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 241 HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright © 2007 by John Wiley & Sons, Inc. 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 (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 (5-2) ln lnkk A A N S E E21 () = () − () ln SnE SnE maam +↔+ 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.) Here, A S is the solute cross-sectional area, A E is the molecular area of the strong solvent, N E is the mole fraction of the strong solvent in the mobile phase, k 2 is retention factor of the solute in the binary mobile-phase mixture, and k 1 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 (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: (5-4) where d is a constant. Comparison of the two models reveals that both predict a linear log k 2 versus log N E 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 log logkdq N E2 =− SqEA SAqE ma m +↔+--** THEORY OF RETENTION IN NORMAL-PHASE CHROMATOGRAPHY 243 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 A S and N E 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: (5-5) where a′ is an adsorbent activity factor, e 0 1 and e 0 2 are solvent strengths for solvent 1 and 2, and A S 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: (5-6) When a nonlocalizing, nonpolar solvent such as hexane is employed as a weak solvent, the equation can be further simplified so that log logkkaA S21 1 0 2 0 21 =− ′ − () +− () εε ∆∆ log logkkaA S21 1 0 2 0 =− ′ − () εε 244 NORMAL-PHASE HPLC (5-7) assuming hexane does not induce any secondary solvent effects and its solvent strength is zero. Here k h 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 k 2 versus e 0 2 plot, provided that the molecular cross 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 log logkkaA hS22 0 2 =− ′ +ε∆ EFFECT OF MOBILE PHASE ON RETENTION 245 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 246 NORMAL-PHASE HPLC TABLE 5-1. NPC Solvent Strength (e 0 ) and Selectivity a of Various Solvents Employed in HPLC Solvent ε 0 Localization Basic? UV b 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 MTBE d 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. 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 EFFECT OF MOBILE PHASE ON RETENTION 247 Figure 5-2. Snyder’s selectivity triangle for solvents. (Reprinted from reference 9, 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 248 NORMAL-PHASE HPLC Figure 5-3. Selected solvents for mobile-phase optimization in NPC. (Reprinted from reference 11, 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 SELECTIVITY 249 Figure 5-4. Procedure for selectivity optimization in NPC based on mixtures with hexane of nonlocalizing solvent (CH 2 Cl 2 ), 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.) 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 250 NORMAL-PHASE HPLC [...]... Lewis base-modified zirconia for normal phase chromatography, J Chromatogr A 691 (19 95), 205–212 22 U Trüdinger, G Müller, and K K Unger, Porous zirconia and titania as packing materials for high-performance liquid chromatography, J Chromatogr A 535 (1990), 111–125 23 P L Smith and W T Cooper, Retention and selectivity in amino, cyano and diol normal bonded phase high-performance liquid chromatographic... Chromatographia 52 (2000), 564–568 33 D E McKinney, D J Clifford, L Hou, M R Bogdan, and P G Hatcher, High performance liquid chromatography (HPLC) of coal liquefaction process streams using normal-phase separation with diode array detections, Energy Fuels 9 (19 95), 90–96 34 Y Liu and R Warmuth, A “through-shell” binding isotope effect, Angew Chem Int Ed 44 (20 05), 7107–7110 35 M M Mendes-Pinto, A C Ferreira,... aminosilane stationary phase for the high-performance liquid chromatographic separation of polynuclear aromatic compounds, Anal Chem 49 (1977), 2306–2310 J Yan, J W Hubbard, G McKay, and K K Midha, New micro-method for the determination of lamotrigine in human plasma by high-performance liquid chromatography, J Chromatogr B 691 (1997), 131–138 J Wölfling, G Schneider, and A Péter, High-performance liquid chromatographic... of homogeneous d-glucooligosaccharides and -Polysaccharides (degree of polymerization up to about 35) by high-performance Liquid chromatography and thin-layer chromatography, J Chromatogr 321 (19 85), 145–157 55 K Koizumi, T Utamura, and S Hizukuri, Two high-performance liquid chromatographic columns for analyses of malto-oligosaccharides, J Chromatogr 409 (1987), 396–403 56 R LoBrutto, in J Cazes (ed.),... 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... 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... applications for extremely hydrophobic molecules, analytes prone to hydrolysis, carbohydrates, and saturated/unsaturated compounds In the future, with the advent of new stationary 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, ... Chromatogr 238 (1982), 269–280 16 P Jandera, Comparison of various modes and phase systems for analytical HPLC, in K Valkó (ed.), Handbook of Analytical Separations, Separation Methods in Drug Synthesis and Purification, Vol 1, Elsevier, New York, 2000, pp 1–71 17 C Laurent, H Billiet, and L De Galan, On the use of alumina in HPLC with aqueous mobile phases at extreme pH, Chromatographia 17 (1983), 253–258 18... Preparation and evaluation of octadecyl titania as column-packing material for highperformance liquid chromatography, Microchem J 49 (1994), 362–367 19 J Nawrocki, M Rigney, A McCormick, and P W Carr, Chemistry of zirconia and its use in chromatography, J Chromatogr A 657 (1993), 229–282 20 H J Wirth and M T W Hearn, High-performance liquid chromatography of amino acids, peptides and proteins CXXX Modified... validated and determined to be reproducible based on precision, 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 intermediate in the manufacturing of various dye products but also is used as a catalyst in . normal-phase chromatography 241 HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright © 2007 by John Wiley & Sons,. 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