Chromatography: General Aspects

A strategy employed in chemical analysis of real world samples utilizes the separation of the sample into its constituents from a mixed state. Once separated each component will subsequently be identified and quantified.

Chromatography refers to a group of techniques used for the separation of a mixture of substances into its components. The objective in analytical chromatography is to accomplish the separation of mixtures in the smallest of space and shortest of time. In the practice of chromatography (GC and LC) the separation of analytes is accomplished by allowing the analytes to interact between two immisible phases  (biphase system), that are in contact with each other. Further, one phase is held stationary (stationary phase) and the other (mobile phase) is moved over it. The general features of GC and LC are very similar.

The technique of separation involves, first, the introduction (injection) of the analyte mixture into the mobile phase. The analytes will move through the biphase system at different rates; each analyte will separate from the others. An animation will illustrate this process.

Any component (analyte) injected into a biphase system is not at equilibrium at the point of its introduction to the system. The analyte will tend to reach an equilibrium state by distributing between the phases and the extent of distribution is dictated by thermodynamics. This phenomenon is known as partitioning. The characteristic of partitioning is that the concentrations of the analyte in the two phases are unequal; hence the ratio of the concentrations of the analytes in the two phases (= K; partition coefficient) differ from unity.

The value of K for a given analyte, i, determined by the thermodynamic properties of the analyte in the two phases and the temperature [Ki = f(i, mobile phase, stationary phase, T)]. In general each analyte has its own K value under a given set of circumstances. For an analyte i, partition coefficient is defined as;

where Ci,s and Ci,m are the concentrations of analyte i in stationary phase and mobile phase, respectively. The uniqueness of values of K of the analytes (at least in principle) forms the basis for the separation of analytes from each other in chromatographic systems. A given analyte when subjected to a biphase system, as described, distributes between the stationary phase and the mobile phase, establishing an 'equilibrium'. Those 'molecules' of an analyte entering the stationary phase will be 'held' in the stationary phase and those in the mobile phase are moved away. The movement of the mobile phase disturbs the equilibrium state established. The tendency will then be to establish a 'new equilibrium state'. This 'new equilibrium' state will bring out/in material from stationary phase/mobile phase as it needed. The result of this process is to move the analyte from the starting point to a new position within the biphase system. The chromatographic processs is dynamic.

A continuation of this distribution/redistribution will result in the movement of the analyte within the chromatographic column (space). The rate of movement however is dependent on the K value. Because each analyte in a mixture has a unique K value and will move at different rates through the biphase system. This results in the physical separation of the components from each other in time and space. The dominant mechanisms for distribution of analytes between the phases are partitioning and adsorption. There exists separation techniques that involve a single phase; in such cases the mechanisms of separation are different.

General Features of GC and LC:

The separation region consists of a ststionary phase packed into a column, through which the mobile phase is forced through.

Mobile phase moved over the stationary phase at a linear velocity, u.

Introduction of sample on to the mobile phase.

Analytes set up a dynamic equilibrium (distributes) between phases, u.

Mobile phase moves the analytes downstream.

Analytes when in mobile phase moves at mobile phase velocity.

Stationary phase 'retains' the analytes differentially and in the stationary phase the analutes are practically immobile.

Each analyte migration velocity (<u) is unique to the analyte.

Each analyte moves as a band.

Each band broadens as it moves.

Consider a system where both phases are fluids. When an analyte is put in a given stationary phase/mobile phase system, the analyte distributes between them. K measures partitioning of an analyte between to different phases. The stationary phase will retain the analytes to different degrees depending on the value of analyte partition coefficient thus generating differential velocities. Larger partition coefficient implies a higher affinity of the analyte to the stationary phase. The differential migration rates of analytes arises due to the differences in K values of analytes. Differential migration velocities of analytes permits separation of mixtures in chromatography in time and stationary phase.

The mobile phase is responsible for the movement of the analytes downstream. The mobile phase is said to 'elute' the analytes from the stationary phase. When the analyte is in the mobile phase it moves at u, but retardation from stationary phase results in an effective migration velocity < u.

From the molecular point of view, analytes are attracted to the stationary phase by way of their intermolecular forces. Thus the differences inter molecular forces between the analytes and the stationary phase is the key for chromatographic separation .Analytes preferring (interacting more attractively with stationary phase, larger K) the stationary phase are held on to for a longer time, and therefore are retarded proportionally.

To effect a separation,

(a) each analyte band must move at a unique migration velocity.

(b) for (a) to happen the stationary phase must retard the analytes differentially.

(c) for (b) to happen partition coefficients of analytes must be different in the system.

For a given analyte, the K depend on the stationary phase/mobile phase system and the temperature. K varies from analyte to analyte under same physical conditions.

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