 25 cm X 4.6 mm i col-umn. The a value of the enantiomers is1.28. Related compounds such asaiprenolol, atenoloL betaxolol, bufralol,bupranolol. oxprenolol. practolol, pin-dolol, pronethalol, and propanoioi canbe resolved with slight modifications ofmobile phases.

The Whelk-O 1 (Regis Technologies)column originally was developed for sepa-rating the enantiomers of naproxen (Fig-

ure 2). Naproxen can be resolved by usinga normal-phase method with 80:20:0.5hexane-isopropanol-acetic acid on a 25cm X 4.6 mm Whelk-O 1 column at aflow rate of I mL/min in 16 min (5).AJternatively, 60:40 methanoI-0,1%phosphate can be used at the same flowrate with the same run time. The a valueis 2.1 in the normal-phase mode and 1.7in the reversed-phase mode. Other aryl-propionic acids chat have been resolved inthe normal-phase mode are ibuprofen,keroprofen, and flubiprofen. VariousPharmaceuticals that have been resolvedon this column are cyclothjazide, ben-droflumethiazide, oxazepam, mepheny-toin, bupivaciine, and/i-chloro-warfarin.

An improved version of this column,Whelk-O 2, has recendy been designed toimprove the resistance of stationary phaseto hydrolysis, while using scrong organicmodifiers such as trifluoroacetic acid.

Polysaccharide columns (Type 2): Asmentioned previtiusly. [he resolving capac-ity of polysaccharides such as cellulose wasfirst realized by Dalgliesh with the observa-tion that a racemic amino acid could occa-sionally give two spots in paper chro-matography. Optical resolution of aminoacids is possible on cellulose by TLC aswell. This has led to the use of celluloseand cellulose derivatives, as well as amy-lose, for chiral separations by liquid chro-matography. Cellulose, a linear polymer,has rhe chemical constitution of a linearpoly-P-d-1,4-glucoside (see Figure 3).

Cellulose forms long chains of at least1500 (+ )-d-glucQse units per molecule.The molecular weight of cellulose ranges

from 2.5 X 105 to 1 X lOf*Each of the the (+)-d-glucose repetitiveunit contains five chiral centers and threehydroxyl groups. All the ring sub-sriruents are equatorial.

Broadly substituted cellulose columnscan be divided into two major cate-gories: cellulose esters and cellulose car-bamates. The popular phases (ChiralpakAD, Chiralpak AS, Chiralcel OD, Chi-calcel OJ, Chiralcel OB, and ChiralcelOC, Chiral Technologies, Exton, Penn-sylvania) are now available on 5-fJL,m sil-ica. These columns have found a widerange of applicability. Figure 4 providesa guide to selection of a variety ofcolumns, based upon fiinctionaliries ofthe solute. The compounds are dividedinto four major categories based uponwhether they contain a given functional-ity, for example, aromatic carbonyl, ter-tiary nitrogen, or hydroxyl.

Aromatic compounds containing car-bonyl groups and no hydroxyls or terti-ary nitrogen are more readily separatedon Chiracel Oj, OD, and OC columns,based upon practical experience with alarge number of compounds. If they wereto contain a hydroxyl group as well andno tertiary nitrogen, the choice would beincreased to four columns, wbere Chira-cel OB and OT replace OD and OC. If,on rhe other hand, tertiary nitrogen alsowere present, che choice would again benarrowed co three columns, where Chira-cel OJ is replaced by AD. It should bepointed out that information on selec-tion of columns on this basis is some-what empirical, though based upon afairly large base of practical data.

The coated polysaccharide CSPs arelimited somewhat in regard ro rhe sol-vents that can be used in the mobilephase and sample diluents. Newimmobilized CSPs (Chiralpak IA, Chi-ralpak IB, and Chiralpak IC) were cre-ated to be more stable to a broad rangeof mobile phase diluents, as well as toelevated temperatures.

Inclusion CSPs (Type 3): Chiral dis-crimination on a stationary phase can beachieved by creation of chiral cavities, inwhich stereoselective guest-host interac-tions influence rhe resolution. Includedin this group are cyclodextrins, crownethers, polyacrylates, and polyacry-lamides. The applicability of thisapproach has been extended by utilizing

1 1 2 4 LCGC NORTH AMERICA VOLUME 25 NUMBER ] 1 NOVEMBER 2007 www. chrorr^atographyonline.cow

Table III: Comparison of various CSPs (1)

Bru5h-type

Resolves enantiomers

High efficiency +4-

Coiumn stability

Mobile phase compatibility + +

Inversion of elution order Yes

High capacity + +

Analysis time +

No No No NoHammMBOBSK

Yes Yes

macrocyciic antibiotics as chiral station-ary phases instead of cyclodextrins byArmstrong and colleagues (8-10).

Cyclodextrin ColumnsCyclodextnns are produced by the partialdegradation of starch and the enzymaticcoupling of cleaved glucose units intocrystalline, homogeneous toroidal struc-tures of different molecular size. Alpha-,P-, and 7-cyclodextrins have been mostwidely characterized; chey contain 6, 7,and 8 glucose units, respectively, and arechiral. Por example, p-cyclodextrin has 35stereogenic centers, and the toroidal struc-ture has a hydrophilic surface resultingfrom the 2-, 3-, and 6-position hydroxyigroups making them water-soluble.

The cavity in cyclodextrins is composedof the giucoside oxygen and methyienehydrogen, giving it an apolar character. Asa result, cyclodextrins can include polarmolecules of appropriate dimensions intheir cavities and bind them throughdipole-dipoie interactions, hydrogenbonding, or London dispersion forces. Ingeneral, cydodextrins are stable from pH 3to 14. Listed in the foiiowing section is agenerai plan that can be utiiized for makinginitial choices. [i-Cyclodcxtrin has beenused in the largest number of applicationsbecause it has been fotmd tiseftil for low-molecular weight analytes in the pharma-ceutical and environmental areas. Thesecoiumns can be operated in the foiiowingmodes: reversed phase or normal phase.

To achieve a chiral separation in thereversed-phase mode, it is essential that theanalyte have at least one aromatic ring.Exceptions are heterocyciic anaiytes and t-boc amino acids. For cyclodextrin inclu-sion, the molecular weight of rhe polyaro-

matic ring structure is not as critical as itsbulk. The most important consideration isproper fit of the molecule in the cyclodex-trin cavity. This fit is a function of both sizeand shape of the anaiyte reiative to tiie cav-ity. For example, an anaiyte like norgestrel,a five-ring steroid structure, is better sepa-rated on a 7-cyclodextrin column, whilethe enantiomer of a naphthalene-like struc-ture or single substituted aromatic ringwould fit better on a P-cyclodextrin col-umn. For chiral recognition, solventstrength is independent in most casesbecause it affects only displacement of theanaiyte from the cavity (1).

Normai-phase separations on cyciodex-trin columns have generally been carriedout with mobile phases such as hexane-Jso-propanol, acetonitriie-methanol,methanot, or ethanol. TT-TT hydrogen bond-ing forces primarily influence enantiomericseparations. It is possible to override inclu-sion complexation in favor of interactingdirectly with secondary hydroxyi groupsacross the lai^er opening of a cyclodextrintoroid or the appendant carbamate, acetate,or hydroxypropyl lunctionai groups, p-Blockers such as propanolol, timoiol, andatenolol, and compounds iike warfarin canbe separated.

Bonded DerivatizedCyclodextrinsA number of ironded cyclodextrins are cur-rently avaiiabie. The carbamate coupling ofthe TT bases, 1-naphthyiethyi to a bondedcyclodextrin, creates a compiex environ-ment that has demonstrated diverse chiralseparations. It has been labeled as multi-modal chiral stationary phase because it canbe used in a normal- as well as a reversed-phase mode with appropriate modifiers.

Anaiyte structure, solubility, and stabilitydictate the proper selection of the mobilephase. For example, if the analyte is TTacidic, normai-phase soivetits can be used.If the anaiyte is not -FT acidic but containstwo hydrogen bonding groups, one on ornear the stereogenic center, polar modifiersmust be used. Of the three carbamatesavailable, the ^-naphthylethyt carbamatehas shown the greatest selectivity and versa-tility. Because the naphthylethyl carbamatedoes play a role in enantioselectivity, the Rform can be usefiii if the separation doesnot occur on the S form. A variety of com-pounds such as catelol, iabetolol, nadoiol,metoproiol, pindoiol, propanoioi, timoiol,oxazepam., suprofen, comuachlor. andwarfarin have been resolved on cyciodex-tnn columns (1).

Macrocyciic AntibioticsThe chiral seiectors in this group indudevancomycin, ristocetin A, teicoplanin,avoparcin, rifamycin B, and thiostrepon.Chirobiotic V is based upon covaientiybonding the amphoteric giycopeptidevajicomycin to 5-mm silica gei. These Hg-ands are iinked to assure their stabilitywhile retaining essential components forchiral interactions. For exampie, van-comycin contains 18 chirai centers sur-rounding three pockets or cavities. Fivearomatic ring structures bridge these cavi-ties. Hydrogen donor and acceptor sitesare readily available close to the ring struc-tures. It has been ciaimed that the selectiv-ity of this coiumti is similar to a-1 acidglycoprotein (AGP), and it is stable when0- 100% organic modifier is used.

Broad selectivity has been demonstratedby vancomycin in both reversed-phase andnormai-phase solvents, and limited selec-

1 1 2 6 LCGC NORTH AMERICA VOLUME 25 NUMBER11 NOVEMBER 2007 www. chromatographyonline.com

Warfarin

0. . 0 0

Chirobiotic T Chirobiotic V

Figure 5: Comparison of separations of warfarin on Chirobiotic T and V (1).

tivity with polar organic mobiie phases.Since vancomycin contains peptide, carbo-hydrate, and other ionizable groups, itwould be expected to offer different selec-tivity in these modes. The structure of van-comycin indicates that ail the typlcai inter-aaions outlined for protein phases andother celiuiosic polymer type phases arepossible with this phase. The potentialinteractions and their relative strengths areas foiiows: TT-TT interactions —- very strong;inclusion -— weak becatise of shallow pock-ets; dipoie stacking — medium to strong;steric interactions — weak; and anionic orcationic binding — strong.

Compared with cyclodextrins, the shal-low pockets for inclusion yield weakerenergies. This leads to faster kinetics,which can in turn lead to faster separa-tions. Reversed-phase conditions favorinclusion and hydrogen bonding. Underthese conditions, changes in pPi can pro-duce cationic or anionic interactions.Dipoie stacking and TT-IT compiexationare favored in normal-phase solvents. Polar

ot^anic mobiie phases enhance the poten-tial for ail of the previous interactions.Anaiytes such as acids, amides, esters, andneutral compounds can be resolved.

Chirobiotic T (Astec, Whippany, NewJersey) is based upon bonding the ampho-teric glycoside teicoplanin to a 5-mm sil-ica gel through covaient linkage,Teicoplanin contains 20 chiral centers sur-rounding four pockets or cavities. Hydro-gen donor and acceptor sites are readilyavailable dose to seven aromatic rings.

Separations of warfarin enantiomers onChirobiotic T and V columns is shown inFigure 5 with the same mobile phase(10:90 acetonitile-I% triethylammo-nium acetate, pH 4.1). Much better reso-lution is observed with Chirobiotic V.

Recently Chirobiotic V2 and T2 havebeen created to produce higher selectivityand higher capacity.

Selection of mobile phases: The mobilephase functions equally in reversed-phase ornormal-phase solvents because of the com-plex structure of the macrolide and ioniz-

abie groups. In the reversed-phase mode,optimization is accomplished by controllingthe type and amount of organic modifier,type of buffer, and pH. Efficiency and selec-tivity are affected by ionic strength, buffertype, flow rate, and temperature. Of thevarious organic modifiers, tetrahydrofurangives greater selectivity and efficiency, lypi-cai composition of organic modifier-buffer(pH 4.0 to 7.0) is 10:90. Alcohols as modi-fiers generally ret]uire higher concentration,for example, 20% for comparable retentionto acetonitrile or tetrahydrofuran. Ammo-nium nitrate and triethylamine acetatebuffers have been found useful. In generai.anaiytes act more favorably at a pH wherethey are not ionized. Lower column tem-peratures are favored because of enhance-ment of weaker bonding forces.

In normal-phase separations, peak effi-ciency and resolution can be improvedwith ethanol as the polar niodifiet ofhexane. A good starting point might be20% ethanol. In most cases, ethanolworks better than isopropanol.

Applications: A wide range of aminoacid derivatives have been resolved onthese columns. Anaiytes sucb as neutralmolecules, amides, acids, esters, and cyclicamines show considerable enantioselectiv-ity. Other amines have been separated withvarying degrees of success. Benoxaprofen,ibuprofen, fenoterol, mephobarbital,naproxen, warfarin, albuteroi, citulline,DOPA (3,4-dihydroxy-phenylaianine),and phenylaianine have been resolved onchirobiotic columns (1).

Ligand exchange (Type 4): Davankovand Kurganov (]6) were the first to indi-cate that cross-linked resins with fixed lig-ands, (7?)-N,N'-dibenzyi-l,2-propanedi-amine in the form of copper(II) complexes,display iiigh enantioselectivity for alanine,serine, and leucine. Various amino acids,including baclofen (1), can be resolved ona reversed-phase C18 column with a ciiiralmobile phase of aqueous cupric acetate andN,N-di-n-propyl-l-alanine (DPA) contain-ing 1^% acetonitrile (17,18). Cationexchange chromatography can then beused to break the Cu-DPA-badofen com-plex on a Dowex-50W column (DowChemicai, Midland, Michigan) to yieidsmaii quantities of the optical isomers formechanistic studies. A number of ligandexchangers are sold by Daicel (Chirai Tech-noiogies), including Chiralpak WH, Chi-ralpak WM, and Chiralpak WE. These

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columns are useful for resolution of aminoacids and their derivatives (15). The mobilephase is simply aqueous copper sulfate.C^hiralpak Ma ( + ) is another Iigand-excliange colunm that is useRil for hydrox-ycarboxylic acids.

Investigations have revealed that a ligandfxchangc column can be a simpler andmore useful approach for separating enan-tiomers of baclofen (1), a drug similar tophcnyliilanine. The selection of ChiralpakWH column, a column designed to serveas a ligand exchanger (19), considerably•shortened the method development time.Icis fairly well known chat the separation ofamino acids is significandy affected by theniolarity of CuSOj, used for the separation.However, the effea of organic solvents suchas methanol on the retention of free aminoacids is not well known, except for valine,which shows a decrease in retention timewith an increase in methanol concentrationup to 20%, Temperature has a significanteffect on retention — for example, theretention time of phenylaJanine decreasessignificantly with increasing temperature.These considerations led to an optimalmobile phase (containing 0.25 mM copperstilfate, run at a flow rate of 1.5 mlVmin atSO "C) for the resolution of d- and l-fbrmsof haclofen from the racemate (1).

Protein phases (Type 5): Chiral AGP(C'hromlech, AB, Norsborg, Sweden) is asecond-generation chiral selector that ishased upon the al-acid glycoprotein as thechiral stationary phase. The process ofimmobilizing AGP on porous spherical 5-nim siiica has been patented. This CSP hasbeen found useful for resolving a broadrange of compounds such as racemicumines, acids, and nonprotolytic com-pounds without derivatization. A numberol examples of drugs such as aiprenolol,arenolol, bupivicane, chlorthalidone,disopyraniide, ephedrine, ethotoin,telodipinc, fcnoprofen, hexobarbital. meto-prolol, pheniramine, and verapamil havebeen resolved on Chirai AGP (I). The res-olution ability of this colutnn is due to theunique nature of the chiral stationary phaseand the fact that enantioselectivity . It isgenerally recognized that the greatest selec-tivity can be induced by choosing propermobile phase composition. For bioanalyti-cal work, this CSP h;is been recommendedhighly. The typical mobile phase for thiscolumn is phosphate buffer with anorganic rnodifler. Enantioselectivity and

retention can be regulated by changing themobile phase composition with respect toany of the following parameters: pH,organic modifier, modifier concentration.For the types of variotis modifiers that havebeen tiscd and their respective concentra-tions, sec Table 10.6 in reference 1. Typicaloperating conditions entail the use of 10mM buffer at pH 7.0, with or without anorganic modifier. Modifier concentrationsas high as 15% isopropanol or 10% ace-tonitriie have been used. The pH can aifectthe resolution of different analytes. A num-ber of other protein-ba.sed columns are alsoavailable for certain applications. It shouldbe noted that the protein columns tend tobe less stable and have low capacity.

Fable III can offer some help in selec-tion of a suitable CSP on the basis of re.s-olution, efficiency, capacity, column sta-bility, mobile phase compatibility, andanalysis time.

A Fast Approach to CSPSelectionA significan savings in time can be madeby use of a chiral database searchable thatis based upon molecular structures. Chris-tian Roussel and others (24) have providedCHIRBASE, a molecular-oriented factualdatabase that includes tens of diousands ofentries, and the entire system can besearched by ISIS software (ISIS Software.Inc., Richmond, Virginia). The list pro-vides comprehensive structural, experi-mental, and bibliographic information onsuccessful and unsuccessful chiral separa-tions (25). Assistance can be sought fromthe major column manufacturers (forexample, Astec/Supelco, Chiral Technolo-gies, Regis Technologies, and ChromTechAB, Norsborg, Sweden) who offer practi-cal background information on theircolumns as well as technical assistance indeveloping analytical and/or preparativeenantiomeric separations.

With over 170 CSPs commercially avail-able, reliable and rapid enantioselectivityprediction of a new chiral molecule withany CSP still remains elusive. Of the com-mercially available CSPs, the polysaccha-rides and macroc)clic glycopeptides havebeen favored by various groups for thescreening .strategies in HPLC. Two screen-ing strategies have proposed by PerHn andcolleagues (26,27) for the enantiometicseparation of drugs using polysaccharidecolumns in the isocratic normal- and

reversed-phase modes. In both modes,three complementary columns are usedthat have broad enantiorecognition abilitiesfor a wide range of pharmaceutical com-pounds. These columns are the ChiralcelC)D-H, Chiralpak AD and Chiralcel OJfor the normal phase. The compounds are.•screened on each column iLsing two mobilephases containing 90:10 (v/v) of «-hexane-2-propanol or w-hexane-ethanol at a flow rate of 1 mL/min. Diethy!amine (0.1% v/v) is added to the mobilephase for the analysis of basic compounds;however, for the acidic compounds 0. 1 %(v/v) trifluoroacetic acid is used instead.This strategy was applied to a set of 36drugs. The study also has shown that forbasic compounds, the screening on theChiralcel OD-H and the Chiralpak ADcolumns is usually sufficient to achieve theseparation. Resoltition of the enantiomerswas observed for 32 compounds on at leastone column. Short analysis times (that is,20 min or less) were usually achieved.

In the reversed-phase strategy, the threecolumns (Chiralcel OD-RH, ChiralpakAD-R and Chiralcel OJ-R) are used withtwo mobiles phases. The compounds arefirst analyzed with a mobile phase consist-ing of an aqueous phosphate buffer, pH2.0, containing 100 mM potassium hexa-fluorophospbate, mixed with acetonitrile(60t40 v/v). The chaotropic agent hexa-fluorophosphate is added to the mobilephase to achieve the separation of basicanalytes at low pH. According to Perrinand colleagues (27), most enantiomerscan be separated with this approach. If noor very litde enantioselectivity is achievedfor some components, all three columnsare investigated with a basic mobile phaseconsisting of an aqueous 20 mM boratebuffer, pH 9.0, mixed with acetonitrile(60:40 v/v) at a flow rate of 0.5 mL/min.Enantioselective separation is achieved onat least one of the columns for 89 % Oiout of 37) of the drugs analyzed. Analysistimes are usually less than 30 min.

Another screening strategy using thesame type of columns but with normal-phase gradient elution h;is been pr()po.sed(28). Each compound is analyzed on fourcolumns: Chiralcel OD-H, Chiralpak AD,Chiralpak AS. and Chiralcel OJ. «-Hexane-2--propanol and an n-hexane^-ethanol gradient elution system areused to screen the compounds except forthe Chiralpak Al^ column, where only the

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former system is used. To speed-up theanalysis, a column-switching device is used.The gradien: is run from 20% to 70%alcohol in 20 min with a flow rate of 0.75mL/min. Baseline resolution is achievedwith this strategy for 85% of the substancestested (more than 800 compounds havebeen investigated). When rhe baseline sep-aration is not achieved, isocratic optimiza-tion of the separations is necessary.

Four polysaccharide-based CSPs andthree macrocyclic glycopeptide based CSPshave been evaluated for rapid screening forof over 55 chiral compounds of pharma-ceutical interest (29). The polysaccharidecolumns are employed in the normal phaseand polar organic modes and showed over-all enantioselectivity for 87% of the com-pounds tested. The macrocyclic glycopep-tide columns are employed in thereversed-phase and polar organic modeand showed enantioselectivity for 65% ofthe analytes. Wlien the results from boththe polysaccharide and the macrocyclicglycopeptide screen are combined, theyshow enantioselectivity for 53 out of 55enantiomeric pairs (96%). This shows thatthe two screens are complementary — thatis, both types of columns should beincluded to achieve the highest probabilityof success. The screens can be automatedusing a column switcher that allows fordifferent combinations of CSP and mobilephase to be tested overnight. It is claimedthat the method development can be com-pleted within 24 h for a given compound.The column coupling approach has beenapplied on macrocyclic glycopepcide CSPs.It dlows evaluation of this entire class ofchimi selectors with a single coupled col-umn for the ability to separate a molecule.Even if a partial separation is obtained onthe coupled column, a baseline separationis potentially possible with one of thecolumns in this class (30).

A parallel multicolumn screeningapproach has been published by Zhangand colleagues (31). The modifiedHPLC system allows simultaneousscreen of five CSPs in parallel using a reg-ular HPLC autosampier and a pumpwith five UV detectors.

Future chiral screening program willfocus on various separation techniquesbesides HPLC (32). These include SFC,capillary electrophoresis (CE), and GC.This could lead to a unified strategy forchiral method development screening.

Summary and ConclusionsThe selection of an appropriate column(CSP) is the most important step inmethod development of chiral com-pounds. Selectivity, mode of operation,compatibility, robustness, efficiency, load-ability, and reproducibility also need to beconsidered. The trial-and-error approachgenerally used in chira! separations can beextremely time-consuming. Some of thepreviously mentioned books can be helpfulin column selection. Most chiral separa-tions can be achieved on two types of CSPs:polysaccharide-based and macrocyclic gly-copeptides. These columns can be operatedin the reversed-phase, normal-phase, andpolar organic modes with complementaryenantioseiectivities. The chiral databases aswell as the column screening approach dis-cussed previously can shorten the selectionprocess. Improved columns need to bedesigned based upon careful evaluations ofmolecular architechure of CSP as it canprovide clues relating to which portion oftheir structure offers the desired selectivity.

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