Introduction
High-performance liquid chromatography (HPLC) is a separation technique that can be used for the analysis of organic molecules
and ions. HPLC is based on mechanisms of adsorption, partition and ion exchange, depending on the type of stationary phase
used. HPLC involves a solid stationary phase, normally packed inside a stainless-steel column, and a liquid mobile phase.
Separation of the components of a solution results from the difference in the relative distribution ratios of the solutes between the
two phases.
HPLC can be used to assess the purity and/or determine the content of many pharmaceutical substances. It can also be used to
determine enantiomeric composition, using suitably modified mobile phases or chiral stationary phases. Individual separation
mechanisms of adsorption, partition and ion exchange rarely occur in isolation since several principles act to a certain degree
simultaneously.
Apparatus
The apparatus consists of a pumping system, an injector, a chromatographic column, stationary and mobile phases, connecting
tubing and fittings, a detector and a data collection device (computer, integrator or recorder).
Pumping system
HPLC pumping systems are required to deliver metered amounts of mobile phase at a constant flow rate. Pumping systems that
deliver solvent from one or more reservoirs are available. Pressure fluctuations should be minimized, e.g. by passing the
pressurized solvent through a pulse-dampening device. Tubing and connections should be capable of withstanding the pressures
developed by the pumping system. Many HPLC pumps are fitted with a facility for "bleeding" the system of entrapped air bubbles.
Computer- or microprocessor-controlled pumping systems are capable of accurately delivering a mobile phase of either constant
(isocratic elution) or varying (gradient elution) composition, according to a defined program. In the case of gradient elution,
solvent mixing can be achieved on either the low- or high-pressure side of the pump(s).
Depending on a number of factors including column dimensions, particle size of the stationary phase, the flow rate and
composition of the mobile phase, operating pressures of up to 42 000 kPa (about 6000 psi) can be generated.
Injector
The sample solution is usually introduced into the flowing mobile phase at or near the head of the column using an injection
system based on an injection valve design which can operate at high pressure. Such an injection system has a fixed-loop or a
variable volume device which can be operated manually or by an auto-sampler. Partial filling of loops may lead to poorer injection
volume precision
Chromatographic column
Columns are usually made of polished stainless steel, are between 50 and 300 mm long and have an internal diameter of
between 2 and 5 mm. They are commonly filled with a stationary phase with a particle size of 3–10 μm. Columns with internal
diameters of less than 2 mm are often referred to as microbore columns. Ideally the temperature of the mobile phase and the
column should be kept constant during an analysis. Most separations are performed at ambient temperature but columns may be
heated using, for instance, a water-bath, a heating block or a column oven in order to achieve better efficiency.
Stationary phases
Separation of pharmaceuticals is usually achieved by partition of compounds in the test solution between the mobile and the
stationary phases. HPLC systems consisting of polar stationary phases and non-polar mobile phases are described as normal phase chromatography; those with non-polar stationary phases and polar mobile phases are called reversed-phase
chromatography.
There are many types of stationary phases used in HPLC including:
- Unmodified silica, alumina or porous graphite, used in normal-phase chromatography, where separation is based on
differences in adsorption;
- A variety of chemically-modified supports prepared from polymers, silica or porous graphite, used in reversed-phase HPLC,
where separation is based principally on partition of the molecules between the mobile phase and the stationary phase;
- 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;
- Porous silica or polymers.
Most separations are based on partition mechanisms using chemically-modified silica as the stationary phase and polar solvents as the mobile phase (reversed-phase HPLC). The surface of the support, e.g. the silanol groups of silica, is reacted with various
silane reagents to produce covalently bonded 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.
For the separation of enantiomers, special chemically-modified or coated stationary phases (chiral chromatography) are
available, which contain usually one of the following structures as a chiral selector: polymeric structures including proteins,
polysaccharide, crown ethers, cyclodextrins or macrocyclic glyco peptides. The enantiomers of a chiral test substance may differ
in their affinity to the chiral selector and, therefore, may be retained differently by the stationary phase.
As a guide silica-based, reversed-phase columns are generally considered to be stable in mobile phases with an apparent pH in
the range 2.0–8.0, but the column manufacturer's instructions should be consulted before using the column. Columns containing
particles of polymeric materials such as styrene-divinylbenzene copolymer are stable over a wider pH range.
Analysis using normal-phase HPLC with unmodified silica, porous graphite or polar chemically-modified silica (e.g. cyanopropyl
or diol) as the stationary phase and a non-polar mobile phase is employed in certain cases.
For analytical separations the particle size of the most commonly used stationary phases varies between 3 μm and 10 μm. The
particle shape may be spherical or irregular, of different porosities and specific surface area. In the case of reversed phase, the
extent of bonding of the stationary phase is expressed as the carbon-loading. Furthermore, stationary phases may be "endcapped", i.e. the number of residual silanol groups is reduced by methylation. These parameters contribute to the
chromatographic behavior of a particular stationary phase. Tailing of peaks, particularly for basic substances, can occur when
residual silanol groups are present.
Mobile phases
The choice of mobile phases is based on the desired retention behavior and the physicochemical properties of the analyte as
well as the type of detector chosen.
For normal-phase HPLC using unmodified stationary phases lipophilic solvents should be employed. The presence of water in
the mobile phase must be avoided as this will reduce the efficiency of the stationary phase. In reversed-phase HPLC aqueous
mobile phases, with and without organic modifiers, are used.
The mobile phase should be filtered through suitable membrane-type filters to remove particles or undissolved material.
Multicomponent mobile phases should be prepared by measuring the required volumes (unless masses are specified) of the
individual components, followed by manual or mechanical mixing. Alternatively, the solvents may be delivered by the individual
pumps or proportioning valves of the liquid chromatograph and mixed according to the desired proportion. Solvents are normally
degassed by sparging with helium or by means of sonification before pumping to avoid the formation of gas bubbles in the
detector cell.
Mobile phases may contain other components, e.g. a counter-ion for ion-pair chromatography or a chiral selector for chiral
chromatography using an achiral stationary phase.
Connecting tubing and fittings
The full efficiency of an analytical column may never be achieved because of the design limitations of pumps, injectors and
detectors. The connections between injector/column, column/detector and/or detector/detector may compromise the overall
efficiency of the system. Any fittings should be of the "zero dead volume" (ZDV) type. It is recommended that minimum lengths of
capillary tubing with a maximum internal diameter of 0.25 mm be used for these fittings to minimize band broadening.
Detectors
Ultraviolet/visible (UV/vis) absorption spectrophotometers are commonly used detectors in pharmaceutical analysis. In specific
cases fluorescence spectrophotometers, differential refractometers (RI), electrochemical detectors, evaporative light-scattering
detectors (ELSD), charged aerosol detectors (CAD), mass spectrometers (MS) or other special detectors may be used. Where an
analyte possesses a chromophore that absorbs UV/vis radiation, the UV/vis detector is the first choice because of its favorable
signal to noise ratio. Such a detector is not suitable for detecting analytes with very weak chromophores.
A variant on the UV/vis type of detector, which can furnish detailed spectral information, is the diode array spectrophotometer.
This type of detector acquires absorbance data over a certain UV/vis range and can provide chromatograms at multiple,
selectable wavelengths, together with spectra for the eluted peaks. In addition, the detector and accompanying computer
programs can be used to assess the spectral homogeneity of peaks, which may provide information on the chromatographic
purity of the peaks. This can be especially useful in method development and validation.
Data collection devices
Signals from the detector may be collected on chart recorders or electronic integrators that vary in complexity and in their ability
to process, store and reprocess chromatographic data. The data storage capacity of these devices is usually limited.
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