PART I HPLC THEORY AND PRACTICE 1 INTRODUCTION Yuri Kazakevich and Rosario LoBrutto 1.1 CHROMATOGRAPHY IN THE PHARMACEUTICAL WORLD In the modern pharmaceutical industry, high-performance liquid chromatog- raphy (HPLC) is the major and integral analytical tool applied in all stages of drug discovery, development, and production. The development of new chem- ical entities (NCEs) is comprised of two major activities: drug discovery and drug development. The goal of the drug discovery program is to investigate a plethora of compounds employing fast screening approaches, leading to gen- eration of lead compounds and then narrowing the selection through targeted synthesis and selective screening (lead optimization). This lead to the final selection of the most potentially viable therapeutic candidates that are taken forward to drug development. The main functions of drug development are to completely characterize candidate compounds by performing drug metabolism, preclinical and clinical screening, and clinical trials. Concomi- tantly with the drug development process, the optimization of drug synthesis and formulation are performed which eventually lead to a sound and robust manufacturing process for the active pharmaceutical ingredient and drug product. Throughout this drug discovery and drug development paradigm, rugged analytical HPLC separation methods are developed and are tailored by each development group (i.e., early drug discovery, drug metabolism, pharmokinetics, process research, preformulation, and formulation). At each phase of development the analyses of a myriad of samples are performed to adequately control and monitor the quality of the prospective drug candidates, excipients, and final products. Effective and fast method development is of 3 HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright © 2007 by John Wiley & Sons, Inc. paramount importance throughout this drug development life cycle. This requires a thorough understanding of HPLC principles and theory which lay a solid foundation for appreciating the many variables that are optimized during fast and effective HPLC method development and optimization. 1.2 CHROMATOGRAPHIC PROCESS Chromatographic separations are based on a forced transport of the liquid (mobile phase) carrying the analyte mixture through the porous media and the differences in the interactions at analytes with the surface of this porous media resulting in different migration times for a mixture components. In the above definition the presence of two different phases is stated and consequently there is an interface between them. One of these phases pro- vides the analyte transport and is usually referred to as the mobile phase, and the other phase is immobile and is typically referred to as the stationary phase. A mixture of components, usually called analytes, are dispersed in the mobile phase at the molecular level allowing for their uniform transport and interac- tions with the mobile and stationary phases. High surface area of the interface between mobile and stationary phases is essential for space discrimination of different components in the mixture. Analyte molecules undergo multiple phase transitions between mobile phase and adsorbent surface. Average residence time of the molecule on the sta- tionary phase surface is dependent on the interaction energy. For different molecules with very small interaction energy difference the presence of sig- nificant surface is critical since the higher the number of phase transitions that analyte molecules undergo while moving through the chromatographic column, the higher the difference in their retention. The nature of the stationary and the mobile phases, together with the mode of the transport through the column, is the basis for the classification of chro- matographic methods. 1.3 CLASSIFICATION The mobile phase could be either a liquid or a gas, and accordingly we can subdivide chromatography into liquid chromatography (LC) or gas chromatography (GC). Apart from these methods, there are two other modes that use a liquid mobile phase, but the nature of its transport through the porous stationary phase is in the form of either (a) capillary forces, as in planar chromatography (also called thin-layer chromatography, TLC), or (b) elec- troosmotic flow, as in the case of capillary electrochromatography (CEC). The next classification step is based on the nature of the stationary phase. In gas chromatography it could be either a liquid or a solid; accordingly, we 4 INTRODUCTION distinguish gas–liquid chromatography (long capillary coated with a thin film of relatively viscous liquid or liquid-like polymer; in older systems, liquid-coated porous particles were used) and gas–solid chromatography (capillary with thin porous layer on the walls or packed columns with porous particles). In liquid chromatography a similar distinction historically existed, since to a significant extent the development of liquid chromatography reflected the path that was taken by gas chromatography development. Liquid–liquid chro- matography existed in the early 1970s, but was mainly substituted with liquid chromatography with chemically bonded stationary phases. Recently, liquid–liquid chromatography resurfaced in the form of countercurrent chro- matography with two immiscible liquid phases of different densities [1]. The other form of LC is liquid–solid chromatography. Liquid chromatography was further diversified according to the type of the interactions of the analyte with the stationary phase surface and according to their relative polarity of the stationary and mobile phases. Since the invention of the technique, adsorbents with highly polar surface were used (CaCO 3 —Tswett, porous silica—most of the modern packing mate- rials) together with relatively non-polar mobile phase. In 1964, Horvath intro- duced a chemically modified surface where polar groups were shielded and covered with graphitized carbon black and later with chemically bonded alkyl chains. The introduction of chemically modified hydrophobic surfaces replaces the main analyte—surface interactions from polar to the hydrophobic ones, while mobile phase as an analyte carrier became polar. The relative polarity of the mobile and stationary phases appears to be “reversed” compared to the historically original polar stationary phase and non-polar mobile phase used by M. S. Tswet. This new mode of liquid chromatography became coined as reversed-phase liquid chromatography (RP), where “reversed-phase” referred to the reversing of the relative polarity of the mobile and stationary phases. In order to distinguish this mode from the old form of liquid chromatography, the old became known as normal-phase (NP). The third mode of liquid chromatography, which is based on ionic interac- tions of the analyte with the stationary phase, is called ion-exchange (IEX). The separation in this mode is based on the different affinity of the ionic ana- lytes for the counterions on the stationary phase surface. Specific and essentially stand-alone mode of liquid chromatography is asso- ciated with the absence or suppression of any analyte interactions with the sta- tionary phase, which is called size-exclusion chromatography (SEC). In SEC the eluent is selected in such a manner that it will suppress any possible analyte interactions with the surface, and the separation of the analyte molecules in this mode is primarily based on their physical dimensions (size).The larger the analyte molecules, the lower the possibility for them to penetrate into the porous space of the column packing material, and consequently the faster they will move through the column. The schematic of this classification is shown in Figure 1-1. CLASSIFICATION 5 1.4 HISTORY OF DISCOVERY AND EARLY DEVELOPMENT (1903–1933) Chromatography as a physicochemical method for separation of complex mixtures was discovered at the very beginning of the twentieth century by Russian–Italian botanist M. S. Tswet. [2]. In his paper “On the new form of adsorption phenomena and its application in biochemical analysis” presented on March 21, 1903 at the regular meeting of the biology section of the Warsaw Society of Natural Sciences, Tswet gave a very detailed description of the newly discovered phenomena of adsorption-based separation of complex mix- tures, which he later called “chromatography” as a transliteration from Greek “color writing” [3]. Serendipitously, the meaning of the Russian word “tswet” actually means color. Although in all his publications Tswet mentioned that the origin of the name for his new method was based on the colorful picture of his first separation of plant pigments (Figure 1-2), he involuntarily incor- porated his own name in the name of the method he invented. The chromatographic method was not appreciated among the scientists at the time of the discovery, as well as after almost 10 years when L. S. Palmer [4] in the United States and C. Dhere in Europe independently published the description of a similar separation processes. More information on history of early discovery and development of chromatography could be found in refer- ence 5. Twenty-five years later in 1931, Lederer read the book of L. S. Palmer and later found an original publications of M. S. Tswett, and in 1931 he (together with Kuhn and Winterstein) published a paper [6] on purification of xantophylls on CaCO 3 adsorption column following the procedure described by M. S. Tswet. In 1941 A. J. P. Martin and R. L. M. Synge at Cambridge University, in UK discovered partition chromatography [7] for which they were awarded the Noble Prize in 1952. In the same year, Martin and Synge published a seminal paper [8] which, together with the paper of A. T. James and A. J. P. Martin [9], laid a solid foundation for the fast growth of chromatographic techniques that soon followed. 6 INTRODUCTION Figure 1-1. Classification of chromatographic modes. Chromatography was discovered by Tswet in the form of liquid–solid chro- matography (LSC), but its development continued for over 50 years primar- ily in the form of gas chromatography and partially as thin-layer and liquid–liquid chromatography. Rebirth of liquid chromatography in its modern form and its enormously fast growth had driven this to be the dominant ana- lytical technique in the twenty-first century which can be attributed in the most part to the pioneering work of Prof. C. Horvath at Yale University. In the mid- 1960s Prof. Horvath, who previously worked on the development of a porous layer open-tubular columns for gas chromatography, had decided to use for liquid chromatography small glass beads with porous layer on their surface to facilitate the mass transfer between the liquid phase and the surface. Columns packed with those beads developed a significant resistance to the liquid flow, and Prof. Horvath was forced to build an instrument that allowed develop- ment of a continuous flow of the liquid through the column [11]. This was the origin of high-performance liquid chromatography (HPLC), and the actual name for this separation method was introduced by Prof. Horvath in 1970 at the Twenty-first Pittsburgh Conference in Cleveland, where he gave this title HISTORY OF DISCOVERY AND EARLY DEVELOPMENT (1903–1933) 7 Figure 1-2. Tswet’s original drawings of his experiments. From M. S. Tswet, “Chromophils in the plant and animal world” [10]. See color plate. to his invited talk. Later in 2001, he further defined the meaning of the word “performance” as “an aggregate of the efficiency parameters” shown in Figure 1-3. The first separation on a chemically modified surface with an aqueous eluent, which later got the name “reversed-phase,” was also invented by Horvath Figure 1-4, he demonstrated the first reversed-phase separation of fatty acids on pellicular glass beads covered with graphitized carbon black. 1.5 GENERAL SEPARATION PROCESS M. S.Tswet defined the fractional adsorption process, with the explanation that molecules of different analytes have different affinity (interactions) with the adsorbent surface, and analytes with weaker interactions are less retained [3]. In modern high-performance liquid chromatography the separation of the analytes is still based on the differences in the analyte affinity for the 8 INTRODUCTION Figure 1-3. Components of performance as defined by C. Horvath. (Reprinted from reference 12, with permission.) Figure 1-4. Separation of fatty acids on pellicular graphitized carbon black from the mixture of ethanol and 10 −4 M aqueous NaOH. Refractive index detection. (Reprinted from reference 13, with permission.) stationary phase surface, and the original definition of the separation process given at its inception almost 100 years ago still holds true. Liquid chromatography has come a long way with regard to the practical development of HPLC instrumentation and the theoretical understanding of different mechanisms involved in the analyte retention as well as the devel- opment of adsorbents with different geometries and surface chemistry. 1.5.1 Modern HPLC Column The separation of analyte mixtures in modern HPLC is performed in the device called the “column.” Current HPLC columns in most cases are a stain- less steel tube packed with very small (1–5µm) particles of rigid porous mate- rial. Packing material is retained inside the column with special end-fittings equipped with porous frits allowing for liquid line connection (to deliver mobile phase to the column). Stainless steel or titanium frits have a pore size on the level of 0.2–0.5µm, which allows for the mobile phase to pass through while small particles of packing material are retained inside the column. The column is the “heart” of the chromatographic system; and it is the only device where actual separation of the analyte mixture takes place. Detailed discussion of HPLC columns and stationary phases is given in chapter 3. 1.5.2 HPLC System Typical HPLC system consists of the following main components: Solvent Reservoirs. Storage of sufficient amount of HPLC solvents for con- tinuous operation of the system. Could be equipped with an online degassing system and special filters to isolate the solvent from the influ- ence of the environment. Pump. This provides the constant and continuous flow of the mobile phase through the system; most modern pumps allow controlled mixing of dif- ferent solvents from different reservoirs. Injector. This allows an introduction (injection) of the analytes mixture into the stream of the mobile phase before it enters the column; most modern injectors are autosamplers, which allow programmed injections of dif- ferent volumes of samples that are withdrawn from the vials in the autosampler tray. Column. This is the heart of HPLC system; it actually produces a separa- tion of the analytes in the mixture.A column is the place where the mobile phase is in contact with the stationary phase, forming an interface with enormous surface. Most of the chromatography development in recent years went toward the design of many different ways to enhance this interfacial contact (a detailed discussion is given in Chapter 3). Detector. This is a device for continuous registration of specific physical (sometimes chemical) properties of the column effluent. The most GENERAL SEPARATION PROCESS 9 common detector used in pharmaceutical analysis is UV (ultraviolet), which allows monitoring and continuous registration of the UV absorbance at a selected wavelength or over a span of wavelengths (diode array detection). Appearance of the analyte in the detector flow- cell causes the change of the absorbance. If the analyte absorbs greater than the background (mobile phase), a positive signal is obtained. Data Acquisition and Control System. Computer-based system that controls all parameters of HPLC instrument (eluent composition (mixing of dif- ferent solvents); temperature, injection sequence, etc.) and acquires data from the detector and monitors system performance (continuous moni- toring of the mobile-phase composition, temperature, backpressure, etc.). 1.6 TYPES OF HPLC The four main types of HPLC techniques are NP, RP, IEX, and SEC (Section 1.2). The principal characteristic defining the identity of each technique is the dominant type of molecular interactions employed. There are three basic types of molecular forces: ionic forces, polar forces, and dispersive forces. Each specific technique capitalizes on each of these specific forces: 1. Polar forces are the dominant type of molecular interactions employed in normal-phase HPLC (see Chapter 5). 2. Dispersive forces are employed in reversed-phase HPLC (see Chapter 4). 3. Ionic forces are employed in ion-exchange HPLC (see Chapter 4, Section 4.10). The fourth type of HPLC technique, size-exclusion HPLC (see Chapter 6), is based on the absence of any specific analyte interactions with the stationary phase (no force employed in this technique). An introduction to the basic principles and typical application areas of each of the above-mentioned HPLC modes is given below. 1.6.1 Normal-Phase Chromatography (NP HPLC) Normal-phase HPLC explores the differences in the strength of the polar interactions of the analytes in the mixture with the stationary phase. The stronger the analyte–stationary phase interaction, the longer the analyte reten- tion. As with any liquid chromatography technique, NP HPLC separation is a competitive process. Analyte molecules compete with the mobile-phase mol- ecules for the adsorption sites on the surface of the stationary phase. The stronger the mobile-phase interactions with the stationary phase, the lower the difference between the stationary-phase interactions and the analyte interac- tions, and thus the lower the analyte retention. 10 INTRODUCTION [...]... application of normal-phase HPLC for the separation of a mixture of different lipids Detailed discussion of normal-phase chromatography process, mechanism, and retention theories, as well as types and properties of used stationary phases, is given in Chapter 5 1.6.2 Reversed-Phase HPLC (RP HPLC or RPLC) As opposed to normal-phase HPLC, reversed-phase chromatography employs mainly dispersive forces (hydrophobic... conformation and degree of the solvation varies with the variation of the solvent properties Detailed discussion of all aspects of size exclusion chromatography is given in Chapter 6 HPLC DESCRIPTORS (Vr, k, N, etc.) 15 1.7 HPLC DESCRIPTORS (Vr, k, N, etc.) 1.7.1 Retention Volume Modern HPLC is a routine tool in any analytical laboratory Standard HPLC system represents a separation output in the form... reversed, such that the surface of the stationary phase in RP HPLC is hydrophobic and mobile phase is polar, where mainly water-based solutions are employed Reversed-phase HPLC is by far the most popular mode of chromatography Almost 90% of all analyses of low-molecular-weight samples are carried out using RP HPLC One of the main drivers for its enormous popularity is the ability to discriminate very... different affinities of the analyte ions for the oppositely charged ionic centers in the resin or adsorbed counterions in the hydrophobic stationary phase Consider the exchange of two ions A+ and B+ between the solution and exchange resin E−: A · E + B+ ⇔ B · E + A+ The equilibrium constant for this process is shown in Eq (1 -1): K= [ A + ][ BE ] [ AE ][ B + ] (1 -1) which essentially determines the relative... allow for increase in separation ruggedness and reproducibility, compared to bare silica Selection of using normal-phase HPLC as the chromatographic method of choice is usually related to the sample solubility in specific mobile phases Since NP uses mainly nonpolar solvents, it is the method of choice for highly hydrophobic compounds (which may show very stronger interaction in reversed-phase HPLC) ,... 14, with permission.) explained from an energetic point of view: Dispersive forces employed in this separation mode are the weakest intermolecular forces, thereby making the overall background interaction energy in the chromatographic system very low compared to other separation techniques This low background energy allows for distinguishing very small differences in molecular interactions of closely... used in RP HPLC are chemically modified porous silica The properties of silica have been studied for many years [15, 16], and the technology of manufacturing porous spherical particles of controlled size and porosity is well-developed Chemical modification of the silica surface was also intensively studied in the last 30 years, mainly as a direct result of growing popularity of reversedphase HPLC [16,... the intensive research and enormous growth of commercially available packing materials and columns, there is still no 13 TYPES OF HPLC consensus on which properties the optimum RP stationary phase should have for the selective analysis of diverse sets of compounds such as pharmaceutical compounds that have a plethora of various ionizable functionalities, varying hydrophobicities, and different structural... added to the mobile phase in relatively small amounts Since polar forces are the dominant type of interactions employed and these forces are relatively strong, even only 1 v/v% variation of the polar modifier in the mobile phase usually results in a significant shift in the analyte retention Packing materials traditionally used in normal-phase HPLC are usually porous oxides such as silica (SiO2) or alumina... radius, the shorter the retention Historically, two different names are used for this method In 1959 the molecular sieving principle was applied for the separation of biochemical polymers on dextran gels, and it was called gel-filtration chromatography (GFC) (uses aqueous-based eluents with salts) In 1961 the same principle was applied for the molecular weight determination of synthetic polymers, and the name . molecular forces: ionic forces, polar forces, and dispersive forces. Each specific technique capitalizes on each of these specific forces: 1. Polar forces are. fast method development is of 3 HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright © 2007 by John Wiley & Sons,