Overview of Analytical Separations
We examined several methods for separating an analyte from potential interferents. For example, in a liquid–liquid extraction the analyte and interferent are initially present in a single liquid phase. A second, immiscible liquid phase is in- troduced, and the two phases are thoroughly mixed by shaking. During this process the analyte and interferents partition themselves between the two phases to differ- ent extents, affecting their separation. Despite the power of these separation tech- niques, there are some significant limitations.
Suppose we have a sample containing an analyte in a matrix that is incompatible with our analytical method. To determine the analyte’s concentration we first sepa- rate it from the matrix using, for example, a liquid–liquid extraction. If there are additional analytes, we may need to use additional extractions to isolate them from the analyte’s matrix. For a complex mixture of analytes this quickly becomes a te- dious process.
Furthermore, the extent to which we can effect a separation depends on the distribution ratio of each species in the sample. To separate an analyte from its ma- trix, its distribution ratio must be significantly greater than that for all other com- ponents in the matrix. When the analyte’s distribution ratio is similar to that of an- other species, then a separation becomes impossible. For example, let’s assume that an analyte, A, and a matrix interferent, I, have distribution ratios of 5 and 0.5, re- spectively. In an attempt to separate the analyte from its matrix, a simple liquid– liquid extraction is carried out using equal volumes of sample and a suitable extrac- tion solvent. Following the treatment outlined, it is easy to show that a single extraction removes approximately 83% of the analyte and 33% of the inter- ferent. Although it is possible to remove 99% of A with three extractions, 70% of I is also removed. In fact, there is no practical combination of number of extractions or volume ratio of sample and extracting phases that produce an acceptable separa- tion of the analyte and interferent by a simple liquid–liquid extraction.
The problem with a simple extraction is that the separation only occurs in one di- rection. In a liquid–liquid extraction, for example, we extract a solute from its ini- tial phase into the extracting phase. Consider, again, the separation of an analyte and a matrix interferent with distribution ratios of 5 and 0.5, respectively. A single liquid–liquid extraction transfers 83% of the analyte and 33% of the interferent to the extracting phase (Figure 12.1). If the concentrations of A and I in the sample were identical, then their concentration ratio in the extracting phase after one extraction is
Thus, a single extraction improves the separation of the solutes by a factor of 2.5. As shown in Figure 12.1, a second extraction actually leads to a poorer separation. After combining the two portions of the extracting phase, the concentration ratio decreases to
We can improve the separation by first extracting the solutes into the extracting phase, and then extracting them back into a fresh portion of the initial phase (Figure 12.2). Because solute A has the larger distribution ratio, it is extracted to a greater extent during the first extraction and to a lesser extent during the second ex- traction. In this case the final concentration ratio of in the extracting phase is significantly greater.
The process of extracting the solutes back and forth between fresh portions of the two phases, which is called a counter- current extraction, was developed by Craig in the 1940s.1* The same phenomenon forms the basis of modern chromatography.
Chromatographic separations are accomplished by continuously passing one sample-free phase, called a mobile phase, over a second sample-free phase that re- mains fixed, or stationary. The sample is injected, or placed, into the mobile phase. As it moves with the mobile phase, the sample’s components partition themselves between the mobile and stationary phases. Those components whose distribution ratio favors the stationary phase require a longer time to pass through the system. Given sufficient time, and sufficient stationary and mobile phase, solutes with simi- lar distribution ratios can be separated.
The history of modern chromatography can be traced to the turn of the cen- tury when the Russian botanist Mikhail Tswett (1872–1919) used a column packed with a stationary phase of calcium carbonate to separate colored pigments from plant extracts. The sample was placed at the top of the column and carried through the stationary phase using a mobile phase of petroleum ether. As the sample moved through the column, the pigments in the plant extract separated into individual col- ored bands. Once the pigments were adequately separated, the calcium carbonate was removed from the column, sectioned, and the pigments recovered by extrac- tion. Tswett named the technique chromatography, combining the Greek words for “color” and “to write.” There was little interest in Tswett’s technique until 1931 when chromatography was reintroduced as an analytical technique for biochemical separations. Pioneering work by Martin and Synge in 19412 established the impor- tance of liquid–liquid partition chromatography and led to the development of a theory for chromatographic separations; they were awarded the 1952 Nobel Prize in chemistry for this work. Since then, chromatography in its many forms has become the most important and widely used separation technique. Other separation meth- ods, such as electrophoresis, effect a separation without the use of a stationary phase.
Analytical separations may be classified in three ways: by the physical state of the mobile phase and stationary phase; by the method of contact between the mobile phase and stationary phase; or by the chemical or physical mechanism responsible for separating the sample’s constituents. The mobile phase is usually a liquid or a gas, and the stationary phase, when present, is a solid or a liquid film coated on a solid surface. Chromatographic techniques are often named by listing the type of mobile phase, followed by the type of stationary phase. Thus, in gas–liquid chro- matography the mobile phase is a gas and the stationary phase is a liquid. If only one phase is indicated, as in gas chromatography, it is assumed to be the mobile phase.
Two common approaches are used to bring the mobile phase and stationary phase into contact. In column chromatography, the stationary phase is placed in a narrow column through which the mobile phase moves under the influence of gravity or pressure. The stationary phase is either a solid or a thin, liquid film coating on a solid particulate packing material or the column’s walls. In planar chromatography the stationary phase coats a flat glass, metal, or plastic plate and is placed in a developing chamber. A reservoir containing the mobile phase is placed in contact with the stationary phase, and the mobile phase moves by capillary action.
The mechanism by which solutes separate provides a third means for charac- terizing a separation (Figure 12.3). In adsorption chromatography, solutes sepa- rate based on their ability to adsorb to a solid stationary phase. In partition chro- matography, a thin liquid film coating a solid support serves as the stationary phase. Separation is based on a difference in the equilibrium partitioning of solutes between the liquid stationary phase and the mobile phase. Stationary phases consisting of a solid support with covalently attached anionic (e.g., –SO3–) or cationic (e.g., –N(CH3)3+) functional groups are used in ion-exchange chro- matography. Ionic solutes are attracted to the stationary phase by electrostatic forces. Porous gels are used as stationary phases in size-exclusion chromatogra- phy, in which separation is due to differences in the size of the solutes. Large solutes are unable to penetrate into the porous stationary phase and so quickly pass through the column. Smaller solutes enter into the porous stationary phase, increasing the time spent on the column. Not all separation methods require a stationary phase. In an electrophoretic separation, for example, charged solutes migrate under the influence of an applied potential field. Differences in the mo- bility of the ions account for their separation.