26.1: A General Description of Chromatography

In chromatography we pass a sample-free phase, which we call the mobile phase, over a second sample-free stationary phase that remains fixed in space (Figure \(\PageIndex{1}\)). We inject or place the sample into the mobile phase. As the sample moves with the mobile phase, its components partition between the mobile phase and the stationary phase. A component whose distribution ratio favors the stationary phase requires more time to pass through the system. Given sufficient time and sufficient stationary and mobile phase, we can separate solutes even if they have similar distribution ratios.

Figure \(\PageIndex{1}\). In chromatography we pass a mobile phase over a stationary phase. When we inject a sample into the mobile phase, the sample’s components both move with the mobile phase and partition into the stationary phase. The solute that spends the most time in the stationary phase takes the longest time to move through the system.

Classification of Chromatographic Methods

There are many ways in which we can identify a chromatographic separation: by describing the physical state of the mobile phase and the stationary phase; by describing how we bring the stationary phase and the mobile phase into contact with each other; or by describing the chemical or physical interactions between the solute and the stationary phase. Let’s briefly consider how we might use each of these classifications.

We can trace the history of chromatography to the turn of the century when the Russian botanist Mikhail Tswett used a column packed with calcium carbonate and a mobile phase of petroleum ether to separate colored pigments from plant extracts. As the sample moved through the column, the plant’s pigments separated into individual colored bands. After effecting the separation, the calcium carbonate was removed from the column, sectioned, and the pigments recovered. Tswett named the technique chromatography, combining the Greek words for “color” and “to write.” There was little interest in Tswett’s technique until Martin and Synge’s pioneering development of a theory of chromatography (see Martin, A. J. P.; Synge, R. L. M. “A New Form of Chromatogram Employing Two Liquid Phases,” Biochem. J. 1941, 35, 1358–1366). Martin and Synge were awarded the 1952 Nobel Prize in Chemistry for this work.

Types of Mobile Phases and Stationary Phases

The mobile phase is a liquid or a gas, and the stationary phase is a solid or a liquid film coated on a solid substrate. We often name chromatographic techniques by listing the type of mobile phase followed by the type of stationary phase. In gas–liquid chromatography, for example, the mobile phase is a gas and the stationary phase is a liquid film coated on a solid substrate. If a technique’s name includes only one phase, as in gas chromatography, it is the mobile phase.

Contact Between the Mobile Phase and the Stationary Phase

There are two common methods for bringing the mobile phase and the stationary phase into contact. In column chromatography we pack the stationary phase into a narrow column and pass the mobile phase through the column using gravity or by applying pressure. The stationary phase is a solid particle or a thin liquid film coated on either a solid particulate packing material or on the column’s walls.

In planar chromatography the stationary phase is coated on a flat surface—typically, a glass, metal, or plastic plate. One end of the plate is placed in a reservoir that contains the mobile phase, which moves through the stationary phase by capillary action. In paper chromatography, for example, paper is the stationary phase.

Interaction Between the Solute and the Stationary Phase

The interaction between the solute and the stationary phase provides a third method for describing a separation (Figure \(\PageIndex{2}\)). In adsorption chromatography, solutes separate based on their ability to adsorb to a solid stationary phase. In partition chromatography, the stationary phase is a thin liquid film on a solid support. Separation occurs because there is a difference in the equilibrium partitioning of solutes between the stationary phase and the mobile phase. A stationary phase that consists of a solid support with covalently attached anionic (e.g., \(-\text{SO}_3^-\) ) or cationic (e.g., \(-\text{N(CH}_3)_3^+\)) functional groups is the basis for ion-exchange chromatography in which ionic solutes are attracted to the stationary phase by electrostatic forces. In size-exclusion chromatography the stationary phase is a porous particle or gel, with separation based on the size of the solutes. Larger solutes are unable to penetrate as deeply into the porous stationary phase and pass more quickly through the column.

Figure \(\PageIndex{2}\). Four examples of interactions between a solute and the stationary phase: (a) adsorption on a solid surface, (b) partitioning into a liquid phase, (c) ion-exchange, and (d) size exclusion. For each example, the smaller, green solute is more strongly retained than the larger, red solute.

There are other interactions that can serve as the basis of a separation. In affinity chromatography the interaction between an antigen and an antibody, between an enzyme and a substrate, or between a receptor and a ligand forms the basis of a separation.

Elution Chromatography on Columns

Of the two methods for bringing the stationary phase and the mobile phases into contact, the most important is column chromatography. In this section we develop a general theory that we may apply to any form of column chromatography.

Figure \(\PageIndex{3}\) provides a simple view of a liquid–solid column chromatography experiment. The sample is introduced as a narrow band at the top of the column. Ideally, the solute’s initial concentration profile is rectangular (Figure \(\PageIndex{4}\)a). As the sample moves down (or throught) the column, the solutes begin to separate (Figure \(\PageIndex{3}\)b,c) and the individual solute bands begin to broaden and develop a Gaussian profile (Figure \(\PageIndex{4}\)b,c). If the strength of each solute’s interaction with the stationary phase is sufficiently different, then the solutes separate into individual bands (Figure \(\PageIndex{3}\)d and Figure \(\PageIndex{4}\)d).

Figure \(\PageIndex{3}\). Progress of a column chromatographic separation of a two-component mixture. In (a) the sample is layered on top of the stationary phase. As mobile phase passes through the column, the sample separates into two solute bands (b–d). In (e) and (f), we collect each solute as it elutes from the column.

Figure \(\PageIndex{4}\). An alternative view of the separation in Figure \(\PageIndex{3}\) showing the concentration of each solute as a function of distance down the column.

We can follow the progress of the separation by collecting fractions as they elute from the column (Figure \(\PageIndex{3}\)e,f), or by placing a suitable detector at the end of the column. A plot of the detector’s response as a function of elution time, or as a function of the volume of mobile phase, is known as a chromatogram (Figure \(\PageIndex{5}\)), and consists of a peak for each solute.

Figure \(\PageIndex{5}\). Chromatogram for the separation shown in Figure \(\PageIndex{3}\) and Figure \(\PageIndex{4}\), showing the detector’s response as a function of the elution time.

There are many possible detectors that we can use to monitor the separation. Later sections of this chapter describe some of the most popular.