HPLC for Pharmaceutical Scientists 2007 (Part 5)

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High-performance liquid chromatography (HPLC) is a separation tool par excellence for the analysis of compounds of wide polarity. Since its inception approximately four decades ago, HPLC has revolutionized numerous disciplines of science and technology. Among the various modes of HPLC, reversed-phase and normal-phase chromatography (NPC) are employed most commonly in separation. Normal-phase chromatography was the ﬁrst liquid chromatography mode, discovered by M. S. Tswett in 1903, and it is well established as evidenced by a plethora of books and articles that have been published in recent years. In this chapter we describe a simpliﬁed overview of the theory...

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Nội dung Text: HPLC for Pharmaceutical Scientists 2007 (Part 5)

5 NORMAL-PHASE HPLC Yong Liu and Anant Vailaya 5.1 INTRODUCTION High-performance liquid chromatography (HPLC) is a separation tool par excellence for the analysis of compounds of wide polarity. Since its inception approximately four decades ago, HPLC has revolutionized numerous disci- plines of science and technology. Among the various modes of HPLC, reversed-phase and normal-phase chromatography (NPC) are employed most commonly in separation. Normal-phase chromatography was the ﬁrst liquid chromatography mode, discovered by M. S. Tswett in 1903, and it is well estab- lished as evidenced by a plethora of books and articles that have been pub- lished in recent years. In this chapter we describe a simpliﬁed overview of the theory and practice of normal-phase chromatography. 5.2 THEORY OF RETENTION IN NORMAL-PHASE CHROMATOGRAPHY Unlike the more popular reversed-phase chromatographic mode, normal- phase chromatography employs polar stationary phases, and retention is mod- ulated mainly with nonpolar eluents. The stationary phase is either (a) an inorganic adsorbent like silica or alumina or (b) a polar bonded phase con- taining cyano, diol, or amino functional groups on a silica support. The mobile phase is usually a nonaqueous mixture of organic solvents. As the polarity of the mobile phase decreases, retention in normal-phase chromatography HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright © 2007 by John Wiley & Sons, Inc. 241
242 NORMAL-PHASE HPLC Figure 5-1. Hypothetical representation of the adsorption mechanism of retention in normal-phase chromatography. S denotes sample molecule, E denotes molecule of strong polar solvent, and X and Y are polar functional groups of the stationary phase. Prior to retention, the surface of stationary phase is covered with a monolayer of solvent molecules E. Retention in normal-phase chromatography is driven by the adsorption of S molecules upon the displacement of E molecules. The solvent mole- cules that cover the surface of the adsorbent may or may not interact with the adsorp- tion sites, depending on the properties of the solvent. (Reprinted from reference 1, with permission.) increases. Figure 5-1 illustrates the mechanism of retention in NPC [1]. Reten- tion is governed by the extent to which the analyte molecules displace the adsorbed solvent molecules on the surface of the stationary phase. This reten- tion model based on adsorption was ﬁrst proposed by Snyder [2–5] to describe retention on silica and alumina adsorbents and later extended to explain reten- tion on polar bonded phases, such as diol-, cyano-, and amino-bonded silica. Snyder assumed a homogeneous surface so that adsorption energies for solute and solvent molecules are constant. The stoichiometry of solute–solvent com- petition can be given by Sm + nEa ↔ Sa + nEm (5-1) m and a refer to solute (S) and solvent (E) molecules in the mobile and adsorbed phases, respectively. n is the coefﬁcient that takes into account dif- ferent adsorption cross sections for solute and solvents; that is, adsorption of a solute molecule displaces n solvent molecules in the adsorbed monolayer. For a binary mobile-phase system consisting of a weak nonpolar solvent and a strong polar solvent, adsorption of the weak solvent can be ignored. There- fore, solute retention can be expressed by AS ln(k2 ) = ln(k1 ) − ln( N E ) (5-2) AE
THEORY OF RETENTION IN NORMAL-PHASE CHROMATOGRAPHY 243 Here, AS is the solute cross-sectional area, AE is the molecular area of the strong solvent, NE is the mole fraction of the strong solvent in the mobile phase, k2 is retention factor of the solute in the binary mobile-phase mixture, and k1 is the retention factor in the strong solvent alone. Yet another adsorption-based retention model similar to that of Snyder was proposed by Soczewinski [6] to describe the retention in NPC. It assumes that retention in NPC is the product of competitive adsorption between solute and solvent molecules for active sites on the stationary phase surface. The sta- tionary-phase surface consists of a layer of solute and/or solvent molecules, but, unlike the former, the latter model assumes an energetically heteroge- neous surface where adsorption occurs entirely at the high-energy active sites, leading to discrete, one-to-one complexes of the form Sm + qEa -A* ↔ S - A* + qEm (5-3) A* is an active surface site and q refers to the number of substituents on a solute molecule that are capable of simultaneously interacting with the active site. This equation takes into account the possibility of an analyte mol- ecule’s interaction with multiple sites. Based on this model, the solute reten- tion factor can be expressed by the following equation, which is similar to Snyder’s: log k2 = d − q log N E (5-4) where d is a constant. Comparison of the two models reveals that both predict a linear log k2 versus log NE plot. Snyder’s model predicts that the slope of this line should be the ratio of the molecular areas of solute and solvent, whereas Soczewinski’s model predicts that the slope is the number of strongly adsorbing substituent groups (number of adsorption sites on the analyte) on the solute. In practice, it was found that equations (5-1) and (5-2) are most reliable for less polar solvents and solute molecules on alumina or silica stationary phases only. Neither of the models is entirely satisfactory in the forms presented, par- ticularly for predicting retention behavior on bonded stationary phases. These phases contain strongly adsorbing active sites as assumed in Soczewinski’s model, but the solute molecular area and not just polar substituents are known to play an important role in competitive adsorption as assumed by Snyder. Furthermore, secondary solvent effects resulting from solute–solvent interac- tions in both the mobile and adsorbed phases are not taken into considera- tion in either model. These effects, such as hydrogen bonding, give rise to some of the most useful changes in retention and often are an important source of chromatographic selectivity [7, 8]. Another experimental deviation from equations (5-1) or (5-2) was deter- mined to be due to the localization of solvent molecules onto the adsorption sites of stationary phase resulting from silanophilic interactions. When the
244 NORMAL-PHASE HPLC polar substitution groups of a solvent molecule interact strongly with the polar groups on the surface of the column packing, they become attached or local- ized onto the stationary-phase surface. An important consequence of solvent localization is the apparent change in the solvent strength value of a polar solvent. (Solvent strength is presented by e 0, which is determined empirically by using polyaromatic hydrocarbons that do not localize but lie ﬂat on a surface. Solvent with larger value of e has stronger elution power [1].) Con- sequently, the solvent strength does not vary linearly with the concentration of the stronger solvent for a binary mixture where one solvent is stronger than the other [7]. There is competition between the two solvents for the active sites of the adsorbent and the stronger solvent will preferentially adsorb, resulting in a more concentrated adsorbed layer of the stronger solvent. For instance, the dependence of solvent strength for several binary mixtures on alumina as adsorbent shows a large increase in solvent strength due to a small increase in the concentration of a polar solvent at low concentrations. But at the other extreme, a relatively large change in the concentration of the polar solvent affects the solvent strength of the mobile phase to a lesser extent. In the case of low concentration of polar solvent before the localization on the surface of stationary phase reaches saturation, a small change of the polar solvent con- centration can greatly affect the number of polar active sites on the column packing. As a consequence, signiﬁcant variations of analytes retention are observed. Once the polar active sites of the stationary phase are localized com- pletely, change of polar solvent concentration will have a smaller impact on analyte retention. These deﬁciencies were addressed by revising Snyder’s model as follows [8]. To account for the preferential adsorption of solute and solvent onto the strong sites, empirical AS and NE values larger than those calculated from mol- ecular dimensions are used based on experimental observation. The revised model acknowledges the tendency of polar molecules to localize on the strongly adsorbing active site and expresses solute retention in terms of the solvent strength as follows: log k2 = log k1 − a′AS (ε 1 − ε 0 ) 0 2 (5-5) where a′ is an adsorbent activity factor, e 0 and e 0 are solvent strengths for 1 2 solvent 1 and 2, and AS is the analyte cross-sectional area on the adsorbent surface. The “analyte” cross-sectional area can be predicted from molecular dimensions. Secondary solute–solvent interactions are incorporated into the revised model by adding extra terms denoted by ∆ for each of the solvents as follows: log k2 = log k1 − a′AS (ε 1 − ε 0 ) + ( ∆ 2 − ∆ 1 ) 0 2 (5-6) When a nonlocalizing, nonpolar solvent such as hexane is employed as a weak solvent, the equation can be further simpliﬁed so that
EFFECT OF MOBILE PHASE ON RETENTION 245 log k2 = log kh − a′AS ε 0 + ∆ 2 2 (5-7) assuming hexane does not induce any secondary solvent effects and its solvent strength is zero. Here kh is the analyte retention factor in pure hexane. Equa- tion (5-7) has been found useful to understand the fundamental principles governing the retention behavior as far as solute, solvent, and bonded-phase properties are concerned. For instance, by ﬁtting equation (5-7) to the exper- imental NPC data, the extent of solute localization can be determined by com- paring the slopes of a log k2 versus e 0 plot, provided that the molecular cross 2 section can be estimated accurately. 5.3 EFFECT OF MOBILE PHASE ON RETENTION Selection of suitable mobile-phase system is critical in NPC to achieve the desired separation [4]. In general, a suitable solvent should have the follow- ing properties: low viscosity, compatibility with detection system (for instance, solvent should be transparent at wavelength of detection if UV is used as detector), available in pure state, low ﬂammability and toxicity, highly inert, and adequate solubility for solutes. Unlike RPLC, analytes become less retained as solvent strength (solvent polarity) increases. Solvent strength in NPC can be represented by e 0, and values of e 0 for some commonly used NPC solvents are listed in Table 5-1 for silica as column packing [1]. Relative solvent strength for other NPC column packings such as alumina and polar bonded phases follow the same trend as in the table; that is, larger values of e 0 are obtained for more polar solvents. Ideally, the mobile-phase strength should be chosen to maintain analyte retention factor within the optimum range of 1 ≤ k′ ≤ 5 with selectivity values sufﬁcient to reach a satisfactory resolution. In general, binary mobile phases, such as a mixture of a nonselective solvent hexane with a polar solvent, are used for NPC separations. If separation cannot be achieved by adjusting mobile phase strength (change the concentration of one of the components in a binary mixture), then variation of polar solvent nature has to be pursued. Snyder has developed a useful scheme to classify solvents (nonelectrolytic solvents) nature based on their interactions with solutes and the stationary phase [9]. This approach should not be taken as con- crete rules but rather as a phenomological approach. The property of a solvent is characterized by the three most important parameters, which are its proton- acceptor (Xe), proton-donor (Xd), and dipole-donor (Xn) afﬁnity. Each of these contributes to the overall polarity of the solvent, which in turn is related to its chromatographic strength. Rohrschneider determined the values of these parameters from distribution coefﬁcients of test solutes such as ethanol, dioxane, and nitrobenzene [10]. A medium polar solvent—such as chloroform, which has a polarity of 4.31—involves 31% proton acceptor, 35% proton donor, and 34% dipole interactions. If the parameter values of the solvents are plotted on a triple coordinate system, various solvents can be grouped into
246 NORMAL-PHASE HPLC TABLE 5-1. NPC Solvent Strength (e0) and Selectivity a of Various Solvents Employed in HPLC Solvent ε0 Localization Basic? UVb Hexane, heptane, octane 0.00 No c 201 1,1,2-Triﬂurotrichloroethane 0.02 No c 235 (Freon FC-113) Chloroform 0.26 No c 247 1- or 2-Chloropropane 0.28 No c 225 Methylene chloride 0.30 No c 234 2-Propyl ether 0.32 Minor c 217 1,2-Dichloroethane 0.34 No c 234 Ethyl ether 0.38 Yes Yes 219 MTBEd 0.48 Yes Yes 225 Ethyl acetate 0.48 Yes No 256 Dioxane 0.51 Yes Yes 215 Acetonitrile 0.52 Yes No 192 THF 0.53 Yes Yes 230 1- or 2-Propanol 0.60 Yes e 214 Methanol 0.70 Yes e 210 a Silica used as absorbent. b Minimum UV wavelength; assumes that maximum baseline absorbance (100% B) is 0.5 AU. c Solvent basicity is irrelevant for nonlocalizing solvents. d Methyl t-butyl ether. e Different selectivity due to presence of proton donor group. Source: Reprinted from Ref. 1, with permission. eight classes (Figure 5-2) [9]. Solvents within each class should show similar selectivity for a set of components, while the nature of solvents from different classes are quite different and may impart differences in selectivity for the same set of components. In NPC method development, replacing solvents belonging to the same selectivity class cannot offer substantial variation in chromatographic separation. Therefore, it is recommended to select solvents that are placed close to the apices of the triangle for maximum selectivity. Common solvents in group I are isopropyl ether and MTBE, group VII sol- vents include dichloromethane and 1,2-dichloroethane, and chloroform and ﬂuoro-alcohols constitute group VIII solvents. Solvent mixtures having the same elution strength but different selectivities are called isoelutropic mobile phases. Binary mixtures, however, have only limited abilities for controlling mobile- phase selectivity. Therefore, ternary and even quaternary mobile phases that contain two or more different polar solvents along with a nonpolar solvent are often used to achieve the required selectivity. If the ratio of the concentration of two polar solvents is constant but the sum of the their concentration is being changed with respect to that of the nonpolar solvent, the effect on retention is much the same as when the concentration of the single strong solvent
EFFECT OF MOBILE PHASE ON RETENTION 247 Figure 5-2. Snyder’s selectivity triangle for solvents. (Reprinted from reference 9, with permission.) changed in a binary mobile phases. On the other hand, if the sum of the two polar solvents stays constant but the ratio is variable, larger effects on the selectivity of separation are observed than in the system where the ratio is constant. This is attributable to changes in dipole–dipole and proton–donor– acceptor interactions between polar solvents and the analytes. Such selectiv- ity tuning is the main purpose of using ternary mobile phases in NPC. A phenomenological approach for the appropriate selection of ternary mobile mixture based on Snyder’s solvent selectivity triangle concept combined with a statistical approach can be applied [11–15]. As can be seen in Figure 5-4, a seven-run design is used. A primary binary solvent mixture such as hexane- MTBE with the solvent strength that is convenient for the separation is ﬁrst selected. This binary mixture represents one corner of the selectivity triangle. Two other binary mixtures, namely, hexane-dichloromethane and hexane- chloroform, having the composition with the same solvent strength, are then tested. As shown in Figure 5-3, the area bound by the sides of the triangle formed by MTBE, dichloromethane, and chloroform deﬁnes the selectivity domain in which the optimum mobile-phase composition will be found. Next, separations are performed with three different ternary mobile-phase systems produced by mixing an equal volume of each of the binary solvents. Thus, the three experiments are set in the middle of triangle. Finally, the analysis is carried out by mixing in the three binary mixtures in equal ratio. By compar- ing the seven chromatograms obtained in the above experiment, optimum
248 NORMAL-PHASE HPLC Figure 5-3. Selected solvents for mobile-phase optimization in NPC. (Reprinted from reference 11, with permission.) solvent composition for the separation can be easily identiﬁed. Figure 5-4 demonstrates the triangle reduction method whereby the same procedure is repeated, starting from a smaller triangle—for instance, as deﬁned by apices 2, 4, and 5, which corresponds to an area where the resolution is the highest— until an optimum mobile-phase mixture is determined for adequate resolution of the separated mixtures. Furthermore, optimum solvent composition can also be obtained by regression analysis with data obtained from the seven runs experiment [14]. Separation of acidic or basic analytes on NPC generally results in signiﬁ- cant peak tailing due to the strong hydrogen-bonding interactions with silanol group on the stationary phase. Therefore, acidic or basic additive such as TFA (triﬂuoroacetic acid) or DEA (diethylamine) are often included in the mobile- phase system to minimize the hydrogen-bonding interactions. 5.4 SELECTIVITY 5.4.1 Effect of Analyte Structure In NPC, analytes retentions generally increase in the following sequence: alkane < alkenes < aromatic hydrocarbons ≈ chloroalkanes < sulﬁdes < ethers < ketones ≈ aldehyde ≈ esters < alcohols < amides < phenols, amines, and < carboxylic acids [16]. The retention also depends to some extent on the
SELECTIVITY 249 Figure 5-4. Procedure for selectivity optimization in NPC based on mixtures with hexane of nonlocalizing solvent (CH2Cl2), a basic-localizing solvent (MTBE), and a nonbasic localizing solvent (ACN or ethyl acetate). All mobile phases are of equal strength. (Reprinted from reference 1, with permission.) hydrocarbon part of the solutes. Unlike RPLC, however, analytes become less retained as the size of alkyl chains increases. Furthermore, the separation in homologous series is less satisfactory than in RPLC. According to Soczewin- ski’ model, analyte can have multiple interaction sites simultaneously when the adsorption sites interacts with a speciﬁc steric position of functional groups in the solute molecules with multiple functional groups. On the other hand, molecules with other positions of functional groups may have weaker or absent multiple sites interaction with the stationary phase (e.g., ortho versus meta versus para positions on an aromatic solute). This feature makes the use of NPC very suitable for the separation of positional isomers. In addition, dif- ference in the retention and selectivities of molecules of similar polarities, but different shapes, such as rigid planar, rod-like, or of a ﬂexible chain structure, are often observed in NPC. 5.4.2 Types of Stationary Phases In order to accomplish the desired separation, the selection of appropriate sta- tionary phase and eluent system is imperative. The most commonly used sta- tionary phases in normal-phase chromatography are either (a) inorganic adsorbents such as silica and alumina or (b) moderately polar chemically bonded phases having functional groups such as aminopropyl, cyanopropyl, nitrophenyl, and diol that are chemically bonded on the silica gel support [16]. Other phases that are designed for particular types of analytes have also
250 NORMAL-PHASE HPLC proved to be successful. These include modiﬁed alumina [17], titania [18], and zirconia [19–21]. Since the stationary phase in normal phase chromatography is more polar than the mobile phase, analyte retention is enhanced as the relative polarity of the stationary phase increases and the polarity of the mobile phase decreases. Retention also increases with increasing polarity and number of adsorption sites in the column. This means that retention is stronger on adsor- bents with larger speciﬁc surface areas (surface area divided by the mass of adsorbents). Generally, the strength of interaction with analytes increases in the following order: cyanopropyl < diol < aminopropyl < silica ≈ alumina sta- < tionary phases. However, strong selective interactions may change this order. The use of silica columns is less convenient for analytical applications. However, isomer and preparative separation favors the use of unmodiﬁed silica. Basic analytes are generally very strongly retained by the silanol groups in silica gel, and acidic compounds show increased afﬁnities to aminopropyl silica columns. Aminopropyl and diol-bonded stationary phases prefer com- pounds with proton–acceptor or proton–donor functional groups as in alco- hols, esters, ethers, and ketones, whereas dipolar compounds are usually more strongly retained on cyanopropyl silica than on aminopropyl or diol silica. Alumina phase has unique application in the separation of compounds with different numbers or spacing of unsaturated bonds. This is because alumina favors interaction with π electrons and often yields better selectivity than silica [16]. Despite the many desirable properties of silica, its limited pH stability (between 2 and 7.5) is also a major issue in NPC when strong acidic or basic mobile-phase additives are used to minimize interactions. Hence, other inor- ganic materials such as alumina, titania, and zirconia, which not only have the desired physical properties of silica but also are stable over a wide pH range, have been studied. Recently, Unger and co-workers [22] have chosen a com- pletely new approach where they use mesoporous particles based not only on silica but also on titania, alumina, zirconia, and alumosilicates. These materi- als have been used by the authors to analyze and separate different classes of aromatic amines, phenols, and PAHs (polyaromatic hydrocarbons). Bonded stationary phases for NPC are becoming increasingly popular in recent years owing to their virtues of faster column equilibration and being less prone to contamination by water. The use of iso-hydric (same water con- centration) solvents is not needed to obtain reproducible results. However, predicting solute retention on bonded stationary phases is more difﬁcult than when silica is used. This is largely because of the complexity of associations possible between solvent molecules and the chemically and physically het- erogeneous bonded phase surface. Several models of retention on bonded phases have been advocated, but their validity, particularly when mixed solvent systems are used as mobile phase, can be questioned. The most com- monly accepted retention mechanism is Snyder’s model, which assumes the competitive adsorption between solutes and solvent molecules on active sites
APPLICATIONS 251 of the silica surface. Several studies have shown that this model is applicable for diol- [23, 24], cyano- [23, 25], and aminopropyl-bonded silica [26, 27]. 5.5 APPLICATIONS 5.5.1 Analytes Prone to Hydrolysis NPC is ideally suited for the analysis of compounds prone to hydrolysis because it employs nonaqueous solvents for the modulation of retention. An example of the use of NPC in the analysis of a hydrolysable analyte was demonstrated by Chevalier et al. [28] for quality control of the production of benorylate, an ester of aspirin. A major issue in benorylate production is the potential formation of impurities suspected of causing allergic side effects; therefore monitoring of this step is critical to quality control. The presence of acetylsalicylic anhydride prohibited the use of RPLC since it can be easily hydrolyzed in the water-containing mobile phase. However, an analytical method based on the use of normal-phase chromatography with alkylnitrile- bonded silica as the stationary phase provided an ideal solution to the analy- sis. Optimal selectivity was achieved with a ternary solvent system: hexane–dichloromethane–methanol, containing 0.2 v/v% of acetic acid to prevent the ionization of acidic function and to deactivate the residual silanols. The method was validated and determined to be reproducible based on pre- cision, selectivity, and repeatability. Another application that demonstrates the advantages of using NPC for the separation of analytes prone to hydrolysis is the reaction monitoring for the formation of 9,10-anthraquinone [29]. Anthraquinone is an important inter- mediate in the manufacturing of various dye products but also is used as a cat- alyst in the isomerization of vegetable oils. It is produced in large amount by Friedel–Crafts reaction of phthalic anhydride with benzene in the presence of AlCl3 catalyst. The development of a normal-phase HPLC method was warranted due to the presence of phthalic anhydride, which is unstable in water. Analysis in organo- aqueous solvent systems that are used in RPLC would lead to an on-column reaction forming the respective carboxylic acid degradation product. Figure 5-5 shows the chromatogram obtained for the separation of 9,10-anthraquinone from the reactants and impurities on a silica column. The method was suc- cessfully applied to monitor the reaction conversion and also to determine the stability of 9,10-anthraquinone at the speciﬁed storage conditions.
252 NORMAL-PHASE HPLC Figure 5-5. Typical chromatogram of a reaction mixture collected during the course of reaction of phthalic anhydride with benzene in the presence of AlCl3, as catalyst. Peaks: 1, benzene; 2, anthraquinone; 3, phthalic anhydride; 4, maleic anhydride; 5, unknown. Chromatographic conditions: Column: Spherisob silica, 250 × 4.6 mm, 10 µm; mobile phase, n-heptane–ethanol–chloroform–acetic acid (89 : 5 : 5 : 1, v/v/v/v); ﬂow rate, 1 mL/ min; detection, UV at 254 nm; temperature, 27°C. (Reprinted from reference 29, with permission.) In addition, sometimes a normal-phase HPLC method at subambient tem- perature must be applied for analytes that are extremely prone to hydrolysis. In the synthesis of leukotriene D4 antagonist, accurate quantitation of mesy- late intermediate is essential for process optimization. Owing to its inherent instability, analysis of mesylate intermediate must be carried out under normal-phase conditions with nonprotic solvents; however, signiﬁcant cycliza- tion of mesylation was still observed in such condition at room temperature. The authors concluded that the on-column reaction of the mesylate was silica- catalyzed cyclization. By conducting the normal-phase HPLC analysis at −30°C, it was demonstrated that on-column cyclization was adequately inhibited [30]. 5.5.2 Extremely Hydrophobic Compounds NPC has been used in the analysis hydrophobic compounds such as polyaro- matic hydrocarbons [31–33]. An interesting example of an application of NPC involving extremely hydrophobic compounds was recently offered by Liu and
APPLICATIONS 253 Figure 5-6. A schematic of hemicarcerplex. (Reprinted from reference 34, with permission.) Warmuth [34] when they adopted NPC for the analysis of supermolecules, such as hemicarcerplexes. Hemicarcerplexes are complexes formed with hemi- carcerand host and guest molecules. As shown in Figure 5-6, hemicarcerands possess a very hydrophobic structure with molecular weight over 2000 and is insoluble in protic solvents. A normal-phase HPLC method was developed using a silica column with dichloromethane and diethylether as the mobile- phase system. The authors demonstrate that the chromatographic retention of hemicarcerplexes is mainly dominated by its size. Furthermore, a linear rela- tionship between the logarithmic retention factor and the size of the hemi- carcerplexes was observed for linear guest molecules independent of their polarity. 5.5.3 Separation of Isomers With more and more complex molecules being investigated as drug candi- dates, isomer separation has become increasingly challenging. Despite being a workhorse analytical tool, reversed-phase chromatography is limited in its ability to distinguish between isomers [35–39]. On the other hand, NPC has established itself as the technique of choice for the separation of positional isomers as well as stereoisomers due to the speciﬁc nature of interactions. The separation of positional isomers of alkyl-substituted polyaromatic hydrocar- bons (PAHs) in the petroleum industry is an example where NPC has been employed successfully. Alkyl substitution signiﬁcantly increases PAH reten- tion in RPC so that alkyl-substituted PAHs with low ring number have reten- tion times close to some of the nonsubstituted higher-ring-numbered PAHs, resulting in co-elution.Wise et al. [40] reported that aminopropyl-bonded silica
254 NORMAL-PHASE HPLC phase yields a separation sequence of PAHs solely based on the number of conjugated rings independent of the type of alkyl substitution. Separations involving cis/trans isomers can also be accomplished by employing NPC. An example of this application is the separation of tricyclic antidepressant doxepin, which is marketed as a mixture of geometric isomers in a cis/trans ratio of 15 : 85 [41]. When a spherisorb silica column is used with a hexane–methanol–nonylamine mobile-phase system, the cis isomer of doxepin elutes ﬁrst. The structures of the two isomers and the chromato- graphic separation are shown in Figure 5-7. NPC has also been successfully employed in the separation of cis/trans isomers of steroids. Four diastereomers Figure 5-7. (a) Structures of cis and trans isomers of Doxepin and (b) normal-phase chromatographic separation of isomers of doxepin. I, cis-doxepin; II, trans-doxepin; III, nortriptyline; IV, cis-N-desmethyldoxepin; V, trans-N-desmethyldoxepin. Chromato- graphic conditions: Column: Spherisob silica, 150 × 4.5 mm, 3 µm. Hexane : methanol : nonylamine, 95 : 5 : 0.3 (v/v/v); ﬂow rate, 1.0 mL/min; detection, 254 nm; temperature, 23°C. (Reprinted from reference 41, with permission.)
APPLICATIONS 255 Figure 5-8. Structures of cis and trans isomers of steroids. (Reprinted from reference 42, with permission.) consisting of two pairs of cis/trans isomers (see Figure 5-8 for structures) were separated using a silica column and hexane-dichloromethane-2-propanol mobile-phase system as shown in Figure 5-9 [42]. Other interesting examples of positional isomer separation involving NPC are (a) the separation of dihydrodipyridopyridopyrazines, a new family of antitumor agents, on a silica Nucleosil 50 Å-10 µm column [43] and (b) the separation of celecoxib isomers by Chiralpak AD column [44]. NPC was also employed successfully for the resolution of (a) four conﬁgurational isomers of a steroidal calyx pyrrole [45], (b) regio- and stereoisomers of eicosanoids [46], (c) retinal and retinol isomers [47], and (d) several E/Z isomers pairs of vitamin A [48]. In certain cases, such as the separation of PAHs obtained from a coal liquefaction process, using reversed-phase HPLC is complicated as sample preparation is elaborate. This is due in large part to the fact that most complex fuel-related materials contain compounds that are not usually soluble in acetonitrile, the solvent of choice in reversed-phase HPLC. Here, NPC, which employs a variety of solvents, offers an alternative to the analysis of such samples. Separation of ﬁve well-studied coal liquefaction process stream samples was achieved and 19 isomers were resolved when NPC was used [33]. The method employed a tetrachlorophthalimidopropyl-modiﬁed silica column (TCPP) with a charge-transfer mechanism. One of the most challenging tasks in isolating secondary metabolites from fermentation broths is the removal of numerous structural analogs of the desired product formed by the host organism. Pneumocandin B0 is a potent antifungal agent produced as a recently discovered secondary metabolite by the fermentation of Zalerion arboricola [49]. Pneumocandin B0 is the product of interest, with a molecular weight of 1069 Da. Pneumocandin C0, which differs from B0 only by a single carbon shift of hydroxyl group, is a key impurity co- produced by the fermentation. This impurity is proved to be intractable by reversed-phase chromatography or crystallization.The isomer was successfully
256 NORMAL-PHASE HPLC Figure 5-9. Chromatograms of isomers 3–6. Chromatographic conditions: Column: APEX silica, 250 × 4.6 mm, 5 µm (Jones chromatography); mobile phase, hexane- dichloromethane-2-propanol. (a) 82 : 10 : 8 (v/v/v), (b) 84 : 10 : 6 (v/v/v); ﬂow rate, 1 mL/min; detection, 280 nm; temperature, ambient. (Reprinted from reference 42, with permission.) separated in NPC mode by employing LiChrospher Silica stationary phase and an ethyl acetate/methanol/water mixture (86/7/7) mobile-phase system. 5.5.4 Carbohydrates NPC has also found some applications in the ﬁeld of carbohydrate analysis. Typical stationary phases used for this application are alkyl amine-, diol-, or polyol-bonded silicas [50–53]. Alkyl amino-bonded silicas are commonly used for the separation of saccharides and oligosaccharides in various matrixs, such as food or biological ﬂuids. Although water is used as part of mobile phase, the retention behavior of carbohydrate follows the NPC retention behavior.
REFERENCES 257 Carbohydrates are eluted in the order of increasing polarity, and retention decreases when water content increases. With an aminopropyl silica column, Koizumi et al. [54] showed the resolution of d-glycooligosaccharides up to a degree of polymerization of 30–35 under isocratic conditions, with a binary mixture of acetonitrile/water. Similarly, with the use of a polyamine polymer resin-bonded silica or an amino-bonded silica, separation of maltooligosac- charides up to a degree of polymerization of 28 was achieved [55]. 5.5.5 Separation of Saturated/Unsaturated Compounds The surface of the silica may be dynamically coated with transition metals, and the selectivities observed can be attributed to the complexes between the metal ions and the analyte species [56]. The use of silver-impregnated silica (adsorption of salts of transition metals on the silica surface) has been used for the analysis of saturated and unsaturated fatty acid methyl esters (FAME) and triacylglycerols (TAG) [57]. The retention of the unsaturated FAME and TAG can be attributed to the stability of the complex that is formed between the π electrons of the carbon–carbon double bonds and the silver ions. The predominant interaction for saturated analytes is with the polar silanol groups. The secondary interactions are those of the silver ions with the unpaired elec- trons of the carbonyl oxygens of the analytes. The amount of silver adsorbed onto the silica and the pH (employment of acidic or basic modiﬁers) have been determined to have an effect on the retention and resolution of certain acidic and basic compounds and fatty acids [58]. 5.6 CONCLUSIONS In normal-phase chromatography, polar stationary phases are employed and solutes become less retained as the polarity of the mobile-phase system increases. Retention in normal-phase chromatography is predominately based upon an adsorption mechanism. Planar surface interactions determine suc- cessful use of NPC in separation of isomers. The nonaqueous mobile-phase system used in NPC has found numerous applications for extremely hydrophobic molecules, analytes prone to hydrolysis, carbohydrates, and sat- urated/unsaturated compounds. In the future, with the advent of new station- ary phases being developed, one should expect to see increasingly more interesting applications in the pharmaceutical industry. REFERENCES 1. L. R. Snyder, J. J. Kirkland, and J. L. Glajch, Non-ionic samples: Reversed- and normal phase HPLC, in Practical HPLC Method Development, 2nd ed., Wiley, New York, 1997, pp. 266–289.
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