THPP 2022 1

Appendix 4.2 Liquid chromatography

(THP 2021, Appendix 3.5)

The term Liquid chromatography (LC), in the Pharmacopoeia, is referred to as

HPLC, both high–pressure and high–performance. It is a separation technique based on a
soild stationary phase and a liquid mobile phase. Separations are achieved by partition (mass
distribution), adsorption, ion-exchange, or stereochemical-interaction processes, depending
upon the type of stationary phase used. Unless otherwise specified, all information below is
valid for LC as well as for LC using reduced particle-size columns (e.g., sub-2 m). The
latter requires instrumentation characterized by the capability to apply higher pressures
(typically up to 100 MPa, i.e., about 15,000 psi), lower extra-column band broadening,
improved gradient mixing and a higher sampling rate in the detection system.
Apparatus
A liquid chromatograph consists of a reservoir containing the mobile phase, a pump
to force the mobile phase through the system at high pressure, an injector to introduce the
sample into the mobile phase, a chromatographic column, a detector, and a data acquisition
system.
PUMPING SYSTEMS LC pumping systems deliver metered amounts of mobile
phase from the solvent reservoirs to the column through high-pressure tubing and fittings.
Microprocessor-controlled pumping system are capable of accurately delivering a mobile
phase of either constant (isocratic elution) or varying composition (gradient elution),
according to a defined programme. In the case of gradient elution, pumping systems which
deliver solvent(s) from several reservoirs are available and solvent mixing can be achieved
on either the low- or hight-pressure side of the pump(s).
INJECTORS After dissolution in mobile phase or other suitable solution,
compounds to be chromatographed are injected into the mobile phase. Fixed-loop and
variable volume devices operated manually or by an auto-sampler are used.
STATIONARY PHASES For most pharmaceutical analyses, separation is achieved
by partition of compounds in the test solution between the mobile phase and the stationary
phase. A system consisting of a polar stationary phase and a nonpolar mobile phase is
described as normal-phase chromatography, while the opposite arrangement, a polar mobile
phase and a nonpolar stationary phase, is called reversed-phase chromatography.
There are many types of stationary phase materials employed in LC, including:
− silica, alumina or porous graphite, used in normal-phase chromatography, where
the separation is based on differences in adsorption and/or mass distribution (partition
chromatography),
− resins or polymers with acid or basic groups, used in ion-exchange
chromatography, where separation is based on competition between the ions to be separated
and those in the mobile phase,
2 THPP 2022

− porous silica or polymers, used in size-exclusion chromatography, where

separation is based on differences between the volumes of the molecules, corresponding to
steric exclusion,
− a variety of chemically modified supports prepared from polymers, silica or
porous graphite, used in normal-phase (adsorption chromatography) and reversed-phase LC,
where the separation is based principally on partition of the molecules between the mobile
phase and the stationary phase,
− special chemically modified stationary phases, e.g., cellulose or amylose
derivatives, proteins or peptides, cyclodextrins, etc., for the separation of enantiomers
(chromatography on chiral stationary phases).
Most separations are based upon partition mechanisms utilizing chemically
modified silica as the stationary phase and polar solvents as the mobile phase. The surface
of the support, e.g., the silanol groups of silica, is reacted with various silane reagents to
produce covalently bound silyl derivatives covering a varying number of active sites on the
surface of the support. The nature of the bonded phase is an important parameter for
determining the separation properties of the chromatographic system.

Commonly used bonded phases are shown below:

octyl (C8) = Si-[CH2]7-CH3
octadecyl (C18) = Si-[CH2]17-CH3
phenyl (C6H5) = Si-[CH2]n-C6H5
cyanopropyl (CN) = Si-[CH2]3-CN
aminopropyl (NH2) = Si-[CH2]3-NH2
diol = Si-[CH2]3-O-CH(OH)-CH2-OH

Unless otherwise stated by the manufacturer, silica-based reversed-phase columns

are considered to be stable in mobile phases having an apparent pH in the range of 2.0 to
8.0. Columns containing porous graphite or particles of polymeric materials such as
styrenedivinylbenzene copolymer are stable over a wider pH range. Analysis using normal-
phase chromatography with unmodified silica, porous graphite or polar chemically modified
silica, e.g., cyanopropyl or diol, as the stationary phase with a nonpolar mobile phase is
applicable in certain cases. For analytical separations, the partical size of the most commonly
used stationary phases varies between 2 m and 10 m. The particles may be spherical or
irregular, of varying porosity and specific surface area. These properties contribute to the
chromatographic behaviour of a particular stationary phase. In the case of reversed phases,
the nature of the stationary phase, the extent of bonding, e.g., expressed as the carbon
loading, and whether the stationary phase is end-capped (i.e., part of the residual silanol
groups being silylated) are additional determining factors. Tailing of peaks, particularly of
basic substances, can occur when residual silanol groups are present. In addition to porous
particles, superficially porous or monolithic materials may be used.
THPP 2022 3

COLUMNS Columns, made of stainless steel, lined stainless steel, and polymers of
varying length and internal diameter are used for analytical chromatography. Columns with
internal diameters of less than 2 mm are often referred to as microbore columns. The
temperature of the mobile phase and the column must be kept constant during an analysis.
Most separations are performed at room temperature, but some require a different
temperature for optimal performance.
In LC procedures, a guard column may be used with the following requirements,
unless otherwise indicated in the individual monograph: (a) the length of the guard column
must be not more than 15% of the length of the analytical column, (b) the internal diameter
must be the same or smaller than that of the analytical column, and (c) the packing material
should be the same as the analytical column and contain the same bonded phase. In any case,
all system suitability requirements specified in the official procedure must be met with the
guard column installed.
MOBILE PHASES
For normal-phase chromatography, less polar organic solvents are generally
employed. The residual water content of the solvents used in the mobile phase is to be strictly
controlled to obtain reproducible results. In reversed-phase LC, aqueous phases, with or
without organic solvents, are employed.
Components of the mobile phase are usually filtered to remove particles greater than
0.45 m (or 0.2 m when the stationary phase is made of sub-2 m particles and when
special detectors, e.g., light scattering detectors, are used). Solvents are normally degassed
before pumping by sparging with helium, sonication and / or using on-line membrane /
vacuum modules to avoid the creation of gas bubbles in the detector cell. Solvents for the
preparation of the mobile phase are normally free of stabilizers and, if an ultraviolet detector
is employed, are transparent at the wavelength of detection. Solvents and other components
employed are to be of appropriate quality. Adjustment of the pH, if necessary, is effected
using only the aqueous component of the mobile phase and not the mixture. If buffer
solutions or saline solutions are used, adequate rinsing of the system is carried out with a 5%
v/v mixture of the organic part of the mobile phase in water to prevent crystallization of salts
after completion of the analysis. Mobile phases may contain other components, e.g., a
counter-ion for ion-pair chromatography or a chiral selector for chromatography using an
achiral stationary phase.
DETECTORS
Ultraviolet / visible (UV / Vis) spectrophotometers, including diode array detectors,
are the most commonly employed detectors. Fluorescence spectrophotometers, differential
refractometers (RI), electrochemical detectors (ECD), light scattering detectors, charged
aerosol detectors (CAD), mass spectrometers (MS), radioactivity detectors or other special
detectors may be used.
4 THPP 2022

Procedure
Criteria for assessing the suitability of the system are described in the
“Chromatographic Separation Techniques” (THP 2021, Appendix 3.9). The extent to which
adjustments of parameters of the chromatographic system can be made to satisfy the criteria
of system suitability are also given in this Appendix.
In general, the steps involved in the liquid chromatographic technique are as
follows:
1. Equilibrate the column and detector with the mobile phase at the specified flow
rate until a stable baseline is achieved.
2. Prepare the sample and the standard solutions. The solutions must be free from
soild particles.
3. Inject the standard / sample solution through the injector, or use an autosampler.
4. Perform the isocratic / gradient programme.
5. Record the chromatogram.
6. Analyze as specified in the monograph.
In quantitative work, particularly where the use of an internal standard is not
specified in the monograph, the use of a fixed-volume loop injector is recommended. In
certain exceptional cases the use of peak heights alone is prescribed in the monograph; where
this is the case peak heights should be used irrespective of the symmetry factor.
The column is usually made of stainless steel and its dimensions are stated in the
monograph as length × internal diameter. The nominal diameter of the particles of the
stationary phase is stated in parentheses following the name of stationary phase prescribed
in the monograph. In most cases reference is made to a particular commercial brand that has
been found to be suitable for the purpose, but such statements do not imply that a different
but equivalent commercial brand may not be used. The separation should be carried out at a
constant room temperature unless otherwise specified in the monograph. When using mobile
phases of high pH with a silica-based column, it is advisable to use a pre-column before the
analytical column.
Unless otherwise specified in the monograph, the detector consists of a photometric
detector fitted with a low-volume flow cell (about 10 L is suitable); the wavelength setting
is specified in the monograph.