Column Chromatography

  1. Objective

The aim of this experiment is to separate two substances using column chromatography. As an example, methylene blue and methyl orange will be separated using an alumina packed column. The separated substances will then be analyzed spectrophotometrically using a visible spectrophotometer.

  1. Principle and Theory

Chromatography is a technique in which compounds to be separated are distributed between a mobile phase and a stationary phase. In such a system, different distributions based on selective adsorption give rise to separation. There are different types of chromatography, such as paper, thin layer, or column chromatography, each with its own strengths and weaknesses.

Column chromatography is one of the most useful methods for the separation and purification of both solids and liquids when carrying out small-scale experiments. The separation can be liquid/solid (adsorption) or liquid/liquid (partition) in column chromatography. The stationary phase, a solid adsorbent, is usually placed in a vertical glass column and the mobile  phase, is added from the top and let flow down through the column by either gravity or external pressure (Figure 1).


Figure 1. Column chromatography involves a mobile phase flowing over a stationary phase.

Column chromatography is advantageous over most other chromatographic techniques because it can be used in both analytical and preparative applications. It can be used to determine the number of components of a mixture and as well as the separation and purification of those components.

Column chromatography isolates desired compounds from a mixture in such a way that the mixture is applied from the top of the column.  The columns are usually glass or plastic with sinter frits to hold the packing. The liquid solvent (eluent) is passed through the column by gravity or by the application of air pressure. The eluent, instead of rising by capillary action up a thin layer, flows down through the column filled with the adsorbent. Equilibrium is established between the solute adsorbed on the adsorbent and the eluting solvent flowing down through the column. Stationary phases are almost always adsorbents. Adsorbent is a substance that causes passing molecules or ions to adhere to the surface of its particles. The mobile phase is a solvent that flows past the stationary phase, dissolving the molecules of the compounds to be separated some of the time.

Because the different components in the mixture have different interactions with the stationary and mobile phases, they will be carried along with the mobile phase to varying degrees and a separation will be achieved. The individual components, or elutants, are collected as solvent drips from the bottom of the column.

Many compounds are not visible to the eye when dissolved in a solvent or adsorbed on a adsorbent. Visualization processes make these substances visible. The used techniques for this purpose include UV lights that cause fluorescence or phosphorescence and chemical reactions that give colored compounds.

2.1. Adsorbent

Silica gel (SiO2) and alumina (Al2O3) are two adsorbents commonly used by organic chemists for column chromatography. These adsorbents are sold in different mesh sizes, indicated by a number on the bottle label: “silica gel 60” or “silica gel 230-400” are a couple of examples. This number refers to the mesh of the sieve used to size the silica, specifically, the number of holes in the mesh or sieve through which the crude silica particle mixture is passed in the manufacturing process. If there are more holes per unit area, those holes are smaller, thus only smaller silica particles are allowed to pass the sieve. The larger the mesh size, the smaller the adsorbent particles are.

Adsorbent particle size affects the way the solvent flows through the column. Smaller particles (higher mesh values) are used for flash chromatography; larger particles (lower mesh values) are used for gravity chromatography.

Alumina is quite sensitive to the amount of water which is bound to it; the higher its water content, the less polar sites it has to bind organic compounds, and thus the less “sticky” it is. This stickiness or activity is designated as I, II, or III with I being the most active. Alumina comes in three forms: acidic, neutral, and basic. The neutral form of activity II or III, 150 mesh, is most commonly employed.

2.2. Solvent

The polarity of the solvent, which is passed through the column, affects the relative rates at which compounds move through the column. Polar solvents can more effectively compete with the polar molecules of a mixture for the polar sites on the adsorbent surface and will also better solve the polar constituents. Consequently, a highly polar solvent will move even the highly polar molecules rapidly through the column. If a solvent is too polar, movement becomes too rapid, and little or no separation of the components of a mixture will result. On the other hand, if a solvent is not polar enough, no compounds will elute from the column. Proper choice of an eluting solvent is thus crucial for a successful application of column chromatography as a separation technique since compounds interact with the silica or alumina largely due to polar interactions.

2.3. Elution Chromatography

Elution involves transporting a species through a column by continuous addition of fresh mobile phase. Single portion of sample contained in the mobile phase is introduced from the top of the column whereupon the components of that sample distribute themselves between two phases. Introduction of additional mobile phase (the eluent) forces the solvent containing a part of the sample down the column where further partition between the mobile phase and fresh portions of the stationary phase occurs (time t1). Simultaneously, partitioning between the fresh solvent and the stationary phase takes place at the site of the original sample. Continued additions of solvent carry solute molecules move down the column in a continuous series of transfers between the mobile and the stationary phases. Because the movement of the sample can occur in the mobile phase, however, the average rate at which a solute zone migrates down the column depends upon the fraction of time it spends in that phase. This fraction is small for solutes that are strongly retained by the stationary phase and it is large where retention in the mobile phase is more likely. Ideally the resulting differences in rates cause the components in the mixture to separate into bands, or zones located along the length of the column (t2). Isolation of the separated species is then accomplished by passing a sufficient quantity of mobile phase through the column to cause the individual zones to pass out the end, where they can be detected or collected ( times t3 and t4) (Figure 2).

2.3.1. Chromatograms

If a detector that responds to the presence of analyte (dye) is placed at the end of the column and its signal is plotted as a function of time (volume of added mobile phase), a series of peaks is obtained. Such a plot, called a chromatogram, is useful for both qualitative and quantitative analysis.

2.3.2. Migration Rates of Solutes The Partition Coefficient

An analyte is in equilibrium between the two phases;


where the equilibrium constant K is called the partition coefficient.

CS: molar concentration of analyte in stationary phase

CM: molar concentration of analyte in mobile phase


figure 2. Diagram showing the separation of a mixture of components A and B by column chromatography Retention Time

The time it takes after sample injection for the analyte peak to reach the detector is called



Paul C. 2002. “The Essence of Chromatography,” Elsevier. (e-book,

Skoog D. A. and Leary J. J. 1991. “ Principles of Instrumental Analysis”, 4th Edition, Saunders Collage Publishing.

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