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

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-

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],

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.