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Chromatography ( chromatography )

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;v Chromatography Theory Biological molecules can be separated from one an- other by exploiting differences in their size, charge, or affinity for a particular ligand or solid support. Chromatography is the laboratory technique that allows separation of molecules based on their dif- ferential migration through a porous medium. Al- though there are many different types of chro- matography, the principle behind the separation of the molecules is the same: a mixture of compounds will have different affinities for the stationary phase (solid support or matrix) on which it is adsorbed and the mobile phase (buffer or solvent) passing through the stationary phase. General Theory Modern preparative and analytical chromatography is most often performed in a column format. Here, the porous matrix or solid support is enclosed in a durable cylinder or column saturated with aqueous buffer or organic solvent (Fig. 2-1). The column is loaded with a solution containing a mixture of com- pounds at the top of the column by allowing it to flow into the porous medium. After the compounds have entered the solid matrix, they can be differ- entially eluted from the column either by continu- ous buffer flow or by changing the nature of the mobile phase passing through the porous matrix. This process of eluting compounds from the col- umn is termed “development.” As the different compounds emerge from the column, the eluted so- lution can be separated into multiple “fractions” or “cuts” that can be analyzed for the presence of a molecule of interest. Partition Coefficient and Relative Mobility. As stated earlier, molecules adsorbed on a solid sup- port (stationary phase) will partition between it and a mobile phase passing through the stationary phase. To predict the behavior of different mole- cules during chromatography, one must be able to define the affinity of a compound for the station- ary and/or mobile phase. The term used to describe the affinity of a compound for the stationary phase is the partition coefficient (􏰋). It is defined as the frac- tion of the compound that is adsorbed on the sta- tionary phase at any given point in time. The par- tition coefficient can have a value between 0 and 1. The greater the value of 􏰋, the greater the affinity of the compound for a particular stationary phase. For example, a molecule with 􏰋 0.4 will be 40% adsorbed on the stationary phase at any given point in time. It has less affinity for the stationary phase than another molecule with 􏰋 0.7 (70% adsorbed at any given point in time). Mathematically, the par- tition coefficient can be expressed as: EXPERIMENT 2 􏰋􏰍􏰍􏰍􏰍􏰍 molecules adsorbed on stationary phase molecules in stationary and mobile phase The affinity of a molecule for the mobile phase is described in terms of relative mobility (Rf). This term describes the rate of migration of the mole- cule relative to the rate of migration of the mobile phase passing through the solid support. Mathe- matically, Rf is equal to 1 􏰌 􏰋. For instance, a mol- ecule with 􏰋 0.4 will be found in the mobile phase 25

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