Tài liệu HPLC for Pharmaceutical Scientists 2007 (Part 1) docx

1.2 CHROMATOGRAPHIC PROCESS Chromatographic separations are based on a forced transport of the liquidmobile phase carrying the analyte mixture through the porous media andthe differences

PART I

HPLC THEORY AND PRACTICE

INTRODUCTION

Yuri Kazakevich and Rosario LoBrutto

1.1 CHROMATOGRAPHY IN THE PHARMACEUTICAL WORLD

In the modern pharmaceutical industry, high-performance liquid raphy (HPLC) is the major and integral analytical tool applied in all stages ofdrug discovery, development, and production The development of new chem-ical entities (NCEs) is comprised of two major activities: drug discovery anddrug development The goal of the drug discovery program is to investigate aplethora of compounds employing fast screening approaches, leading to gen-eration of lead compounds and then narrowing the selection through targetedsynthesis and selective screening (lead optimization) This lead to the finalselection of the most potentially viable therapeutic candidates that are takenforward to drug development The main functions of drug development are to completely characterize candidate compounds by performing drugmetabolism, preclinical and clinical screening, and clinical trials Concomi-tantly with the drug development process, the optimization of drug synthesisand formulation are performed which eventually lead to a sound and robustmanufacturing process for the active pharmaceutical ingredient and drugproduct Throughout this drug discovery and drug development paradigm,rugged analytical HPLC separation methods are developed and are tailored

chromatog-by each development group (i.e., early drug discovery, drug metabolism,pharmokinetics, process research, preformulation, and formulation) At eachphase of development the analyses of a myriad of samples are performed toadequately control and monitor the quality of the prospective drug candidates,excipients, and final products Effective and fast method development is of

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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 Thisrequires a thorough understanding of HPLC principles and theory which lay

a solid foundation for appreciating the many variables that are optimizedduring 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 andthe differences in the interactions at analytes with the surface of this porousmedia resulting in different migration times for a mixture components

In the above definition the presence of two different phases is stated andconsequently there is an interface between them One of these phases pro-vides the analyte transport and is usually referred to as the mobile phase, andthe other phase is immobile and is typically referred to as the stationary phase

A mixture of components, usually called analytes, are dispersed in the mobilephase 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 isessential for space discrimination of different components in the mixture.Analyte molecules undergo multiple phase transitions between mobile phaseand adsorbent surface Average residence time of the molecule on the sta-tionary phase surface is dependent on the interaction energy For differentmolecules with very small interaction energy difference the presence of sig-nificant surface is critical since the higher the number of phase transitions thatanalyte molecules undergo while moving through the chromatographiccolumn, 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 matographic methods

chro-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 modesthat 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) troosmotic flow, as in the case of capillary electrochromatography (CEC).

elec-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

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 porousparticles).

In liquid chromatography a similar distinction historically existed, since to

a significant extent the development of liquid chromatography reflected thepath 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] Theother form of LC is liquid–solid chromatography

Liquid chromatography was further diversified according to the type of theinteractions of the analyte with the stationary phase surface and according totheir relative polarity of the stationary and mobile phases

Since the invention of the technique, adsorbents with highly polar surfacewere used (CaCO3—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 andcovered with graphitized carbon black and later with chemically bonded alkylchains The introduction of chemically modified hydrophobic surfaces replacesthe 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 thehistorically 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 lytes for the counterions on the stationary phase surface

ana-Specific and essentially stand-alone mode of liquid chromatography is ciated with the absence or suppression of any analyte interactions with the sta-

asso-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 analyteinteractions with the surface, and the separation of the analyte molecules inthis mode is primarily based on their physical dimensions (size) The larger theanalyte molecules, the lower the possibility for them to penetrate into theporous space of the column packing material, and consequently the faster theywill move through the column The schematic of this classification is shown inFigure 1-1

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 byRussian–Italian botanist M S Tswet [2] In his paper “On the new form ofadsorption phenomena and its application in biochemical analysis” presented

on March 21, 1903 at the regular meeting of the biology section of the WarsawSociety of Natural Sciences, Tswet gave a very detailed description of thenewly 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 thatthe 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 porated his own name in the name of the method he invented

incor-The chromatographic method was not appreciated among the scientists atthe 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 thedescription of a similar separation processes More information on history ofearly 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 andlater found an original publications of M S Tswett, and in 1931 he (togetherwith Kuhn and Winterstein) published a paper [6] on purification of xantophylls on CaCO3adsorption column following the procedure described

by M S Tswet

In 1941 A J P Martin and R L M Synge at Cambridge University, in UKdiscovered partition chromatography [7] for which they were awarded theNoble Prize in 1952 In the same year, Martin and Synge published a seminalpaper [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 thatsoon followed

Figure 1-1 Classification of chromatographic modes.

Chromatography was discovered by Tswet in the form of liquid–solid matography (LSC), but its development continued for over 50 years primar-ily in the form of gas chromatography and partially as thin-layer andliquid–liquid chromatography Rebirth of liquid chromatography in its modernform 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 mostpart 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 porouslayer open-tubular columns for gas chromatography, had decided to use forliquid chromatography small glass beads with porous layer on their surface tofacilitate the mass transfer between the liquid phase and the surface Columnspacked 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 theorigin of high-performance liquid chromatography (HPLC), and the actualname for this separation method was introduced by Prof Horvath in 1970 atthe Twenty-first Pittsburgh Conference in Cleveland, where he gave this title

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 aqueouseluent, which later got the name “reversed-phase,” was also invented byHorvath Figure 1-4, he demonstrated the first reversed-phase separation offatty 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 thatmolecules of different analytes have different affinity (interactions) with theadsorbent 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

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 10−4M aqueous NaOH Refractive index detection (Reprintedfrom reference 13, with permission.)

stationary phase surface, and the original definition of the separation processgiven at its inception almost 100 years ago still holds true.

Liquid chromatography has come a long way with regard to the practicaldevelopment of HPLC instrumentation and the theoretical understanding ofdifferent 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 thedevice 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-fittingsequipped with porous frits allowing for liquid line connection (to delivermobile 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 throughwhile small particles of packing material are retained inside the column.The column is the “heart” of the chromatographic system; and it is the onlydevice where actual separation of the analyte mixture takes place Detaileddiscussion 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 tinuous operation of the system Could be equipped with an onlinedegassing system and special filters to isolate the solvent from the influ-ence of the environment

con-Pump. This provides the constant and continuous flow of the mobile phasethrough 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 intothe stream of the mobile phase before it enters the column; most moderninjectors are autosamplers, which allow programmed injections of dif-ferent volumes of samples that are withdrawn from the vials in theautosampler tray

Column. This is the heart of HPLC system; it actually produces a tion of the analytes in the mixture.A column is the place where the mobilephase is in contact with the stationary phase, forming an interface withenormous surface Most of the chromatography development in recentyears went toward the design of many different ways to enhance thisinterfacial contact (a detailed discussion is given in Chapter 3)

separa-Detector. This is a device for continuous registration of specific physical(sometimes chemical) properties of the column effluent The most

common detector used in pharmaceutical analysis is UV (ultraviolet),which allows monitoring and continuous registration of the UVabsorbance 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 greaterthan the background (mobile phase), a positive signal is obtained.

Data Acquisition and Control System. Computer-based system that controlsall parameters of HPLC instrument (eluent composition (mixing of dif-ferent solvents); temperature, injection sequence, etc.) and acquires datafrom 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), isbased on the absence of any specific analyte interactions with the stationaryphase (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 polarinteractions of the analytes in the mixture with the stationary phase Thestronger the analyte–stationary phase interaction, the longer the analyte reten-tion As with any liquid chromatography technique, NP HPLC separation is acompetitive process Analyte molecules compete with the mobile-phase mol-ecules for the adsorption sites on the surface of the stationary phase Thestronger the mobile-phase interactions with the stationary phase, the lower thedifference between the stationary-phase interactions and the analyte interac-tions, and thus the lower the analyte retention

Mobile phases in NP HPLC are based on nonpolar solvents (such as hexane,heptane, etc.) with the small addition of polar modifier (i.e., methanol, ethanol).Variation of the polar modifier concentration in the mobile phase allows forthe control of the analyte retention in the column Typical polar additives arealcohols (methanol, ethanol, or isopropanol) added to the mobile phase in rel-atively small amounts Since polar forces are the dominant type of interactionsemployed and these forces are relatively strong, even only 1 v/v% variation ofthe polar modifier in the mobile phase usually results in a significant shift inthe analyte retention.

Packing materials traditionally used in normal-phase HPLC are usuallyporous oxides such as silica (SiO2) or alumina (Al2O3) Surface of these sta-tionary phases is covered with the dense population of OH groups, whichmakes these surfaces highly polar Analyte retention on these surfaces is verysensitive to the variations of the mobile-phase composition Chemically modified stationary phases can also be used in normal-phase HPLC Silicamodified with trimethoxy glycidoxypropyl silanes (common name: diol-phase)

is typical packing material with decreased surface polarity Surface density of

OH groups on diol phase is on the level of 3–4µmol/m2, while on bare silicasilanols surface density is on the level of 8µmol/m2 The use of diol-type sta-tionary-phase and low-polarity eluent modifiers [esters (ethyl acetate) instead

of alcohols] allow for increase in separation ruggedness and reproducibility,compared to bare silica

Selection of using normal-phase HPLC as the chromatographic method ofchoice is usually related to the sample solubility in specific mobile phases.Since NP uses mainly nonpolar solvents, it is the method of choice for highlyhydrophobic compounds (which may show very stronger interaction inreversed-phase HPLC), which are insoluble in polar or aqueous solvents.Figure 1-5 demonstrates the application of normal-phase HPLC for the sepa-ration 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 stationaryphases, is given in Chapter 5

1.6.2 Reversed-Phase HPLC (RP HPLC or RPLC)

As opposed to normal-phase HPLC, reversed-phase chromatography employsmainly dispersive forces (hydrophobic or van der Waals interactions) Thepolarities of mobile and stationary phases are 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 phy Almost 90% of all analyses of low-molecular-weight samples are carriedout using RP HPLC One of the main drivers for its enormous popularity isthe ability to discriminate very closely related compounds and the ease of vari-ation of retention and selectivity The origin of these advantages could be

explained from an energetic point of view: Dispersive forces employed in thisseparation mode are the weakest intermolecular forces, thereby making theoverall background interaction energy in the chromatographic system verylow compared to other separation techniques This low background energyallows for distinguishing very small differences in molecular interactions ofclosely related analytes As an analogy, it is possible to compare two spectro-scopic techniques: UV and fluorescence spectroscopy In fluorescence spec-troscopy, emission registers essentially against zero background light energy,which makes its sensitivity several orders of magnitude higher than in UVspectroscopy, where background energy is very high A similar situation is in

RP HPLC, where its sensitivity to the minor energetic differences inanalyte–surface interactions is very high attributed to the low backgroundinteraction energy

Adsorbents employed in this mode of chromatography are porous rigidmaterials with hydrophobic surfaces In all modes of HPLC with positiveanalyte surface interactions (NP, RP, IEX) the higher the adsorbent surfacearea, the longer the analyte retention and in most cases the better separation.The majority of packing materials used in RP HPLC are chemically modifiedporous 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 inthe last 30 years, mainly as a direct result of growing popularity of reversed-phase HPLC [16, 17] Despite the intensive research and enormous growth

of commercially available packing materials and columns, there is still no

Figure 1-5 Separation of selected representatives of different lipid classes (1)

Paraf-fin, (2) n-hexadecyl palmitate; (3) cholesterol palmitate; (4) stearic acid methyl ester;

(5) glycerol tripalmitate; (6) hexadecyl alchohol; (7) stearic acid; (8) cholesterol; (9)glycerol-1,3-dipalmitate; (10) glycerol-1,2-dipalmitate; (11) glycerol monopalmitate;(12) erucylamide Column LiChrosphere®Diol (125 × 3 mm) 5-µm particles Gradientfrom isooctane (A) to 60% methyl tritbutyl ether (MTBE) in 34 min + 10 min isocratichold (Reprinted from reference 14, with permission.)