Appendix III D. Liquid Chromatography

(Ph. Eur. method 2.2.29)

Liquid chromatography (LC) is a method of chromatographic separation based on the difference in the distribution of species between two non-miscible phases, in which the mobile phase is a liquid which percolates through a stationary phase contained in a column.

LC is mainly based on mechanisms of adsorption, mass distribution, ion exchange, size exclusion or stereochemical interaction.

The apparatus consists of a pumping system, an injector, a chromatographic column (a column temperature controller may be used), a detector and a data acquisition system (or an integrator or a chart recorder). The mobile phase is supplied from one or several reservoirs and flows through the column, usually at a constant rate, and then through the detector.

LC pumping systems are required to deliver the mobile phase at a constant flow rate. Pressure fluctuations are to be minimised, e.g. by passing the pressurised solvent through a pulse-dampening device. Tubing and connections are capable of withstanding the pressures developed by the pumping system. LC pumps may be fitted with a facility for "bleeding" the system of entrapped air bubbles.

Microprocessor controlled systems 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 high-pressure side of the pump(s).

The sample solution is introduced into the flowing mobile phase at or near the head of the column using an injection system which can operate at high pressure. Fixed-loop and variable volume devices operated manually or by an auto-sampler are used. Manual partial filling of loops may lead to poorer injection volume precision.

There are many types of stationary phases 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,

• — 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, 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 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 (chiral chromatography).

Most separations are based upon partition mechanisms utilising 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	=	Si-[CH2]7-CH3	C8
octadecyl	=	Si-[CH2]17-CH3	C18
phenyl	=	Si-[CH2] n -C6H5	C6H5
cyanopropyl	=	Si-[CH2]3-CN	CN
aminopropyl	=	Si-[CH2]3-NH2	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 2.0 to 8.0. Columns containing porous graphite or particles of polymeric materials such as styrene-divinylbenzene 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 non-polar mobile phase is applicable in certain cases.

For analytical separations, the particle size of the most commonly used stationary phases varies between 3  µm and 10  µm. The particles may be spherical or irregular, of varying porosity and specific surface area. These parameters 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. residual silanol groups are silylated) are additional determining factors. Tailing of peaks, particularly of basic substances, can occur when residual silanol groups are present.

Columns, made of stainless steel unless otherwise prescribed in the monograph, of varying length and internal diameter (Ø) 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 columns may be heated to give higher efficiency. It is recommended that columns not be heated above 60  °C because of the potential for stationary phase degradation or changes occurring to the composition of the mobile phase.

For normal-phase chromatography, less polar solvents are employed. The presence of water in the mobile phase is to be strictly controlled to obtain reproducible results. In reversed-phase LC, aqueous mobile phases, with or without organic modifiers, are employed.

Components of the mobile phase are usually filtered to remove particles greater than 0.45  µm. Multicomponent mobile phases are prepared by measuring the required volumes (unless masses are specified) of the individual components, followed by mixing. Alternatively, the solvents may be delivered by individual pumps controlled by proportioning valves by which mixing is performed according to the desired proportion. Solvents are normally degassed before pumping by sparging with helium, sonication 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 stabilisers and are transparent at the wavelength of detection, if an ultraviolet detector is employed. 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 are used, adequate rinsing of the system is carried out with a mixture of water and the organic modifier of the mobile phase (5  per cent  V/V) to prevent crystallisation of salts after completion of the chromatography.

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.

Ultraviolet/visible (UV/Vis) spectrophotometers, including diode array detectors, are the most commonly employed detectors. Fluorescence spectrophotometers, differential refractometers, electrochemical detectors, mass spectrometers, light scattering detectors, radioactivity detectors or other special detectors may also be used.

Equilibrate the column with the prescribed mobile phase and flow rate, at room temperature or at the temperature specified in the monograph, until a stable baseline is achieved. Prepare the solution(s) of the substance to be examined and the reference solution(s) required. The solutions must be free from solid particles.

Criteria for assessing the suitability of the system are described in the chapter on Chromatographic separation techniques (2.2.46). 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 chapter.

The composition and flow rate of the mobile phase are stated in the monograph. It is advisable to use as the mobile phase solvent mixtures that have been de-aerated using a vacuum pump or other suitable means of de-aeration that has no effect on the composition of the mixture.

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. The dimensions are stated as (length × internal diameter). When the monograph prescribes the use of a stationary phase designated by a letter, the relevant stationary phase defined below is intended. The nominal diameter of the particles of the stationary phase is stated in parentheses immediately following the designating letter. 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 ambient 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 µl is suitable); the wavelength setting is specified in the monograph.

The design of a particular chromatograph may require modification of the conditions detailed in the monograph. In such a case the analyst should be satisfied that the modified conditions produce comparable results.

Where no injection volume is specified in the monograph, the analyst should select an appropriate volume for their specific application. The volume chosen is dependent on the response of the analyte, the detector used, the efficiency of the column and the overall performance of the chromatographic system. Where a volume is not indicated, 20 µl is usually appropriate; however this should be checked for suitability under the local operating conditions.

Where no run time is specified in the monograph, the analyst should select an appropriate run time for their specific application. The run time chosen is dependent on the type of test. For example, where a run time is not indicated in a Related substances test the analyst should ensure that the run time is greater than all known or likely secondary peaks; similarly in an Assay the run time should be chosen to allow the baseline to stabilise following the elution of the peak of interest.

Reference may be made to secondary peaks. A secondary peak is a peak in the chromatogram other than the principal peak and any peak due to internal standard, solvent or derivatising agents. Peaks identified as being due to the counter-ion and/or other excipients including preservatives in the material being examined may also be excluded.

Solvents and reagents used in the preparation of solutions for examination should be of a quality suitable for use in liquid chromatography.