Appendix III D. Liquid Chromatography

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.

Unless otherwise specified all information below is valid for standard LC as well as for LC using reduced particle-size columns (e.g. sub-2 µm).

The latter requires instrumentation characterised 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

The apparatus typically 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.

Pumping systems

LC pumping systems are required to deliver the mobile phase at a controlled 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 pumping 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).

Injectors

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.

Stationary phases

There are many types of stationary phases employed in LC, including:

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:

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 2 µm and 10 µm. 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 are 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.

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 some require a different temperature for optimal performance.

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 mobile phases, with or without organic solvents, are employed.

Components of the mobile phase are usually filtered to remove particles greater than 0.45 µm (or 0.2 µm when the stationary phase is made of sub-2 µm particles and when special detectors, e.g. light scattering detectors, are used). 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 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 stabilisers 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 mixture of water and a small proportion of the organic part of the mobile phase (5 per cent V/V) to prevent crystallisation 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.

METHOD

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 2.2.46. Chromatographic separation techniques. 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.

Additional points for monographs of the British Pharmacopoeia

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.

Injection volume

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.

Resolution factor

Unless otherwise stated in the monograph, for monographs with a stated resolution factor read Resolution (Rs) as referred to in Appendix III Chromatographic Separation Techniques.

Run time

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.

Secondary peaks

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.

Materials

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