Liquid Chromatography

The term "liquid chromatography" (LC) is used to describe a number of different chromatographic methods whose common characteristic is the use of a liquid mobile phase. The technique is important in environmental analysis because many environmentally relevant compounds are not volatile enough to be analyzed by GC or are not thermally stable. LC differs fundamentally from GC in the way in which selectivity can be manipulated. In GC, at a given temperature, the relative retention of two peaks is determined only by the nature of the stationary phase and the properties of separated compounds. In order to change the selectivity, it is usually necessary to change the stationary phase of the column.* The nature of the mobile phase affects the speed of the separation and its efficiency, but not the selectivity. On the other hand, in LC, selectivity is determined to a large extent by the composition of the mobile phase. Since the selectivity of an LC system can be changed by changing the nature of the stationary phase or the composition of the mobile phase, LC is generally more flexible than GC. On the other hand, this additional functionality increases the number of parameters which can be manipulated during method optimization, making the process more complicated.

* There are exceptions to this rule. The relative retention of two compounds may change in GC when the temperature changes, sometimes leading to reversed elution order. This is often the case with polar stationary phases.

Another important difference between GC and LC is related to the fact that molecular diffusion is much slower in liquids than it is in gases (the difference between the molecular diffusion coefficients of a given compound in gases and in liquids may reach as much as four orders of magnitude). At the same time, viscosity of liquids is much higher than viscosity of gases. Because of the slow diffusion in liquids, the use of capillary columns of diameters similar to those used in GC is impractical. When used in LC, such columns would have very low efficiencies because equilibration between the mobile phase and the stationary phase would be very slow. To be useful for LC, open tubular columns would have to have much smaller diameters, but then pressures required to drive the mobile phase through the column would become impractical, and the sample capacity of such columns would be very low. Consequently, packed columns with a much smaller number of theoretical plates than open tubular columns used in GC are used almost exclusively in liquid chromatography. Thus, while most separations in GC are carried out under the conditions of moderate selectivity and high efficiency, typical LC separations are performed under the conditions of moderate efficiency and high selectivity.

A. Retention Mechanisms

Historically, the first liquid chromatographic separations were performed using unmodified solid particles as the stationary phases. In this scenario, the solute molecules interact with the particles via adsorption mechanisms. Typical stationary phases used in this technique include silica, alumina, carbon, as well as chemically bonded stationary phases with polar functional groups. The mobile phases in these types of separations are mixtures of nonaqueous polar solvents diluted to the desired strength with a nonpolar solvent, e.g., hexane. Because of once-widespread use of this technique, it is often referred to as normal phase chromatography. Other commonly used names include liquid-solid chromatography or adsorption chromatography. During chromatographic separation by this method, solute molecules continuously become adsorbed to the surface of the stationary phase and then replaced by the solvent molecules, which compete for the active sites on the surface. The relative ability of the solvents to displace solutes from a given adsorbent is described by the solvent strength parameter e0 (also called eluent strength), which is defined as the free energy of adsorption of the solvent per unit surface area. By definition, e0 is set to zero for adsorption of pentane on unmodified silica. It is clear that while the numerical value of e0 depends on the type of the adsorbent, the general trends should be similar for different adsorbents. This is illustrated in Table 3.2,6 which presents an example of a so-called eluotropic series (solvents ranked according to their solvent strength). In general, the greater the eluent strength, the more rapidly the solutes will be eluted from the column.

Normal phase chromatography is generally considered suitable for the separation of nonionic organic compounds soluble in organic solvents. However, the method is not as popular today as it was in the past because of a number of problems associated with the use of adsorbents as stationary phases. Adsorption isotherms are nonlinear, which leads to nonGaussian peaks at high solute concentrations. Retention of polar compounds may be irreproducible, e.g., as a result of irreversible adsorption. Traces of water in the mobile phase may deactivate the adsorbent, leading to irreproducible separations. Some of these problems can be eliminated by using polar, chemically bonded phases. Still, very often better results can be obtained by using other retention mechanisms. Today, normal phase LC remains the method of choice for separation of geometric isomers and class separations.

An alternative to normal phase chromatography is reversed phase LC (RPLC). This method is the most popular today owing to its unmatched simplicity, versatility and scope. In RPLC, the stationary phase is nonpolar, while the mobile phase is polar and usually contains water. The strength of the eluent increases as the polarity of the mobile phase decreases. This reversal of the properties of the stationary and the mobile phases compared to normal-phase chromatography

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