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In this article we discussedabout column chromatography in detail. What is column chromatography, Principle of column chromatography, components of column chromatography, steps involved in column chromatography, its application andvantage and disadvantages.
Column chromatography is a solid, liquid technique in which the stationary phase is a solid and the mobile phase is a liquid or gas. In column chromatography, a mixer is dissolved in a solvent and poured down a column packed with a solid material. Different components within the mixer flow out of the column at different rates depending on their absorption to the solid material. In this way, the different components were isolated and separated. Column chromatography was first developed by the American chemist DT Day in the year 1900, while Miss Swett used the absorption columns in his investigation of plant pigments,
In column chromatography, the statutory phase is packed into a glass or metal column. The mixture of analytes or sample is then applied and the mobile phase, commonly referred to as eluent, is passed through the column either by a use of pumping system or applied gas pressure. The stationary phase is either coated on to discrete small particles and packed into the column or applied as a thin film to the inside wall of the column. As the eluent or mobile phase flows through the column, analytes or samples separates depending on the different degree of adhesion to the silica of each component in the sample or the compound mixer. This is the principle of column chromatography
Components of column chromatography.
There are 6 components of column chromatography they are as follows-
The first component of the column chromatography is statiary phase. Statiary phase is a solid material, usually silica gel having a good absorption property. Statiary phase should be suitable for the analytes to be separate. The statutory phase should not Causeway any hindrance in the flow of the mobile phase.
The 2nd component of the column chromatography is mobile phase. Mobile phase is made-up of solvents that complement the statutory phase. The mobile phases or solvents act as a developing agent to promote separation of the components in the sample to form bands and an eluting agent to remove the components from the column that are separated during the experiment.
The third component of column chromatography is column A. Column material and its dimension are very crucial to support the stationary phase and promote effective separations. For liquid chromatography, the column length is 2 to 50cm long and 4 MM internal diameter and fabricated with stainless steel. For gas chromatography, the column length is 1 to 3M long and two to 4mm internal diameter and fabricated either with glass or stainless steel.
The 4th component of the column chromatography is injector system. Injector system is responsible for delivering test samples to the columns top in a reproducible pattern.
The 5th component of the column chromatography is detector or chart recorder. Detector or chart recorder is used to give a continuous record of the presence of the analytes in the ELU as it emerges from the column. Detection is usually based on the measurement of your physical parameters such as visible or ultraviolet absorption or fluorescence. A peek on the chart recorder represents each separated analyte.
The 6th component of the column chromatography is fraction collector. A fraction collector is used for collecting the separated analytes for further biochemical studies.
There are Four steps involved in column Chromatography
The first step of the column chromatography is preparation of the column. The column mostly consists of a glass tube packed with a suitable stationary phase. A glass wool or cotton wool or an asbestos pad is placed at the bottom of the column before packing the stashery phase. After packing a paper just kept on the top so that the stashery layer is not disturbed during the introduction of the sample or the mobile phase, there are two types of preparing the column. They are dry packing or dry filling is the first type. In this, the amount of absorbent needed is added as a fine dry powder in the column and the solvent flows freely through the column until equilibrium is achieved. The second type is wet packing or wet filling. In this, the slurry of absorbent is prepared along with the mobile phase and is poured into the column. It is recorded as the ideal technique for packaging. Before using the column, it should be washed properly and dried. The column should be free from impurity and uniformly filled with the stashary phase.
Second step of the column chromatography is introduction of the sample. The sample which is usually a mixer of components is dissolved in minimum quantity of the mobile phase. The entire sample is introduced into the column at once and get absorbed onto the top portion of the column. From this zone, individual sample can be separated by a process of elution.
The third step of the column chromatography is elution. By elution technique, the individual components are separated out from the column. The process of elution can be carried out by employing 2 techniques. The first technique is Isocratic elution technique. In this technique, the same solvent, composition or solvent of same polarity is used throughout the process of separation. Example use of chloroform alone. The second technique is gradient elution technique. In this technique solvents of gradually increased polarity or increased elution strength are used during the process of separation. Example, initially benzene, then chloroform, then ethyl acetate, then chloroform.
The 4th and final step of the column chromatography is detection of compounds. If the compounds separated in a column chromatographic procedure are colored, the progress of the separation can be simply be monitored visually. If the compounds undergoing separation are colorless, then small fractions of the elutant are sequentially collected in the tubes that are labeled through TLC. The composition of each fraction is determined. Make some of the factors affecting the column efficiency or dimensions of the column particle, size of the absorbent, nature of the solvent, temperature of the column and pressure.
This Article is Written by Bio Help Learning Team, Bio Help Learning where we specialize in providing cutting-edge skill development and biotechnology services to help individuals and organizations reach their full potential in the field of biotechnology.
