Chromatography is a technique used for the separation and analysis of complex mixtures. The separation phenomenon was first reported by a Russian botanist Mikhail Tswett in 1906. He separated different colored constituents of plant extract by passing them through a column of finely divided calcium carbonate, alumina, and sucrose.
Tswett observed that various pigments such as xanthophylls and chlorophylls appeared as colored bands on the column and he coined the term the chromatography for this technique (Greek word chroma means ‘column’ and graphein means ‘to write’).
Many advances have been made in this field in the last century, notably by Martin and Synge who introduced the concept of gas-liquid chromatography in 1941. They were awarded Noble Prize for their contribution to chromatographic science. Broadly, chromatographic techniques can be divided into planar and column chromatography.
As the name suggests, the planar technique incorporates the use of a flat, planar, two-dimensional surface (where only length and breadth are taken as area) on which chromatograms are formed. This surface is coated with the stationary phase. Paper Chromatography, Thin Layer Chromatography (TLC), and High-Performance Thin Layer Chromatography (HPTLC) are different types of planar techniques. Column Chromatography has a stationary phase held in a column or a narrow tube. The most common types are Gas Chromatography (GC) and High-Performance Liquid Chromatography (HPLC).
Chromatography is a dynamic process and all its types involve two immiscible phases- one is a stationary phase (which stays inside the column or on a planar surface) and the other mobile phase (which moves through the column or over the planar surface).
The mobile phase can be liquid or gas and helps in redistributing the components of a mixture between the two phases, thus aiding in their separation. The stationary phase can be solid, liquid, liquid adsorbed on solid and organic species bonded to a solid surface. Solid forms include non-exchange resins.
Principle of Centrifugation Column chromatography separations involves stationary phase being held in a narrow tube (column). The mobile phase or eluent, along with the mixture of components (solutes) is forced through the tube having stationary phase, under pressure. This process is termed elution.
Eluate is fluid emerging from the end of the column. Columns are either packed or open tubular. The packed column is filled with small particles of stationary phase whereas an open tubular column is usually a narrow capillary tube with stationary phase coated on the inside wall.
Process of Elution
The process of elution and separation of various components by column chromatography can be takes place. The Chromatogram Various solutes emerging from the column are observed with “Detectors”.
The detectors may be specific for the type of solute detected. A chromatogram is a graph showing the detector response plotted against elution time. It is a chromatogram of two components. Such a plot is useful for carrying out both qualitative and quantitative analysis. The positions of the peaks on the time axis are used for qualitative analysis whereas the peak areas reveal the quantitative aspect of the components.
Retention time is the time taken by a solute to reach the detector after the mixture is injected into the column. It is represented by tr. Adjusted retention time, tr given by the expression
t’r = tr – tm
tm is the time taken by unretained species or mobile phase to reach the detector.
It is also referred to as Dead Time. So, the adjusted retention time for a solute is to move through the column beyond the time required by the mobile phase to travel through the length of the column.
The solute components partition themselves in the mobile phase and stationary phase as they move through the column. If the column is run slow enough, the concentration of solute in the stationary phase and mobile phase reaches equilibrium. The equilibrium constant or partition coefficient, K, for this reaction is defined as
K = CS / CM
It is also called the retention factor and is used to describe the rate of migration of the solute on the column. The capacity factor k’ is defined as
k’ = tr – tm / tm
Selectivity Factor or Relative Relation
This term describes the extent of separation between two components. The greater the value of selectivity factor or relative retention, the greater is the separation between closely spaced components in the chromatogram.
Measures of Column Efficiency
Column efficiency depends upon how well the column can separate the components of the sample.
Two factors contribute to this aspect:
• The width of the peaks.
• The difference in retention times between peaks.
Wide peaks result in poor separation and peaks having very close retention times may not be able to resolve completely. We will deal with peak width before elaborating upon the resolution of the peaks.
Effect of peak width
The width of the chromatographic peak, as is recorded on the chromatogram is the culmination of various phenomena taking place in the column. So, we will elaborate on theoretical terms related to the rate of movement of a solute down a column. The efficiency of separation can be attributed to the concept of Height Equivalent to Theoretical Plates (HETP) or simply plate height.
A theoretical plate is an imaginary concept that does not conform to any actual parameter in the column, but for evaluation purposes, it is a useful measure.
Plate height refers to the length in the column within any two adjacent plates. The name is derived from the theory of distillation; in which column is made up of a series of bubble cap tile plates and separation is performed in these discrete stages or plates.
Plate height is the length that can achieve equilibrium of the solute between the stationary phase and mobile phase under a particular set of conditions (flow rate, temperature of column, type of stationary phase, etc.).
Chromatography is a separation technique involving the mobile phase and stationary phase. Components of the mixture are separated based on their affinity towards either of these phases. Column chromatography occurs in the narrow column and depending on whether the mobile phase is a liquid or gas, it is called Liquid Chromatography or Gas chromatography.
Application of Column chromatography
• Separation of mixture of compounds.
• Purification process.
• Isolation of active constituents.
• Estimation of drug in formulation.
• Separation of diastereomers.