Method development
What does the analyst do when the conventional systems fail to separate substances of interest? In such cases there is a need to develop a suitable method for separation. A trial-and error approach is possible, but it is more sensible to take a systematic approach. For TLC, the development technique to be used when analysing a sample is selected based on the number of detectable components in the mixture and their polarity range (Table 13.13). It may not be necessary to separate each and every component from one another. Depending on the aim of the separation it may only be necessary to separate the components of major interest from each other and from the less important components. The less important components may not need to be separated individually. A single development with capillary-controlled flow may be too difficult or impossible for mixtures that contain more than eight to ten components of interest. In addition, if the range of polarities is too wide, multiple develop ment techniques using mobile phase gradients are likely to be required to enable separation of all components. Method development is easier if standards for the relevant compounds are available. Standards simplify zone tracking (i.e. where a component has ‘run’ on a plate) and enable detection characteristics and the possibility of spectroscopic resolution of incompletely separated zones to be established. Standards are also required for calibration if quantification is required and to construct spectral libraries for identification purposes. The expected concentration range of relevant compounds may indicate that derivatisation will be required in order to detect substances at the levels at which they are present in samples and to increase zone separation of neighbouring compounds if one compound is a minor component with similar migration properties to a major component.
A general guide to the selection of stationary phases for TLC separations is summarised in Fig. 1. Silica gel is generally the first choice to separate drugs of low molecular weight that are soluble in moderately polar organic solvents. Reversed-phase chromatography on chemically bonded layers is generally used to separate drugs that are difficult to separate on silica gel because of inadequate retention, inadequate selectivity or zone asymmetry. Ionic compounds and easily ionised compounds are frequently separated by reversed-phase chromatography using buffered mobile phases (weak acids and bases) or ion-pair reagents (strong acids and bases). Only a limited number of stationary phases are available for ion-exchange chromatography, which is not a widely used separation mechanism in TLC. Since the solvent used for the separation is evaporated prior to detection, a wider range of UV-absorbing solvents are commonly used in TLC than is the case for HPLC. Solvents must be of high purity, since nonvolatile impurities and stabilisers remain sorbed to the stationary phase and can cause problems in the detection step. Multicomponent mobile phases can produce a mobile-phase gradient in the direction of devel opment through demixing. If demixing is complete, zones with sharp boundaries are formed, which separate the chromatogram into sections of different solvent composition and therefore selectivity. Demixing effects are less apparent when saturated developing chambers are used. These considerations hinder optimisation strategies based on the composition of the mobile phases as popularised in HPLC. The selection of a mobile phase to separate simple mixtures need not be difficult and can be arrived at quickly by guided trial and error methods. A solvent of the correct strength for a unidimensional development should be selected which migrates the sample components into the RF range 0.2–0.8, or thereabouts and, if of the correct selectivity, distributes the sample components evenly throughout this range. Solvent systems can be screened in parallel using several development chambers, as prescribed in the PRISMA model (see Fig. 13.13) (Nyiredy et al. 1985a,b, 1988; Dallenbach-Tölke et al.1986). To select suitable mobile phases, the first experiments are carried out on TLC plates in unsaturated chambers with ten solvents, chosen from the different selectivity groups, indicated by bold type in Table 13.14. After these screening experiments with single solvents, the solvent strength is either reduced or increased so that the substance zones are distributed in the RF range 0.2–0.8. If the substances migrate into the upper third of the plate, the solvent strength is reduced by dilution with hexane (the strength adjusting solvent). If the substances remain in the lower third of the plate with the single solvents, the solvent strength is increased by the addition of a strong solvent, such as water or acetic acid. A similar procedure is followed in the reversed-phase mode, except that solvent selection is limited to water-miscible solvents and water is used as the strength-adjusting solvent. From these trial experiments, those solvents that show the best separation are selected for further optimisation in the second part of the model.
Figure 1 Mode selection guide for TLC. LSC, liquid–solid chromatography on an inorganic oxide layer; BPC, liquid–solid chromatography on a chemically bonded layer; RPC, reversed-phase chromatography with a chemically bonded layer and an aqueous organic mobile phase; IPC, ion-pair chromatography with reversed-phase separation conditions; PC, precipitation chromatography used to separate polymers based on solubility differences in a mobile-phase solvent gradient.
Figure 2 The PRISMA mobile-phase optimisation model, showing the construction of the prism and the arrangement of selectivity points on the top face or horizontal plane cut through the prism.
Between two and five solvents can be selected to construct the PRISMA model for solvent opti misation. Modifiers required to maintain an acceptable zone shape, such as acids and ion-pair reagents, can be added in a low and constant concentration, so that their influence on solvent strength can be neglected. The PRISMA model (Fig. 2) is a three-dimensional geometrical design that correlates solvent strength with selectivity of the mobile phase. The model consists of three parts: the base or platform (represents the modifier), the regular part of the prism with congruent base and top surfaces, and the irregular truncated top prism (frustum). The lengths of the edges of the prism (SA, SB, SC) correspond to the solvent strengths of the neat solvents (A, B, C). Since the selected solvents usually have different solvent strengths, the lengths of the edges of the prism are generally unequal and the top plane of the prism is not parallel and congruous with its base. If the prism is cut parallel to its base at the height of the lowest edge (determined by the solvent strength of the weakest solvent, solvent C in Figure 13.13, the lower part gives a regular prism, and the top and any planes, which represent weaker solvents diluted with a strength-adjusting solvent, are parallel equilateral triangles. The upper frustum of the model is used for mobile-phase optimisation of polar drugs in normal phase TLC, while the regular part is used to separate moderately polar drugs in normal-phase TLC and all separations by reversed-phase TLC. For polar compounds, optimisation is always started on the top irregular triangle of the model, either within the triangle, when three solvents are selected, or along one side, for binary mobile phases. Any solvent composition on the face of the triangle can be represented by a three-coordinate selectivity point (PS), each co-ordinate corresponding to the volume fraction of the solvent at that position on the triangle (see Fig. 13.13B). Optimisation is commenced by selecting solvent combinations that correspond to the centre point PS =333 and three other points close to the apexes of the triangle PS= 811, 181 and 118. If the separation obtained is insufficient, other selectivity points are tested around the solvent combination that gave the best separation. On changing the selectivity points on the top triangle, the solvent strength changes as well, especially when the solvent strengths of the solvents used to construct the prism are significantly different. The solvent strength should be adjusted with the strength adjusting solvent as required to maintain the separation in the optimum RF range. Failure to obtain the beginning of a separation requires that a new prism be constructed, using a different solvent for at least one of the edges. For reversed-phase TLC, the solvation parameter model provides a convenient computer-aided approach to method develop ment. Suitable water-miscible solvents with a range of selectivity include methanol, propan 2-ol, 2,2,2-trifluoroethanol, acetonitrile (or dioxane), acetone (or tetrahydrofuran) and dimethylformamide (or pyridine). For optimisation of systems (stationary phases and binary mobile phases), preliminary results in the form of system maps (a continuous plot of the system constants against mobile-phase composition) are required. System maps are a permanent record of the system properties used in all calculations and are available for most common layers and indicated solvents for selectivity optimisation. For each computer-simulated separation a retention map is calculated from the system map and displays the computed RF values as a continuous function of the binary mobile-phase composition. A typical retention map for the computer predicted separation of analgesics on an octadecyl siloxane-bonded layer with 2,2,2 trifluoroethanol–water mixtures as the mobile phase is shown in Figure 3. Solvent compositions that result in an acceptable zone separation are identified easily by visual inspection. Computer simulation of retention maps allows those systems (defined as a combination of stationary and mobile phase) likely to provide an acceptable separation to be identified before experimental work commences. The agreement between model predicted and experimental RF values is generally good, typically better than 0.05 RF units. A mixture-design approach is used to extend this method to ternary solvent mixtures.
For drug mixtures of a wide polarity range, stepwise changes in solvent composition are required to achieve a satisfactory TLC separation. Models to calculate migration distances using incremental multiple development with increasing and decreasing solvent-strength gradients have been described, but are complicated and not widely used. Optimised gradients for automated multiple development are usually arrived at by more pragmatic means. Methods based on a universal gradient commence with methanol, end with hexane, and use either dichloromethane or methyl t-butyl ether as the intermediate solvent for separations on silica gel. By scaling and superimposing the chromato gram of the separation above the theoretical gradient profile, those regions of the chromatogram that affect the separation are easily identified. The solvent composition for the initial and final development steps is adjusted to eliminate those portions of the gradient that do not contribute to the separation. The gradient shape is modified to enhance resolution in those regions of the chromatogram that are separated poorly or to minimise regions devoid of sample zones. For moderately complex mixtures this approach is often satisfactory. If, after the above adjustments, the separation is inadequate it is necessary to identify a more selective solvent for problem regions in the gradient. The PRISMA model can be used at this point to identify more selective solvents to incorporate into the gradient as a replacement for the initial, terminal or base solvent.
Figure 3 Retention map for the simulation of the separation of analgesics by reversed-phase TLC on an octadecyl siloxane-bonded layer with 2,2,2-trifluoro ethanol–water as the mobile phase. 1, Chlorphenamine (chlorpheniramine); 2, ibuprofen; 3, naproxen; 4, phenacetin; 5, aspirin; 6, caffeine; 7, paracetamol (acetaminophen).
