Do You Know - How Does Chromatography Work?

Chromatography is essentially a physical method of separation in which the components of a mixture are separated by their distribution between two phases; one of these phases in the form of a porous bed, bulk liquid, layer or film is called stationary phase while the other is a fluid that flows through the stationary phase is called as mobile phase.

Chromatography works because of the differences in the properties of molecules in materials, their mobility, total charge, hydrophobic groups present on the surface, and binding capacity with the stationary phase.

Three components form the basis of the chromatography technique.

  • Stationary phase: This phase is always composed of a “solid” phase or “a layer of a liquid adsorbed on the surface solid support”.
  • Mobile phase: This phase is always composed of “liquid” or a “gaseous component.”
  • Separated molecules

Column Chromatography is one of the most important and widely used techniques for the separation and analysis of complex organic mixtures. It consists of two phases: the mobile phase and the stationary phase. The stationary phase is solid and the mobile phase is liquid. The compound mixture moves along with the mobile phase as it flows through the stationary phase and separation of molecules occurs depending on their affinity with the stationary phase.

High-quality silica gel offers the lowest moisture content, tightest particle size distribution, minimal impurities. This results in greater reproducibility of the separation process. Another parameter that affects the successful completion of the process is mesh size of silica gel used as the stationary phase.

Mesh size of silica gel is given by the value which refers to the number of holes in the mesh that is used to sieve the absorbent. Thus higher mesh values such as "silica gel 230–400" have more holes per unit area and correspondingly smaller particles than "silica gel 60". Typically, 70–230 mesh size of silica gel is used for gravity column chromatography and 230–400 mesh for flash column chromatography.

Scale-up Chromatography: The scale-up of a chromatographic separation is in principle a simple procedure since the process parameters are scalable in a linear fashion and the process is scaled by increasing the column diameter while keeping the bed height, the velocity, and the volumes of the different phases (measured in column volumes) constant.