1.

Introduction
The use of preparative high-performance/pressure liquid chromatography (prep HPLC) has become a
mainstay in the isolation of most
classes of natural products over the last 10 yr. The relative cost of prep
HPLC systems has fallen because of increased competition, with the arrival of numerous column and
equipment manufacturers. In addition, the
constant innovation and new applications within the area of HPLC have
meant that systems, which were ‘‘state of the art’’ some 10 yr ago, are
now within the reach of most research groups. Prep HPLC is a robust, versatile, and usually rapid
technique by which
compounds can be purified from complex mixtures. The main difference
between prep HPLC and other ‘‘lower pressure’’ column chromatographic
system is the consistency and size of the particles in the stationary phase
(see Chap. 5). Particle size distribution is critical when trying to separate
a mixture of two compounds, because the smaller the particle size, better
the separating power (or resolution) between the two compounds. The
‘‘average’’ particle size of prep HPLC stationary phases is typically between
3 and 10 mm, substantially smaller than other stationary phases. The particles are synthesized to be
spherical and the size distribution to be narrow,
which allows the stationary phase to be tightly packed in a highly uniform
and reproducible manner. In addition, this minimizes the occurrence
of voids or channels, which would disrupt the mobile phase traveling
uniformly through the stationary phase and lead to inefficient separation. The small particle size results in
having to use high pressures (up
to 3–4000 psi) to push the mobile phase through the system. However,
the high surface area available for the solutes to interact with the stationary phase results in a
chromatography with high powers of resolution that
are necessary for purifying complex natural product mixtures.
Crude natural product extracts and mixtures can sometimes consist of
hundreds of compounds, and the isolation of particular components presents its own unique problems.
Invariably, a fast and efficient technique is
required to purify out the compounds of interest. However, the end requirement of the pure compounds is
what drives the size and scale of the isolation
technique used. If microgram quantities of compound are needed for initial
bioassay screening, then purification can sometimes be carried out using
analytical-scale HPLC systems, where the column internal diameter (i.d.)
is usually around 4.6 mm. Greater quantities are usually needed for structure
elucidation purposes, and a laboratory-scale prep HPLC system is required
to isolate the milligram quantities needed for NMR or X-ray crystallography. Column internal diameters
usually range from 10 to 100 mm. If gram
quantities are called for, then typically pilot plant-scale prep HPLC systems
are needed (column i.d. > 100 mm) that present their own unique issues,
though the theory behind the isolation process is essentially the same.
The materials and methods used for isolating natural products by prep
HPLC also depend upon the type of compound that is encountered in the
extract, which in turn is dependent upon the extraction procedure. A polar
extract of a plant carried out using aqueous ethanol will differ substantially in compounds encountered
than if the same plant was extracted with n-hexane. Therefore, polarity of the compound mixture is a
major deciding
factor as to which prep HPLC method is to be used.
This chapter concentrates on the practical aspects of carrying out a
lab-scale prep HPLC separation to purify natural products. It covers the
various modes of prep HPLC and selecting the right mode to achieve
separation. Instrumentation setup and detection methods, sample preparation, method development, and
sample work up are also presented.
Discussion of chromatographic theory is kept to a minimum, and further
information can be found in the excellent reviews listed under the
Suggested Readings section at the end of this chapter.
2. Materials
When considering practical aspects of prep HPLC, we also cover
stationary phases, instrumentation, and solvents used.
2.1. Modes of Separation and Stationary Phases
Prep HPLC purification of natural products typically uses one of the following four chromatographic
modes: normal-phase, reversed-phase, gel
permeation chromatography. (GPC), and ion exchange chromatography.
The modes are determined by the stationary phase and the preparative column used, and the solvents
utilized for elution. The mode to be used
depends on the compatibility of the extract or mixture with the different column modes. Table 1 illustrates
the different stationary phases available, and
the separation modes they utilize. The brand of stationary phase also plays a
significant role in the purification process. Not all C18 silicas, for example,
are the same; a separation achieved using a Waters brand column may look
completely different from a Merck brand. This column selectivity therefore
has to be taken into account when considering a separation strategy (1).
2.1.1. Normal-Phase HPLC
Normal-phase chromatography uses a polar stationary phase (usually
silica) and less polar (nonaqueous) eluting solvents. Compounds are separated by adsorption onto the
surface of the polar stationary phase as they
elute down the column and the affinity they have to the eluting nonpolar
solvent. In general, the more polar the compound, the more likely it is to
be adsorbed onto the stationary phase, and less polar compounds will be
eluted first from the column. Increasing the polarity of the eluting solvent
reduces elution time. Normal-phase HPLC is best suited to lipophilic compounds, long chain alkane
derivatives, or where the mixture of interest is sparingly soluble in aqueous conditions. It is often
successful in separating
geometric and positional isomers though not quite so in separating compounds differing only by alkyl
groups. In most cases, normal-phase HPLC has been superseded by reversed-phase HPLC. The eluants
used in normalphase HPLC are usually mixtures of aliphatic hydrocarbons (n-hexane, nheptane),
halogenated hydrocarbons (chloroform, dichloromethane), more
polar oxygenated hydrocarbons (diethyl ether, ethyl acetate, acetone), or
hydroxylated solvents such as isopropanol and methanol (see Note 1). Care
must be taken to control the aqueous content of the solvents as water deactivates silica causing a
breakdown in the separation. This problem is seen
particularly when using the hydroxylated solvents, and they should be
avoided or another separation mode used to maintain the robustness of
the separation system. In addition, the toxic and flammable nature of the
solvents must be taken into account and the prep HPLC system should be
positioned in a fume cupboard. Efforts must made to make sure the system
is ‘‘earthed’’ sufficiently to prevent the possibility of a spark being created by
static electricity causing an explosion.
2.1.2. Reversed-Phase HPLC
As the name indicates, this technique is the reverse of normal-phase
HPLC, whereby the stationary phase is more nonpolar than the eluting
solvent. Examples of reversed stationary phases are given in Table 1
(including a nonsilica-based reversed-phase HPLC sorbent). Silica-based
reversed-phase sorbents are also called ‘‘bonded-phase’’ materials, whereby the silica particles are
derivatized with alkylsilyl reagents. The degree of
silanization (or carbon loading) can result in columns from different manufacturers having substantially
varying chromatographic characteristics,
and in some cases several columns may be used for separating different
mixtures (1). The cost of columns can make it prohibitive to have more
than one or two different brands of prep HPLC column. Therefore, a compromise may have to be struck
between price and optimal separation. The
eluant used in reversed-phase HPLC commonly comprises a mixture of
water and miscible organic solvents, usually acetonitrile (MeCN), methanol (MeOH), or tetrahydrofuran
(THF). In addition, buffers, acids, or
bases may be added to suppress compound ionization or to control the
degree of ionization of free unreacted silanol groups to reduce peak tailing
and improve chromatography. The issues of free silanol groups have been
addressed in numerous other ways to improve chromatography, such as
the use of inert nonsilica supports to remove the silanol issue, or that of
end-capping in an attempt to mop up the free silanol groups. Each of these
innovations adds to the cost of the column and in some ways may not be
necessary for the particular compounds being examined. Reversed-phase HPLC lends itself well to the
purification of most classes of natural products (2). Because of this, it is usually the first technique used
when analyzing and attempting to purify compounds from a complex mixture,
especially when the identity of the compounds of interest is unknown.
2.1.3. Other Modes of Chromatography
Gel permeation chromatography (also called size exclusion chromatography) is predominantly used for
fractionating and purifying proteins
and oligosaccharides but has been used in some cases for separating lower
molecular weight molecules (see Chap. 5). The stationary phase is typically
made of rigid spherical particles of macroporous polystyrene/divinylbenzene copolymers.The
stationaryphaseisinherentlyhydrophobic(similar
to reversed-phase packing materials), and is essentially chemically and
physically inert. The pore size in the particles is strictly controlled.
Compounds are separated by their ability to enter the pores—the smaller
molecules are ‘‘trapped’’ temporarily in the pores, while larger molecules
are not held up and pass through the column relatively unhindered. The
extent of retardation of the molecules is a function of their molecular size,
and as such this type of chromatography has found a use in purifying
biomolecules. While natural product mixtures invariably contain many
compounds of similar molecular weights, gel permeation chromatography
has become a useful adjunct to the other modes of HPLC separation of
natural products where some prior knowledge of the molecular weight
of the various components may be known.
Ion exchange chromatography (see Chap. 6) uses an anionic or cationic
stationary phase for the separation of acids and amines. Compounds with a
net charge bind reversibly to the ionizable groups on the stationary phase
and are eluted through displacement of a stronger ionized species in the
eluent. The support in the stationary phase may be of a silica or a styryldivinylbenzene origin. Again, the
use of ion exchange columns assumes
that there is some prior knowledge of the chemical content of the sample
mixture and as such is not used as a first-line separation method.
2.2. Solvents
Solvents used in HPLC (see Table 2) typically have to be:
1. Of high purity to maintain the integrity of the system and sample.
2. Compatible with the detector and not interfere with the observation of one’s
target compounds, i.e., ‘‘transparent.’’ 3. Compatible with the sample (solubility and nonreactive).
4. Low viscosity to keep system back pressure low.
5. Reasonably priced (a typical prep HPLC run may use a liter or more of
solvent each time).
Furthermore, the solvents need to be ‘‘degassed’’ to remove dissolved
oxygen, which comes out of solution to form microscopic bubbles
under the high pressures seen in the system. These bubbles interfere with
the detector causing sharp spikes to be seen. There are numerous ways
to degas solvents, including applying a vacuum to the solvent or placing
the container of solvent into an ultrasonic bath before use. Most prep
HPLCs however come fitted with in-line degassers and helium ‘‘sparge’’
systems that purge the solvents with helium gas, initially and periodically,
during the use of the instrument and maintain the solvents in a degassed
state.
2.3. Buffers and Ionization Control
As the ionic state of the compounds and the stationary phases are
critical for producing efficient and reproducible chromatography, the pH
of the eluting solvents must be controlled. Keeping compounds in the
unionized form means that they are more likely to interact with the
stationary phase in reversed-phase chromatography. Buffers have been
used extensively in reversed-phase HPLC to do this, and a few of them
are listed in Table 3. Care must be taken with their use to ensure that they do not precipitate out in the presence
of organic solvents and that the salts
are removed from the final purified product (see Note 2). In addition, one
must be careful not to use excessive amount of base as most silica-based
columns are unable to operate at pHs greater than 8.
The use of buffers can be bypassed to some extent by the utilization of
straight acids or bases. This is particularly useful when the compounds are
unknown, and the use of a small amount of acid or base during the method
development phase can help greatly in achieving good chromatography.
Ion suppression of carboxylic acids in samples can be brought about
by the addition of either mineral or organic acids to the mobile phase
(Table 4). Peak tailing, caused by free silanol groups, can lead to poor
chromatography and may be overcome to some extent by adding triethylamine (0.05–0.1% v/v) to the
mobile phase. Care should be taken to ensure
that the acid and/or base is removed quickly after purification takes place
to avoid compound breakdown or unwanted reactions.