optimization of normal-phase chromatographic...

Journal of Chromatography A, 1194 (2008) 80–89

Contents lists available at ScienceDirect

Journal of Chromatography A

journa l homepage: www.e lsev ier .com/ locate /chroma

Optimization of normal-phase chromatographic separation of compounds withprimary, secondary and tertiary amino groups

M. Kagana,∗, M. Chlenova, S. Melnikova, A. Greenfieldb, J. Grossb, R.C. Bernotasc

a Discovery Analytical Chemistry, Chemical Technologies, Wyeth Research, CN 8000, Princeton, NJ 08543, USAb Chemical and Screening Sciences, Wyeth Research, CN 8000, Princeton, NJ 08543, USA
c Chemical and Screening Sciences, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, USA
a r t i c l e i n f o a b s t r a c t

f primcyantaminane wminimine atethyls varchroines olterna

Article history:Received 13 February 2008Received in revised form 7 April 2008Accepted 10 April 2008Available online 24 April 2008

Keywords:BenzylaminesAnilinesEphedrinesTryptaminesSerotonin receptor ligandsSeparationDiolCyanoStationary phaseHPLCPreparative

The retention behavior oHPLC on silica, diol, andanilines, ephedrines, trypand ethoxynonafluorobutand low plate count wereadditive was n-propylamN-N-methyl, and N,N-dimamines on cyano columnpreparative normal-phasesecondary and tertiary ammay provide a practical acandidates.

Normal-phase optimizationAmine modifierEthoxynonafluorobutane

1. Introduction

There are a wide variety of synthetic methods for introducing anamine function into organic molecules. Examples of these methodsare direct amination of organic halides and sulfonates, acylationof amines to form amides followed by reduction to more substi-tuted amines, and more recent mild reductive amination protocols[1–3]. However, many of these approaches are not selective enoughand often lead to complex mixtures of primary, secondary, and ter-tiary amines. The need to test pure compounds in biological assaysrequires the separation of such mixtures into their individual com-ponents.

As the large number of existing drugs and pharmaceuticallyrelevant compounds are of basic nature, their analysis and purifi-cation of drug candidates and their synthetic intermediates with

∗ Corresponding author. Tel.: +1 732 274 4735.E-mail address: [email protected] (M. Kagan).

0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.chroma.2008.04.052

ary, secondary and tertiary amines was studied using normal-phase-o stationary phases. Several classes of amines, including benzylamines,es, and azatryptamines were chromatographed using mixtures of hexaneith methylene chloride and methanol. Peak tailing, diminished selectivityized by the addition of volatile amines to the mobile phase. The optimal

0.1% concentration. On diol columns, the elution order of free primary,amines was predictable, while the elution order of primary and secondaryied depending on the alcohol modifier concentration. The feasibility ofmatography was demonstrated by the separation of a mixture of primary,btained by direct methylation of norephedrine. The procedures describedtive to traditional methods of analysis and purification of potential drug

© 2008 Elsevier B.V. All rights reserved.

primary, secondary and tertiary amino groups still represents achallenge to medicinal chemists. Traditional analytical method-ologies such as silica gel chromatography and reversed-phase(RP) HPLC sometimes suffer from poor peak shape, insufficientselectivity and inadequate retention control for basic compounds.Reduced solubility of organic bases in aqueous mobile phasesand tedious compound recovery are other potential drawbacksof the RP approach to separation and purification of such com-pounds.

Several chromatographic methods for the separation of primary,secondary and tertiary amines have been developed. RP HPLC inacidic aqueous organic mixtures on hydrophobic columns is byfar the most popular technique [4–9]. It has long been recognized[10] that the chromatographic performance of these systems forbasic solutes is diminished because of the interactions of positivelycharged amines with the silica matrix of the column leading totailing of chromatographic peaks, poor selectivity and difficulty inthe control of retention. These problems can sometimes be over-come by the addition of amine additives [11], the use of ion-pairing

http://www.sciencedirect.com/science/journal/00219673

mailto:[email protected]

dx.doi.org/10.1016/j.chroma.2008.04.052

M. Kagan et al. / J. Chromatogr. A 1194 (2008) 80–89 81

Fig. 1. Structures of amine additives NPA,

reagents [12,13] and by application of high pH-tolerant RP pack-ing materials for the separation of amines in their free-base form[14–19]. Such measures are reasonably successful for analyticalmethods, but their practical value for purification of basic com-pounds on a semi-preparative or preparative scale may be limited.

Alternatively, normal-phase (NP) methods can be applied tochromatography of amines on unmodified silica gel. However,basic compounds in general, and amines in particular, are wellknown to show broad tailing on silica as it has a non-homogeneous

Fig. 2. Structures of co

DEA and TEA and compounds 1–17.

adsorbing surface that contains silanol groups and a variety ofother active chemical centers [20]. Problems with peak tailingcan be partially solved, especially for secondary and tertiaryamines, by adding amine additives to the mobile phase [21,22].Hydrophilic interaction chromatography (HILIC) employs bufferedaqueous acetonitrile and methanol eluents on unmodified silicacolumns at neutral [23] or basic pH [24–26] often resulting in highefficiencies and good selectivities of the solutes. Unfortunately,the use of such mobile phases at higher pH can lead to col-

mpounds 18–20.

82 M. Kagan et al. / J. Chromatog

umn degradation [27,28] and may not be practical for preparativepurposes.

Derivatization of silica with cyanopropyl, propylamino and diolgroups produces cyano, amino and diol polar bonded packing mate-rials that have been successfully used for HPLC of amines either inRP [29] or NP mode [30–32]. The presence of an amine additiveappears to be essential to obtain good chromatographic character-istics for basic solutes [33].

Over the last several years we have investigated the chromato-graphic behavior of a broad range of commercially available andin-house synthesized amines using cyanopropyl HPLC columns.These experiments were conducted with gradients of polarorganic solvents in hexane or other nonpolar solvents suchas ethoxynonafluorobutane (ENFB) [34,35]. This environmentallyfriendly solvent is completely miscible with conventional HPLCsolvents including methanol, and has the additional advantageof being compatible with powerful analytical techniques such asLC–MS [36,37].

Table 1Retention factor (k), resolution (Rs) and number of plates (Nt.p.) of the solutes with prinormal-phase conditions

ENFB–MeOH/NPA

DIOL CN

k Rs Nt.p. k Rs Nt.p.

Benzylamines 20% MeOH1a N(CH3)2 0.5 3000 0.7 39002 NCH3 1.5 5.2 2210 2.2 6.2 19003 NH2 2.7 3.4 1600 2.6 1.3 4200

ENFB–MeOH/NPA

DIOL CN


Anilines 5% MeOH9 N(CH3)2 0.3 3900 0.4 390010 NCH3 1.4 9.2 6700 1.1 5.4 441011 NH2 3.8 11.5 7100 2.4 6.8 5900

ENFB–MeOH/NPA

DIOL CN


Ephedrines 10% MeOH12 N(CH3)2 2.3 1500 1.5 46013 NCH3 5.3 4.9 1400 6.9 2.9b 103014 NH2 7.3 2.5 2050 4.2 4.8c 1500

ENFB–MeOH/NPA

DIOL CN


Tryptamines 50% MeOH 60% MeOH15 N(CH3)2 0.7 910 1.8 198016 NCH3 3.4 5.1 680 6.4 6.1b 199017 NH2 4.1 0.7 510 4.5 2.8c 1930

ENFB–MeOH/NPA

DIOL CN

k Rs Nt.p. k Rs Nt.p

5-HT6 modulators 20% MeOH18 N(CH3)2 2.1 3800 1.8 35019 NCH3 4.1 6.1 3000 4.2 7.6 37020 NH2 6.3 3.4 1360 4.7 1.2 420

a Compound No. in Figs. 1 and 2.b Rs between first- and second- and second- and third-eluted peaks, respectively.c Rs between first- and second- and second- and third-eluted peaks, respectively.

r. A 1194 (2008) 80–89

The purpose of this study was to evaluate the optimal columnpacking materials and mobile phases in NP mode of chromatog-raphy for HPLC separation of mixtures of compounds containingprimary, secondary and tertiary amines. Our goals were to developpractical recommendations for medicinal chemists to analyze com-plex synthetic mixtures by LC and LC–MS and to provide NPpreparative HPLC alternatives to existing purification technologiesfor basic amines, such as flash silica gel column chromatographyand preparative RP HPLC.

Initial studies investigated in detail the chromatographicbehavior of a mixture of benzylamine and the corresponding N-methylated derivatives (structures 1–3, Fig. 1) employing silica,cyanopropyl and diol columns and using mixtures of methanol inENFB or methanol and methylene chloride in hexane, both withor without basic additives such as triethylamine, diethylamine orn-propylamine (TEA, DEA and NPA, Fig. 1).

The state of N-alkyl substitution of the physiologically activecompounds containing an amino group is important for their bio-

mary, secondary and tertiary amino groups on the DIOL and CN columns under

Hexane–CH2Cl2–MeOH/NPA

DIOL CN


10% B in A0.4 2930 0.8 57701.8 6.7 2120 3.3 7.4 13003.9 5.2 2100 4.1 1.1 750


DIOL CN


10% B in A0.2 4550 0.3 57000.8 6.6 6010 0.7 4.3 55002.4 10.6 7000 1.4 5.9 6600


DIOL CN


50% B in A0.7 5700 2.6 28502.6 9.0 2850 5.9 5.2b 22504.4 5.2 4700 4.4 2.9c 4700


DIOL CN


50% B in A 80% B in A0.6 1330 1.2 38003.2 5.4 700 4.1 3.6b 20504.5 1.6 750 2.1 4.6c 2000


DIOL CN

. k Rs Nt.p. k Rs Nt.p.

30% B in A0 0.6 3600 0.8 44000 1.5 5.5 4000 2.0 7.0 41000 2.9 6.1 4200 2.5 2.1 2600


Fig. 3. HPLC of benzylamines (1–3) on the SIL column in 20% MeOH in ENFB in thepresence of amine additives: trace (a) no additive, (b) with TEA, (c) with DEA, (d)with NPA. Here and in figures 4–6 and 7–15 numbers at the top of chromatographicpeaks refer to solutes’ structures in Figs. 1 and 2 and * represents an impurity.

logical activity [38]. Therefore, our investigation was expandedto mixtures of more complex molecules incorporating primaryamines along with their N-methyl and N,N-dimethyl analogs. Thesemixtures included structurally diverse benzylamines (4–8), aro-matic amines (9–11), pharmacologically active amines such as
ephedrines (12–14) and tryptamines (15–17), and several com-pounds generated previously in the course of Wyeth researchprograms (18–20, Fig. 2) as serotonin receptor modulators.
2. Experimental

2.1. Instrumentation, solvents, chemicals and model compounds

An Agilent 1100 analytical HPLC system equipped with a G1361binary pump, a G1367A autosampler, a G1316A heated columncompartment and a G1315B diode array detector (DAD) with10-mm flow path (volume 13 �l) was used for analytical HPLCexperiments. Preparative HPLC was carried out on Agilent 1200preparative HPLC system comprising two G1361A isocratic pumps,G2260A autosampler, G1358B DAD (flow path 0.3 mm) and G1364Cfraction collector. Both instrument control and data acquisitionwere done using ChemStation software.

All commercially available compounds (1–17, Fig. 1) and chem-icals were purchased from Sigma–Aldrich (St. Louis, MO, USA).In-house synthesized substances (18–20, Fig. 2) were obtained fromWyeth’s compound bank.

Fig. 4. HPLC of benzylamines (1–3) on the CN column. Conditions as in Fig. 3.

HPLC grade water, methanol (MeOH), acetonitrile (ACN), hexaneand methylene chloride were purchased from EM Science (Gibb-stown, NJ, USA).

Ethoxynonafluorobutane (ENFB) was obtained as 3 M NovecTM

Engineered Fluid HFE-7200 from 3 M Company (St. Paul, MN, USA)[34].

2.2. Normal-phase columns and mobile phases

NP chromatographic experiments were carried out on Kro-masil SIL, Kromasil CN and Kromasil DIOL 0.2 cm × 15 cm columns(referred to as SIL, CN and DIOL columns below) packed with 5 �mparticles (Eka Chemicals, Dobbs Ferry, NY) and operated at 0.4 and0.5 ml/min flow rate and 30 ◦C. All columns were new and exhib-ited performance of 10,000–12,000 theoretical plates for the testcompounds. Their void volume was estimated from manufacturer-supplied test chromatograms.

Before a particular set of experiments, each column was exten-sively washed with 40 column volumes of MeOH and equilibratedwith 40 column volumes of the corresponding mobile phase.

Hexane-based mobile phases were prepared by mixing hexane(solvent A) with a mixture of methylene chloride and MeOH (8:2,v/v, solvent B). Both solvents A and B contained 0.1% (v/v) of amineadditive.

Mobile phases containing 0.01, 0.05, 0.1, 0.2, 0.5 and 1% of NPA ina solution of 1% MeOH in ENFB were prepared manually to study theeffect of increased concentrations of the basic additive on retentionand peak shape of solutes 1–3.

84 M. Kagan et al. / J. Chromatogr. A 1194 (2008) 80–89

Fig. 5. HPLC of benzylamines (1–3) on the DIOL column. Conditions as in Fig. 3.

A preparative Kromasil DIOL column (2 cm × 25 cm, 5 mkm par-ticles) operated at 20 ml/min at room temperature was used forisolation and purification of the products generated by directmethylation of primary amine 14.

2.3. Calculation of chromatographic parameters

Measurements of retention time and peak widths at half-heightswere done manually. Column efficiency values for solutes (Nt.p.)were determined using the formula N = 5.54 [tR/w0.5]2 ([39], p.42). Retention coefficients (k), and resolution (Rs) were calculatedas in ([39], p. 27 and 29, respectively). Results of the calculationsare presented in Table 1.

2.4. Direct methylation of N-desmethylephedrine 14

The direct methylation of primary amine 14 (∼50 mg of thefree-base) was carried out with 1.3 equiv. of methyl iodide in aminimal amount of glyme (1–2 ml) at 60 ◦C for 48 h. The reactionmixture was concentrated under vacuum, dissolved in a mini-mal amount of methylene chloride (0.2–0.4 ml) and purified ona preparative Kromasil DIOL column using a linear gradient ofsolvent B in solvent A (see Section 2.2) for 15 min at 20 mL/min.The structures of the products isolated were confirmed by directcomparison of the retention times with commercially availablecompounds (in case of ephedrines) and by analytical LC–MS (asin Ref. [36]).

Fig. 6. Normal-phase HPLC of mono-N-alkyl-benzylamines 4, 5 and 2 in 1% MeOHin ENFB on the DIOL column (a) and substituted methylbenzylamines 6–8 on the CNcolumn (b).

3. Results and discussion

3.1. Normal-phase HPLC of benzylamine, N-methylbenzylamineand N,N-dimethylbenzylamine on silica and bonded polarstationary phases

It has been long recognized that silica, the chromatographic sup-port of many types of modern HPLC columns, is poorly suited forthe separation of basic organic molecules [20]. The presence ofsilanol groups of various chemical natures and adsorption energiesappears to be the main reason for broad, tailing peaks frequentlyproduced by basic compounds [20,40] resulting from non-linearadsorption isotherms. Columns packed with bonded polar sta-tionary phases maintain more constant surface activity, are lesssensitive to small variations in water content in the mobile phase,and produce less retention than silica allowing separation of highlypolar compounds in a more reproducible manner [41,42].

Addition of basic additives to improve chromatographic behav-ior of basic solutes on silica and polar bonded stationary phaseswas reported earlier [21,22,31–33]. A combination of cyano-bondedstationary phase with a mixture of hexane, methylene chloride, ace-tonitrile and propylamine was found to be one of the most usefulfor a set of solutes consisting of a hundred of basic drug molecules[31].

We observed that N,N-dimethylbenzylamine 1 was weaklyretained by the SIL column in 20% methanol in ENFB without

M. Kagan et al. / J. Chromatog

in the chromatographic process (solutes, packing material and sol-vents) may play an increased role in the mechanism of separationon the CN column. Such observations on chromatographic selec-tivity of the separation systems described could be useful whenone is considering various options to resolve a particular separationproblem.

3.3. Effect of basic additive NPA concentration on retention,resolution and column performance of N-methylbenzylamine 2and benzylamine 3 on CN and DIOL columns

As the presence of an amine additive plays a major role in asolute’s peak shape when chromatographed on bonded polar sta-tionary phases [21], we investigated this effect on the separation of2 and 3, a pair of solutes that could not be resolved on the SIL col-umn. Mobile phases containing 1% MeOH in ENFB with increasingconcentrations of NPA (see Section 2) were used for NP HPLC of amixture of 2 and 3 on the DIOL and CN columns. When concentra-tions of the basic additive were very low, both amines were retainedtoo strongly (Fig. 7, top chart) by both columns. Small increasesin NPA concentration dramatically decreased retention for 2 and

Fig. 7. Effect of additive (NPA) concentration on retention, resolution and columnperformance of benzylamines 3 (BA) and 2 (NBA) on the CN and DIOL columns.

additive and exhibited good peak shape and chromatographic per-formance, whereas N-methylbenzylamine 2 and benzylamine 3,as expected, were retained strongly and co-eluted as broad, tail-ing peaks (Fig. 3, trace a). Addition of 0.1% of TEA, DEA or NPAto the mobile phase resulted in a general reduction in retentionfor all three solutes, with NPA producing some improvement inpeak shape for 3 and 2 and leading to their partial separation(Fig. 3b–d).

Benzylamines 1–3 exhibited decreased retention on the CNcolumn in 20% methanol in ENFB without additive and had an excel-lent peak shape for tertiary amine 1 and poor ones for primaryand secondary amines 3 and 2, respectively (Fig. 4a). Neverthe-less, almost baseline separation of 3 and 2 was obtained despite
low plate count for those solutes. Addition of TEA, DEA or NPAdecreased retention, substantially improved chromatographic per-formance of 3 and 2 and facilitated complete separation (Fig. 4b–d;Table 1).
Excellent resolution of all three benzylamines 1–3 was achievedon the DIOL column in a mobile phase without any additive, despitevery poor chromatographic performance of 3 and 2 (Fig. 5a). Sur-prisingly, addition of TEA increased retention and broadened thepeaks of 3 and 2 (Fig. 5b). In the presence of a secondary amineadditive (DEA), the tertiary and secondary amine solutes 1 and 2eluted as sharp peaks while the primary amine 3 gave a broad peak(Fig. 5c). With primary amine additive NPA presented in the mobilephase, the separation of all three solutes was achieved with highresolution and efficiency (Fig. 5d; see also Table 1). This confirmsthe earlier findings [43] that chemical nature of a basic additiveplays a significant role in the chromatographic behavior of varioussubstituted and unsubstituted amines when a diol-derivatized col-umn is used for their separation. Based on this observation, we usedNPA as a basic additive in all subsequent NP separations on polarbonded stationary phases for the compounds containing variousamino groups.

r. A 1194 (2008) 80–89 85

3.2. Selectivity of methanol–ENFB mobile phase for separation ofstructurally diverse benzylamines 4, 5 and 2 and 6–8 on DIOL andCN columns

After optimizing the separation conditions for benzylamines1–3 on the DIOL and CN columns, we investigated if these systemswere capable of resolving not only permutations of N-methylatedbenzylamines, but also N-alkyl-benzylamines (4 and 5) in additionto constitutional isomers of methyl benzyamines (6–8). N-Butyl,N-ethyl and N-methyl benzylamines (4, 5 and 2) could be easilyseparated on both DIOL and CN columns in 1% MeOH with NPAadded (separation on the DIOL column is shown in Fig. 6a; sim-ilar separation was obtained on the CN column). The elongationof the N-alkyl substituent decreases the ability of the NH-group toform hydrogen bonds with hydroxy-groups of the packing material,resulting in decreased retention for 5 and 4. Similarly, methyl ben-zylamines (6–8) were well separated on the CN column in the samemobile phase, their elution order also being defined by the methylsubstituent’s proximity to the NH2 group (Fig. 6b). Interestingly, noseparation of 6–8 was observed when the same mobile phase wasemployed with the DIOL column. Interactions other than hydrogenbonding between elements of the molecular structures involved

Fig. 8. Comparison of anilines 9–11 separation under NP conditions: mobile phase,5% MeOH in ENFB on the DIOL column.


Fig. 9. Comparison of ephedrines 12–14 separation on the DIOL and CN columns:traces a, b—DIOL and CN columns, correspondingly, 50% of solvent B in A (Section2).

3, increased resolution between them (Fig. 7, middle chart) andimproved their plate count (Fig. 7, bottom chart). NPA concentra-tion of 0.1–0.2% appears to be the optimum for separation of 2 and3. Therefore, in all subsequent chromatographic experiments theconcentration of basic modifier NPA was maintained at 0.1% (v/v).

3.4. Comparison of hexane- and ENFB-based mobile phases forNP separation of benzylamines

While mixtures of MeOH and ENFB may have certain advan-tages over traditional hydrocarbon-based NP mobile phases, theyseem to be better suited for analytical applications due toENFB cost and availability. We compared NP HPLC of benzy-lamines on the DIOL and CN columns in mobile phases made ofhexane–methylene chloride–methanol with chromatographic per-formance of MeOH–ENFB mixtures (both solvents contained 0.1% of
NPA, see Section 2). Hexane-based mobile phases performed verysimilar to ENFB-based systems with respect to retention, resolution,and columns’ performance) for benzylamines 1–3 (see Table 1). Themost likely separation mechanism included molecules of MeOH,amine additive and solutes competing for active sites of the sta-tionary phase. From a practical point of view it is easy to envisionthe development of analytical chromatographic methods (LC orLC–MS) on the DIOL and/or CN columns using methanol–ENFB andthen scaling them up with mixtures of hexane, methylene chloride,methanol and basic additive for preparative HPLC procedures [37].
3.5. NP separation of N,N-dimethylaniline, N-methylaniline andaniline 9–11

The mixture of physiologically active anilines 9–11 was sepa-rated previously on a nitrile column in 0.5% 2-propanol in isooctaneand on a C18 phase in aqueous MeOH [30]. Under NP conditionsthe solute elution order correlated with decreased substitution,with 10 and 11 eluting as fairly broad tailing peaks. RP conditionsrequired the use of buffered methanolic solution at pH 7 resultingin adequate separation of anilines 9–11. We found that compounds9–11 were well resolved when chromatographed on either DIOL or

Fig. 10. Elution order reversal of ephedrine 13 and norephedrine 14 on the CN col-umn. Mobile phase: 2.5, 5, 10 and 20% of solvent B in A (containing 0.5, 1, 2, and 4%of MeOH; traces a, b, c and d, correspondingly), flow rate 1 ml/min.

CN columns in both ENFB- or hexane-based mobile phases (exam-ple in Fig. 8; data in Table 1). Overall, NP under the conditionsdescribed might become a mode of choice for HPLC of permutationsof substituted anilines, especially in light of similar outstanding NPperformance of a novel titanium oxide stationary phase that does
not even require the presence of an amine as an additive [44].
3.6. NP separation of N-methylephedrine, ephedrine andnorephedrine 12–14

Ephedrine and its derivatives are potential central nervous sys-tem stimulants widely used as components of many commonpharmaceutical formulations for asthma, ophthalmic, cold andallergy treatments. Variations in hydroxy group stereochemistryand the state of N-methyl substitution have a direct effect onephedrine’s biological activity [38].

A number of chromatographic methods have been developedto analyze polar basic compounds in extracts from Ephedraespecies, biological fluids and formulations. Early separationsemployed such mobile phases as mixtures of MeOH–aqueousammonia–ammonium nitrate (pH 9) [24] or solutions of ethanolin hexane with ammonia [45] on non-derivatized silica, the for-mer approach made more reproducible with less volatile buffers[46,47]. RP techniques were eventually improved by using phos-phate buffers and triethylamine [48,49], novel base-deactivated


Fig. 11. Comparison of tryptamines 15–17 separation on the DIOL and CN: traces (a)DIOL column, 50% of solvent B in A; (b) CN column, 60% MeOH in ENFB.

C18 [50] and fluorinated [51] stationary phases, as well as 1-alkyl-3-methylimidazolium-based ionic liquids as the eluent [52]. To thebest of our knowledge, there are no reports in the literature on sep-aration of ephedrine-related compounds on polar bonded phasesin organic solvents.

A mixture of norephedrine 14 and its N-substituted analogs N-methylephedrine 12 and ephedrine 13 was successfully resolved onthe DIOL and CN columns in both hexane- and ENFB-based mobilephases (examples in Fig. 9a and b; data in Table 1). The columnperformance was higher in the case of the former mobile phase,which could be explained by possible low solubility of these polarcompounds (ephedrine 13 is soluble in water [49]) in 10% MeOH inENFB. Primary amine 14 eluted before secondary amine 13 from aCN column in both solvent systems. The elution order of 13 and 14could be reversed by decreasing the concentration of methanol in
the mobile phase (Fig. 10).
It appears that at low concentrations of alcohol the process ofhydrogen bonding between solutes and stationary phase deter-mines solutes’ retention, while increased amounts of MeOH causemore complex interactions between molecules of solvent, solutesand the cyano groups present either on the stationary phase or inthe mobile phase. A similar reversal of elution order that dependson concentration of a polar modifier has been described for separa-tion of basic aniline and acidic phenol on a cyano packing materialwith methyl t-butyl ether (MTBE) in hexane [53]. This effect wasexplained by various degrees of acid–base interactions betweensolutes, cyano stationary phase and molecules of localizing mod-ifier MTBE. We, however, had certain difficulties applying similarlogic in order to explain chromatographic behavior of 13 and 14 onthe CN column as both molecules contain strong basic groups.

NP chromatographic conditions seem to be well suited for theanalysis and purification of ephedrine-like molecules. The abilityto alter selectivity of such systems by switching the DIOL and CNcolumns along with LC–MS compatibility of ENFB–MeOH mobilephases can be also exploited in difficult analytical and preparativeapplications.

Fig. 12. Elution order reversal of primary and secondary tryptamines 16 and 17 onthe CN column. Traces a, b and c contain 5, 10 and 25% solvent B in A (1, 2 and 5% ofmethanol; correspondingly), flow rate 1 ml/min.

3.7. NP separation of N-N-dimethyl-5-methoxytryptamine,N-methyl-5-methoxytryptamine and 5-methoxytryptamine15–17

Indoleamine derivatives with psychotomimetic properties are
produced in human body as metabolites of tryptophan and theiranalysis may be important in the diagnosis of some psychiatric dis-orders [54,55]. This analysis can be carried out on silica in methanolcontaining ammonia and ammonia nitrate solution [56] as well asunder ion-exchange [57] and RP HPLC conditions [58].
We used tryptamines 15–17 as model compounds to deter-mine optimal separation conditions for analysis and subsequentpurification of their potential analogs and derivatives. Under NPconditions, the elution order on the DIOL column with both hexane-and ENFB-based mobile phases followed the decrease in N-methylsubstitution (Fig. 11a; Table 1). Primary amine 17 was, however,eluted before secondary amine 16 on the CN column in both mobilephases (example for ENFB-based mobile phase is shown in Fig. 11b;see also Table 1), similar to ephedrines 13 and 14 in Section 3.7.The elution order of 17 and 16 on the CN column could be alsoreversed by decreasing amount of localizing modifier MeOH in themobile phase (Fig. 12). Separation between tryptamines 17 and 16could be enhanced by using a bulkier alcohol as an alcohol modifier(Fig. 13), with selectivity (˛) and resolution Rs gradually improvingin the series MeOH, ethanol, 2-chloroethanol, 2-propanol to ˛ 1.4,2.1, 1.82 and 2.26 and Rs 2.5, 5, 5.3 and 6.05, respectively. Steric


Fig. 14. Comparison of 5-HT6 modulators 18–20 separation on the DIOL and CNcolumns: traces a and b—DIOL and CN columns, correspondingly, 50% of solvent Bin A.

with good efficiency and selectivity, resulting in high resolutionfor solutes 18–20 in both NP solvent systems (Fig. 14a; Table 1).The solutes’ elution order on the CN column was the same as forthe DIOL one (Fig. 14b), with secondary amine 19 eluting beforeprimary amine 20 (opposite to the retention pattern observed withCN column for corresponding ephedrines and tryptamines, see Sec-tions 3.6 and 3.7). In addition, the DIOL column seemed to havebetter selectivity towards separation of the 19 and 20 pair, as theRs values were consistently lower on a CN column in both mobilephases (Table 1).

Fig. 13. Effect of the chemical structure of the alcohol modifier on reso-lution between tryptamines 16 and 17 on the CN column. Mobile phase:hexane–CH2Cl2–alcohol–NPA (75:20:5:0.1), flow rate 1 ml/min. Alcohols: MeOH(trace a), ethanol (b), 2-chloroethanol (c) and isopropanol (d).

factors, and not the alcohol acidity, which is an intrinsic chemicalproperty of the modifier, seem to play a major role in influenc-ing such separation of 16 and 17: the more acidic 2-chloroethanolproduced a similar separation to ethanol, whereas the use of thesignificantly less acidic 2-propanol improved both selectivity and
resolution.
Good selectivity of the CN column, despite modest peaks’ effi-ciency exhibited by the solutes, resulted in good resolution forcompounds 15–17 (Table 1). We believe the combination of theCN column with ENFB and MeOH mobile phases can be success-fully used for analytical NP LC–MS of indoleamines (as in [36,58]),whereas isolation and purification of their synthetic derivatives canbe carried out using preparative NP LC–MS similar to proceduresdescribed in [37].

3.8. NP separation of Wyeth CNS-modulators 18–20

5-HT6 modulators 18–20 were synthesized previously duringa Wyeth 5-HT6 agonist research program [59]. These compoundscan serve as models of “typical” low molecular weight substancesinvestigated as potential drug candidates as they are potent mod-ulators of 5-HT6 receptors. Interestingly, the alkylation state of theamine was generally important to the functional activity since thetertiary and primary amines 18 and 20 were full agonists in a 5-HT6cyclase assay while the secondary amine 19 was a full antagonist.These analogs were successfully separated on the DIOL column,

Fig. 15. Purification of direct methylation reaction of ∼100 mg of norephedrine 14by NP preparative HPLC. Gradient 30–90% of solvent B in A in 15 min. Peaks 1, 2 and 3contain N-methyl ephedrine 12 (9 mg), ephedrine 13 (23 mg) and starting material14 (42 mg), correspondingly.

matog

[

[[[

[[

[[[[[[[[[[[

[[[

[

[[[

[[

[

[

[[41] A.W. Salotto, E.L. Weiser, K.P. Caffey, R.L. Carty, S.C. Racine, L.R. Snyder, J. Chro-

M. Kagan et al. / J. Chro

3.9. Direct alkylation and preparative NP HPLC purification ofCNS-active amines

In order to demonstrate the feasibility of the NP chromato-graphic approach for preparative isolation of basic compounds weapplied it to the separation of the products obtained when a phys-iologically active amine 14 was directly methylated in glyme andseparated on a preparative DIOL column in a gradient of methylenechloride and MeOH in hexane in the presence of basic modifier NPA(Fig. 15). Quick and efficient separation of starting material and boththe mono- and di-methyl derivatives 13 and 12 was achieved.

4. Conclusions

We have demonstrated that normal-phase chromatographicconditions can be successfully used for the HPLC analysis and purifi-cation of mixtures of compounds containing primary, secondaryand tertiary amino groups. HPLC on CN and DIOL columns withhexane or ethoxynonafluorobutane with methylene chloride andmethanol as an eluent were successfully used for separation of N-alkylated benzylamines and their methyl aromatic analogs, as wellas anilines, ephedrines, tryptamines and serotonin receptor ligands.

Columns packed with cyano- and diol-derivatized silicaappeared to be well suited for such separations. Unfavorablechromatographic phenomena, such as peak tailing, diminishedselectivity and low plate count, can be avoided by using mobilephase basic additives. The chemical nature of the amine additive isextremely important since only n-propylamine (primary amine) atan optimal concentration of 0.1% was able to significantly improvepeak shape and reduce tailing for all solutes investigated, especiallyfor compounds containing a primary amino group.

Comparison of the data suggests a more complex mechanismfor separation on the CN compared to the DIOL column. The elutionorder of the solutes on the DIOL column always followed the patternof decreasing amino substitution leading to increased retention,suggesting the NH interaction with the hydroxy groups of the pack-ing material plays a main role in the separation mechanism. Theelution order of the solutes with substituted and free amino groupson the CN column was somewhat less predictable. Compoundscontaining primary amino groups and other structural elementsof the molecule capable of forming hydrogen bonds eluted beforetheir secondary amine counterparts. However, the use of decreas-ing amounts of alcohol modifier in the mobile phase reversed thiseffect.

We also found that in some cases the selectivity of the CN col-umn could be greatly affected by the nature of the localizing alcoholmodifier used. These differences in selectivity can be exploitedexperimentally when a particular separation problem presentsitself. Obvious differences in selectivity observed with the DIOLand CN columns are in a good agreement with earlier [40,41,53]and recent [60] findings and both types of column packing materialshould serve as complementary tools in analytical and preparativechromatographic applications for organic basic compounds.

In addition, the similarity of the chromatographic behaviorof those compounds investigated in NP mobile phases based onethoxynonafluorobutane or hexane may provide flexibility for thedivergent potential chromatographic applications, with the formeremployed for analytical LC and LC–MS experiments and the latterfor preparative work.

The separation techniques used in this study were successfullyapplied for purification of reaction products obtained from synthe-sis of research compounds in sufficient quantities for evaluationof their biological properties. We believe this methodology canbe a viable alternative to conventional strategy for purification ofpotential drug candidates based on reversed-phase HPLC and data

[

[[[[[[

[[

[[[[[

[[

[[

[

r. A 1194 (2008) 80–89 89

comparing both approaches for same groups of compounds will bepublished elsewhere.

References

[1] A.F. Abdel-Mahid, C.A. Maryanoff, K.G. Carson, Tetrahedron Lett. 31 (1990) 5595.[2] C.H. Barney, E.W. Huber, J.R. McCarthy, Tetrahedron Lett. 31 (1990) 5547.[3] A.F. Abdel-Mahid, K.G. Carson, B.D. Harris, C.A. Maryanoff, R.D. Shah, J. Org.

Chem. 61 (1996) 3849.[4] L.R. Snyder, J.L. Glajch, J.J. Kirkland, Practical HPLC Method Development, 2nd

edition, Wiley–Interscience, New York, NY, 1988 (Chapters 3–6).[5] D.V. McCalley, J. Chromatogr. 636 (1993) 213.[6] D.V. McCalley, J. Chromatogr. A 664 (1994) 139.[7] D.V. McCalley, J. Chromatogr. A 769 (1997) 169.[8] D.V. McCalley, J. Chromatogr. A 902 (2000) 311.[9] D.V. McCalley, J. Chromatogr. A 987 (2003) 17.10] P.J. Twitchett, A.C. Moffat, J. Chromatogr. 111 (1975) 149.

[11] R.W. Roos, C.A. Lau-Cam, J. Chromatogr. 370 (1986) 403.12] I.S. Lurie, S.M. Demchuk, J. Liq. Chromatogr. 4 (1981) 337.13] I.S. Lurie, S.M. Demchuk, J. Liq. Chromatogr. 4 (1981) 357.14] J.J. Kirkland, J.B. Adams Jr., M.A. Van Straten, H.A. Claessens, Anal. Chem. 70

(1998) 4344.15] J.J. Kirkland, M.A. van Straten, H.A. Claessens, J. Chromatogr. A 797 (1998) 111.16] A. Espada, A. Rivera-Sagredo, J. Chromatogr. A 987 (2003) 211.

[17] A. Espada, A. Marin, C. Anta, J. Chromatogr. A 987 (2004) 43.18] N.H. Davies, M.R. Euerby, D.V. McCalley, J. Chromatogr. A 1119 (2006) 11.19] N.H. Davies, M.R. Eureby, D.V. McCalley, J. Chromatogr. A 1178 (2008) 71.20] J.J. Kirkland, M.H. Dilks Jr., J.J. DeStefano, J. Chromatogr. 635 (1993) 19.21] M.R. Detavernier, L. Dryon, D.L. Massart, J. Chromatogr. 128 (1976) 204.22] M. Johansson, S. Eksborg, A. Arbin, J. Chromatogr. 275 (1983) 355.23] B.A. Bidlingmeyer, J.K. Del Rios, J. Korpl, Anal. Chem. 54 (1982) 442.24] I. Jane, J. Chromatogr. 111 (1975) 227.25] B. Law, J. Chromatogr. 407 (1987) 1.26] D.V. McCalley, J. Chromatogr. A 1171 (2007) 46.27] H.A. Claessens, M.A. van Straten, J.J. Kirkland, J. Chromatogr. A 728 (1996) 259.28] J.J. Kirkland, J.W. Henderson, J.J. DeStefano, M.A. Van Straten, H.A. Claessens, J.

Chromatogr. A 762 (1997) 97.29] M. De Smet, D.L. Massart, J. Chromatogr. 410 (1987) 77.30] L.-A. Truedsson, B.E.F. Smith, J. Chromatogr. 214 (1981) 291.31] M.R. Detavernier, G. Hoogewijs, D.L. Massart, J. Pharm. Biomed. Anal. 1 (1983)

331.32] M. De Smet, G. Hoogewijs, M. Puttemans, D.L. Massart, Anal. Chem. 56 (1984)

2662.33] T. Hamoir, D.L. Massart, J. Chromatogr. A 673 (1994) 1.34] M. Kagan, J. Chromatogr. A 918 (2001) 293.35] M. Chlenov, M. Kagan, Z. Wang, A. Bach, O. McConnell, E. Trybulski, Poster P222-

T. Book of Abstracts, Proceedings of the 30th International Symposium on Highperformance Liquid Phase Separations and Related Techniques HPLC’2006.

36] M. Kagan, M. Chlenov, C.M. Kraml, J. Chromatogr. A 1033 (2004) 321.37] M. Kagan, M. Chlenov, A. Bach, O. McConnell, J. Liq. Chrom. Relat. Technol. 27

(2004) 1817.38] G. Ma, S.A. Bavadekar, Y.M. Davis, S.G. Lalchandani, R. Nagmany, B.T. Schaneberg,

I.A. Khan, D.R. Feller, J. Pharmacol. Exp. Ther. 322 (1) (2007) 214.39] L.R. Snyder, J.J. Kirkland, J.L. Glajch, Practical HPLC Method Development, 2nd

edition, Wiley–Interscience, New York, NY, 1997.40] E.L. Weiser, A.W. Salotto, S.M. Flach, L.R. Snyder, J. Chromatogr. 303 (1984) 1.

matogr. 498 (1990) 55.42] H.A. Claessens, M. Van Thiel, P. Westra, A.M. Soeterboek, J. Chromatogr. 275

(1983) 345.43] D. Chan Leach, M.A. Stadalius, J.S. Berus, L.R. Snyder, LC–GC 6 (1988) 494.44] J. Winkler, S. Marme, J. Chromatogr. A 888 (2000) 51.45] M. Noguchi, K. Hosoda, S. Hideyo, Yakugaki Zasshi 107 (1987) 372.46] R. Smith, J.O. Rabour, J. Chromatogr. 464 (1989) 117.47] R. Smith, J.P. Westlake, R. Gill, M.D. Osselton, J. Chromatogr. 514 (1990) 97.48] C. Imaz, D. Carreras, R. Navajas, C. Rodriguez, J. Maynar, R. Cortes, J. Chromatogr.

631 (1993) 201.49] P.J. van der Merwe, L.W. Brown, S.E. Hendrikz, J. Chromatogr. B 661 (1994) 357.50] C. Imaz, R. Navajas, D. Carreras, C. Rodriguez, A.F. Rodriguez, J. Chromatogr. A

870 (2000) 23.51] D.S. Bell, H.M. Cramer, A.D. Jones, J. Chromatogr. A 1095 (2005) 113.52] L. He, W. Zhang, L. Zhao, X. Liu, S. Jiang, J. Chromatogr. A 1007 (2003) 39.53] P.L. Smith, W.T. Cooper, J. Chromatogr. 410 (1987) 249.54] A.C. Cottrell, M.F. McLeod, W.R. McLeod, Am. J. Psychiatry 134 (1977) 322.55] T. Forsstrom, J. Tuominen, J. Karkkainen, Scand. J. Clin. Lab. Invest. 61 (2001)

547.56] P.C. White, J. Chromatogr. 169 (1979) 453.57] B.R. Sitaram, R. Talomsin, G.L. Blackman, W.R. McLeod, G.N. Vaughan, J. Chro-

matogr. 275 (1983) 21.58] M. Perkal, G.L. Blackman, A.L. Ottrey, L.K. Turner, J. Chromatogr. 196 (1980) 180.59] R.C. Bernotas, S. Lenicek, S.A. Antane, G.-M. Zhang, D.L. Smith, J. Coupet, L.E.

Schechter, 230th American Chemical Society National Meeting, Washington,DC, USA, August 28–September 1, 2005.

60] C. West, E. Lesellier, J. Chromatogr. A 1110 (2006) 200.

optimization of normal-phase chromatographic...

Documents