Mode Selection

Chromatography provides many different approaches for the separation of mixtures. There are many instances where the same mixture can be adequately separated by more than one approach. In this section we will take a mechanistic look at how solutes are separated by the common chromato-graphic techniques to provide some guidelines for method suitability.

If the only consideration were efficiency and speed, then GC would be the preferred technique. In practice, GC is restricted to thermally stable compounds with a significant vapour pressure at the temperature required for their separation. The upper temperature limit for common GC stationary phases is 200-400°C. Few compounds with a molecular weight greater than 1000 Da have sufficient vapour pressure to be separated in this temperature range, and many low molecular weight compounds are known to be labile at temperatures required for their vaporization. Derivatization techniques extend the scope of GC to otherwise labile compounds by forming thermally stable derivatives, often with increased volatility, and by tagging compounds with specific groups that simplify trace analysis using one of the selective and sensitive group or element-selective detectors available for GC.

Under typical conditions the mobile phase in GC behaves essentially as an ideal gas and does not contribute to selectivity. To vary selectivity either the temperature is changed or a new stationary phase (column) is employed for the separation. Temperature and separation time are closely connected in GC. The range over which temperature can be varied is usually short and will likely provide only a small change in selectivity, but because of the large number of theoretical plates available for a separation in GC, this may be sufficient to provide adequate resolution. Provided that stationary phases that differ in their relative capacity for intermolecular interactions are selected, then larger changes in selectivity can be anticipated by stationary-phase optimization. In modern column technology the most versatile group of stationary phases are the poly(siloxanes), which can be represented by the basic structure -(R2SiO)B-, in which the type and relative amount of individual substituents can be varied to create the desired variation in selectivity (R = methyl, phenyl, 3,3,3-trifluoropropyl, cyanoethyl, fluorine-containing alcohol, etc.) Special phases in which R contains a chiral centre or a liquid-crystalline unit are used to separate enantiomers and geometric isomers. Other common stationary phase include hydrocarbons, poly(phenyl ethers), poly(esters) and poly(ethylene glycols), although many of these phases are restricted to packed column applications because of difficulties in either coating or immobilizing them on the walls of fused-silica capillaries, favoured for the manufacture of open-tubular columns. The solvation parameter model provides a reliable systematized approach for selectivity optimization and the prediction of retention in GLC. For GSC the stationary phase is usually silica, alumina, graphitized carbon, organic polymer or zeolite porous particles (packed columns); or a thin layer dispersed over the inner surface of a capillary column with an open passageway down the centre (porous layer open-tubular column, or PLOT column). These materials are used to separate inorganic gases, volatile halocarbon compounds, low molecular weight hydrocarbons and, in particular, geometric and isotopic isomers.

LC and GC should be considered as complementary techniques. Since the only sample requirement for LC is that the sample has reasonable solubility in some solvent suitable for the separation, and since separations by LC are commonly carried out close to room temperature, thermal stability is not generally an issue. The large number of separation mechanisms easily exploited in the liquid phase provides a high level of flexibility for selectivity optimization. In general, many applications of LC can be categorized as those for which GC is unsuited and includes applications to high molecular weight synthetic polymers, biopolymers, ionic compounds and many thermally labile compounds of chemical interest.

Mode selection within LC is quite complicated because of the number of possible separation mechanisms that can be exploited, as illustrated in Figure 22. Preliminary information on the molecular weight range of the sample, relative solubility in organic solvents and water, and whether or not the sample is ionic, can be used as a starting point to arrive at a suitable retention mechanism for a separation. The molecular weight cutoff at 2000 indicated in Figure 22 is quite arbitrary and reflects the fact that size exclusion packings are readily available for the separation of higher molecular weight solutes, although size exclusion is not used exclusively to separate high molecular weight compounds because of its limited peak capacity. Wide-pore packing materials allow polymers with a molecular weight

Packing Chromatography
Figure 22 Selection of the separation mechanism in LC based on the criteria of sample molecular weight, solubility and conductivity. (Reproduced with permission from Poole CF and Poole SK(1991) Chromatography Today, p. 455, copyright © Elsevier Science B.V.)

exceeding 2000 to be separated by conventional sorption and ion exchange mechanisms.

Liquid-solid chromatography (LSC) is characterized by the use of an inorganic oxide or chemically bonded stationary phase with polar functional groups and a nonaqueous mobile phase consisting of one or more polar organic solvents diluted to the desired solvent strength with a weak solvent, such as hexane. A characteristic of these systems is the formation of an adsorbed layer of mobile-phase molecules at the surface of the stationary phase with a composition that is related to the mobile-phase composition but generally not identical to it. Retention is essentially determined by the balance of interactions the solute experiences in the mobile phase and its competition with mobile-phase molecules for adsorption sites at the surface of the stationary phase. The position and type of polar functional groups and their availability for interaction with discrete immobile adsorption sites is responsible for selectivity differences when silica or alumina are used as stationary phases. The ability of LSC to separate geometric isomers has been attributed to the lock-key type steric fitting of solute molecules with the discrete adsorption sites on the silica surface.

Reversed-phase liquid chromatography (RPC) is characterized by the use of a stationary phase that is less polar than the mobile phase. A chemically bonded sorbent or a porous polymer could be used as this stationary phase, while for most practical applications the mobile phase contains water as one of its major components. RPC is ideally suited to the separation of polar molecules that are either insoluble in organic solvents or bind too strongly to inorganic oxide adsorbents for normal elution. RPC employing acidic, low ionic strength eluents is a widely established technique for the purification and characterization of biopolymers. Other favourable attributes include the possibility of simultaneous separation of neutral and ionic solutes; rapid equilibrium between phases facilitating the use of gradient elution; and the manipulation of secondary chemical equilibria in the mobile phase (e.g. ion suppression, ion pair formation, metal complexation and micelle formation) to optimize separation selectivity in addition to variation in solvent type and composition of the mobile phase. A large number of chemically bonded stationary phases of different chain length, polarity and bonding density are available to complement mobilephase optimization strategies. About 70% of all separations performed in modern LC are by RPC, which gives an indication of its flexibility, applicability and ease of use. The main driving force for retention in RPC is solute size because of the high cohesive energy of the mobile phase compared to the stationary phase, with solute polar interactions, particularly solute hydrogen bond basicity, reducing retention. These findings strongly reflect the properties of water, which is the most cohesive of the solvents normally used in LC, as well as a strong hydrogen bond acid.

Ion exchange chromatography (IEC) is used for the separation of ions or substances easily ionized by manipulation of pH. Stationary phases are characterized as weak or strong ion exchangers based on the extent of ionization of the immobile ionic centres, and as anion or cation exchangers based on the charge type associated with the ionic centres. Thus, sulfonic acid groups are strong, and carboxylic acid groups are weak, cation exchangers. Most of the metal cations in the Periodic Table have been separated by IEC with acids or complexing agents as elu-ents. In clinical laboratories ion exchange has long been employed as the basis for the routine, automated separation of amino acids and other physiologically important amines involved in metabolic disorders and to sequence the structure of biopolymers. Soft, nondenaturing, ion exchange gels are widely used in the large-scale isolation, purification and separation of peptides, proteins, nucleosides and other biological polymers. Metal-loaded ion exchangers and anion exchange chromatography of complexed carbohydrates are well-established separation techniques in carbohydrate chemistry. The combination of pellicu-lar ion exchange columns of low capacity, low concentration eluents with a high affinity for the ion exchange packing, and universal, online detection with a flow-through conductivity detector revolution ized the analysis of inorganic and organic ions in industrial and environmental laboratories. As well as electrostatic interactions, retention in IEC is influenced by hydrophobic sorptive interactions between the sample and stationary phase similar to those in RPC, and size and ionic exclusion effects. Resolution is optimized by choice of the mobile-phase counterion, the ionic strength, pH, temperature, flow rate, and addition of organic modifiers.

In size exclusion chromatography (SEC) retention differences are controlled by the extent to which sample components can diffuse through the pore structure of the stationary phase, as indicated by the ratio of sample molecular dimensions to the distribution of stationary-phase pore size diameters. Since no separation will result under conditions where the sample is completely excluded from the pore volume or can completely permeate the pore volume, the zone capacity of SEC is small compared with that of the other LC techniques. The separation time is predictable for all separations, corresponding (ideally) to a volume of eluent equivalent to the column void volume. No solvent optimization beyond finding a solvent for the sample that is compatible with the stationary phase is required. For synthetic polymers this can result in the use of exotic solvents and high temperatures. SEC is a powerful exploratory method for the separation of unknown samples, since it provides an overall view of sample composition within a predictable time, and is also commonly employed in sample fractionation to isolate components belonging to a defined molecular size range. Analytical separations employ small particles of rigid, polymeric or silica-based gels of controlled pore size to separate samples of different molecular size and to obtain average molecular weights and molecular weight distribution information for polymers.

Fundamentally the retention mechanisms for LC and TLC are identical. TLC is selected over LC when advantage can be taken of the attributes of employing a planar format for the separation. Examples include when a large number of samples requiring minimum sample preparation are to be separated, when post-chromatographic reactions are usually required for detection, or if sample integrity is in question. The use of a disposable stationary phase for TLC allows sample clean-up and separation to be performed simultaneously. Reasons for preferring LC over TLC are its greater separation capacity for mixtures containing more components than can be adequately resolved by TLC; a wider range of stationary phases are available for methods development; a wider selection of detection techniques exist; and automation for unattended operation is more straightforward.

The retention mechanism for MEKC strongly resembles that of RPC with two important differences. Surfactants used to generate the pseudo-stationary phases provide a different type of sorption environment to solvated chemically bonded phases and, therefore, different selectivity. The intrinsic efficiency of MEKC is significantly greater than that of LC and enhances resolution, although the peak capacity is lower owing to the finite migration window for MEKC. A significant number of RPC-type applications are now performed by MEKC, indicating that the method can compete favourably with RPC for some separations. MEKC is inherently a microcolumn technique, providing advantages in coupling to other chromatographic systems and for the analysis of samples only available in small amounts. Disadvantages include sample introduction problems, limited dynamic sample concentration range, and poor limits of detection for trace analysis (because of the very small sample sizes involved). Selectivity optimization is determined largely by the choice of surfactant and the use of mobile- and stationary-phase additives.

Supercritical fluids have solvating properties that are intermediate between those of gases and liquids. In addition, supercritical fluids are compressible so that their density and solvating power can be varied by changing external parameters, such as pressure and temperature. This feature is unique to supercritical fluids and represents a major approach to selectivity optimization. Temperature not only affects density, but may also influence the vapour pressure of low molecular weight solutes, promoting some GC-like character to the retention mechanism. The most common mobile phase is carbon dioxide, a relatively nonpolar fluid. More-polar fluids, such as water, ammonia or methanol, tend to have unfavourable critical constants or are highly corrosive to column or instrument components, limiting their use. Mixed mobile phases can be used to vary selectivity, such as carbon dioxide-methanol mixtures, but miscibility problems and high critical constants for the mixed mobile phases may restrict the range of properties available. SFC can provide faster separations than LC, but it is more restricted than LC in the choice of mobile phases and retention mechanisms to vary selectivity. SFC is compatible with most detection options available for both GC and LC. All practical applications of SFC occur significantly above ambient temperature, which is unsuitable for the separation of some thermally labile compounds and most biopolymers. Supercritical fluids such as carbon dioxide are unable to mask active sites on typical column packings, resulting in unsatisfactory separations of polar compounds owing to adsorption, which pro duces unacceptable peak shapes and poor resolution. However, SFC finds applications in many areas where GC and LC are unsatisfactory, for example in the separation of middle molecular weight compounds, low molecular weight synthetic polymers, fats and oils, enantiomers, and organometallic compounds.

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