Column Chromatography- Definition, Principle, Parts, Steps, Uses

Column chromatography is a technique in which the substances to be separated are introduced onto the top of a column packed with an adsorbent, passed through the column at different rates that depend on the affinity of each substance for the adsorbent and for the solvent or solvent mixture, and are usually collected in solution as they pass from the column at different times. It is a solid-liquid technique in which the stationary phase is a solid and the mobile phase is a liquid or gas. It was developed by the American chemist D.T Day in 1900 while M.S. Tswett, the Polish botanist, 1906 used adsorption columns in his investigations of plant pigments.

• Liquid chromatography (LC): This is a form of column chromatography in which the mobile phase is a liquid or a mixture of liquids and the stationary phase is a solid or a liquid coated on a solid support. LC can be further divided into subtypes such as high-performance liquid chromatography (HPLC), reversed-phase liquid chromatography (RPLC), size-exclusion chromatography (SEC), ion-exchange chromatography (IEC), and affinity chromatography (AC).
• Gas chromatography (GC): This is a form of column chromatography in which the mobile phase is a gas or a mixture of gases and the stationary phase is a solid or a liquid coated on a solid support. GC can be further divided into subtypes such as gas-solid chromatography (GSC), gas-liquid chromatography (GLC), and capillary gas chromatography (CGC).
• Adsorption chromatography: This is a form of column chromatography in which the separation of analytes is based on their differential adsorption on the surface of the stationary phase. The stationary phase is usually a polar solid such as silica gel or alumina and the mobile phase is a non-polar or slightly polar solvent or solvent mixture. Adsorption chromatography can be used to separate organic compounds based on their polarity, functional groups, or molecular size.
• Partition chromatography: This is a form of column chromatography in which the separation of analytes is based on their differential partitioning between two immiscible phases. The stationary phase is usually a liquid that is immiscible with the mobile phase and is coated on an inert solid support. The mobile phase is another liquid that has different solubility properties for the analytes. Partition chromatography can be used to separate organic compounds based on their polarity, acidity, or basicity.
• Ion-exchange chromatography: This is a form of column chromatography in which the separation of analytes is based on their differential interaction with charged groups on the surface of the stationary phase. The stationary phase is usually a resin that contains either positively or negatively charged functional groups and the mobile phase is an aqueous solution that contains counter-ions and buffers. Ion-exchange chromatography can be used to separate ionic compounds based on their charge, size, or affinity for the resin.
• Gel chromatography: This is a form of column chromatography in which the separation of analytes is based on their differential exclusion from the pores of the stationary phase. The stationary phase is usually a gel that has a network of pores of varying sizes and shapes and the mobile phase is a solvent or solvent mixture that can penetrate the gel. Gel chromatography can be used to separate macromolecules such as proteins, nucleic acids, or polymers based on their molecular weight, shape, or charge.

Column chromatography is based on the principle of differential adsorption or partition of the analytes between a stationary phase and a mobile phase. The stationary phase is a solid material that has a large surface area and can retain some molecules more strongly than others. The mobile phase is a liquid or gas that flows through the column and carries the analytes along with it. The analytes have different affinities for the stationary and mobile phases, depending on their chemical and physical properties. Therefore, they move at different rates through the column and get separated into distinct bands or zones.

The separation of analytes in column chromatography depends on several factors, such as:

• The polarity of the analytes and the stationary and mobile phases. Generally, polar analytes have stronger interactions with polar stationary phases and weaker interactions with non-polar mobile phases, and vice versa for non-polar analytes. Therefore, by choosing an appropriate combination of polarity for the phases, one can achieve a good separation of analytes based on their polarity differences.
• The size and shape of the analytes and the stationary phase particles. Generally, smaller and more spherical analytes can diffuse faster through the pores of the stationary phase than larger and more irregular ones. Therefore, by choosing an appropriate size and shape of the stationary phase particles, one can achieve a good separation of analytes based on their size and shape differences.
• The temperature and pressure of the column. Generally, higher temperature and lower pressure increase the mobility of the analytes and decrease their retention time on the column. Therefore, by adjusting the temperature and pressure of the column, one can control the speed and efficiency of the separation.

The principle of column chromatography can be illustrated by a simple example. Suppose we have a mixture of two compounds, A and B, that have different polarities. We choose a polar stationary phase (such as silica gel) and a non-polar mobile phase (such as hexane) for the separation. We load the mixture onto the top of the column and start eluting with hexane. Compound A, being more polar, will have stronger interactions with the silica gel than with hexane, and will therefore stay longer on the column. Compound B, being less polar, will have weaker interactions with the silica gel than with hexane, and will therefore move faster through the column. As a result, compound B will elute first from the column, followed by compound A. We can collect them in separate fractions as they exit the column.

A typical column chromatographic system using a gas or liquid mobile phase consists of the following components:

• A stationary phase: Chosen to be appropriate for the analytes to be separated. It can be a solid, a liquid, or a gel that is packed into a column or coated on its inner wall. The stationary phase should have high surface area, uniform particle size, and good chemical stability. Some examples of stationary phases are silica gel, alumina, cellulose, ion exchange resins, and polyacrylamide gels.
• A column: In liquid chromatography, these are generally 25-50 cm long and 4mm internal diameter and made of stainless steel or glass. In gas chromatography, they are 1-3m long and 2-4mm internal diameter and made of either glass or stainless steel. They may be either of the conventional type filled with the stationary phase, or of the microbore type in which the stationary phase is coated directly on the inside wall of the column. The column should have good mechanical strength, low dead volume, and uniform packing.
• A mobile phase and delivery system: Chosen to complement the stationary phase and hence to discriminate between the sample analytes and to deliver a constant rate of flow into the column. The mobile phase can be a liquid or a gas that carries the analytes through the column. The mobile phase should have low viscosity, low toxicity, and high purity. Some examples of mobile phases are water, organic solvents, helium, nitrogen, and hydrogen. The delivery system consists of pumps or pressure regulators that control the flow rate and pressure of the mobile phase.
• An injector system: To deliver test samples to the top of the column in a reproducible manner. The injector system can be manual or automatic, depending on the type and volume of the sample. The injector system should have low sample loss, low contamination, and high precision. Some examples of injector systems are syringes, loops, valves, and autosamplers.
• A detector and chart recorder: To give a continuous record of the presence of the analytes in the eluate as it emerges from the column. Detection is usually based on the measurement of a physical parameter such as visible or ultraviolet absorption, fluorescence, refractive index, conductivity, mass spectrometry, or radioactivity. A peak on the chart recorder represents each separated analyte. The detector and chart recorder should have high sensitivity, high selectivity, high linearity, and low noise.
• A fraction collector: For collecting the separated analytes for further biochemical studies. The fraction collector can be manual or automatic, depending on the number and volume of fractions to be collected. The fraction collector should have low cross-contamination, low evaporation loss, and high accuracy.

In this way, column chromatography can be performed using different combinations of stationary phases, mobile phases, columns, injectors, detectors, and fraction collectors to achieve optimal separation and purification of various substances.

Column chromatography involves the following steps:

• Preparation of the column: The column is a glass or metal tube packed with a suitable stationary phase, such as silica gel or alumina. A plug of cotton or glass wool is placed at the bottom of the column to prevent the stationary phase from leaking out. The column is then filled with a solvent or a solvent mixture that acts as the mobile phase. The solvent should be chosen to have a low affinity for the stationary phase and a high affinity for the solutes to be separated. The column is then equilibrated with the solvent until it flows out at a constant rate.
• Introduction of the sample: The sample, which is a mixture of solutes to be separated, is dissolved in a small amount of the same solvent used in the column. The sample solution is then carefully applied to the top of the column, either by using a pipette or by injecting it through a septum. The sample should form a narrow band on the top of the stationary phase.
• Elution: Elution is the process of separating the solutes by passing the mobile phase through the column. As the mobile phase flows down the column, it carries along the solutes at different rates depending on their affinity for the stationary phase and the mobile phase. The solutes with lower affinity for the stationary phase and higher affinity for the mobile phase will elute faster than those with higher affinity for the stationary phase and lower affinity for the mobile phase. Elution can be done in two ways:

• Isocratic elution: This means using the same solvent composition or solvent polarity throughout the separation. This is suitable for separating solutes with large differences in their affinities for the stationary phase and the mobile phase.
• Gradient elution: This means changing the solvent composition or solvent polarity gradually during the separation. This is suitable for separating solutes with small differences in their affinities for the stationary phase and the mobile phase, or for separating a large number of solutes.

• Detection and collection of fractions: As the solutes elute from the column, they can be detected and collected in different ways:

• Visual detection: If the solutes are colored, they can be seen as distinct bands moving down the column. The fractions containing each solute can be collected manually by using a valve or a stopcock at the bottom of the column.
• Instrumental detection: If the solutes are colorless or have low visibility, they can be detected by using an appropriate detector attached to the end of the column. The detector can measure a physical property of the solutes, such as absorbance, fluorescence, refractive index, conductivity, etc. The detector can also generate a chromatogram, which is a plot of detector response versus elution time or elution volume. The chromatogram can show peaks corresponding to each solute. The fractions containing each solute can be collected automatically by using a fraction collector that is synchronized with the detector.

Column efficiency is a measure of how well the column can separate the analytes in a mixture. It depends on several factors, such as:

• Dimensions of the column: The length and diameter of the column affect the resolution and retention time of the analytes. Longer columns provide more interactions between the analytes and the stationary phase, resulting in better separation. However, longer columns also increase the pressure drop and the elution time. Smaller diameter columns reduce the diffusion and band broadening of the analytes, leading to higher efficiency. However, smaller diameter columns also require smaller sample volumes and more sensitive detectors.
• Particle size of the adsorbent: The particle size of the stationary phase determines the surface area available for adsorption and the flow rate of the mobile phase. Smaller particles provide more surface area and better separation, but they also increase the pressure drop and decrease the flow rate. Larger particles allow faster flow rates and lower pressure drop, but they also reduce the surface area and the separation efficiency.
• Nature of the solvent: The choice of the mobile phase affects the polarity, viscosity, and elution strength of the solvent. The polarity of the solvent determines its affinity for the analytes and the stationary phase. More polar solvents tend to elute more polar analytes faster, while less polar solvents tend to elute less polar analytes faster. The viscosity of the solvent affects the flow rate and the pressure drop of the mobile phase. Higher viscosity solvents have lower flow rates and higher pressure drop, while lower viscosity solvents have higher flow rates and lower pressure drop. The elution strength of the solvent is related to its ability to displace the analytes from the stationary phase. Higher elution strength solvents can elute more strongly adsorbed analytes faster, while lower elution strength solvents can elute more weakly adsorbed analytes faster.
• Temperature of the column: The temperature of the column affects the solubility, volatility, and diffusion of the analytes in the mobile phase. Higher temperature increases the solubility and volatility of the analytes, resulting in faster elution and lower retention time. However, higher temperature also increases the diffusion and band broadening of the analytes, leading to lower resolution and efficiency. Lower temperature decreases the solubility and volatility of the analytes, resulting in slower elution and higher retention time. However, lower temperature also decreases the diffusion and band broadening of the analytes, leading to higher resolution and efficiency.
• Pressure: The pressure applied to the column affects the flow rate and density of the mobile phase. Higher pressure increases the flow rate and density of the mobile phase, resulting in faster elution and lower retention time. However, higher pressure also increases the diffusion and band broadening of the analytes, leading to lower resolution and efficiency. Lower pressure decreases the flow rate and density of the mobile phase, resulting in slower elution and higher retention time. However, lower pressure also decreases the diffusion and band broadening of the analytes, leading to higher resolution and efficiency.

These factors can be optimized to achieve a balance between speed, resolution, and sensitivity in column chromatography.

Column chromatography is one of the most useful methods for the separation and purification of both solids and liquids. Its major applications include:

• Separation of mixture of compounds: Column chromatography can be used to separate complex mixtures of organic or inorganic compounds based on their different affinities for the stationary and mobile phases. For example, column chromatography can be used to separate pigments from plant extracts, amino acids from proteins, steroids from biological fluids, etc.
• Removal of impurities or purification process: Column chromatography can also be used to remove unwanted impurities or contaminants from a sample. For example, column chromatography can be used to purify organic compounds from synthetic reactions, natural products from crude extracts, enzymes from cell lysates, etc.
• Isolation of active constituents: Column chromatography can be used to isolate and identify the active constituents of a sample that have a specific biological or chemical activity. For example, column chromatography can be used to isolate antibiotics from microorganisms, hormones from glands, alkaloids from plants, etc.
• Isolation of metabolites from biological fluids: Column chromatography can be used to isolate and analyze the metabolites of a drug or a substance that are present in biological fluids such as blood, urine, saliva, etc. For example, column chromatography can be used to detect the presence of drugs or toxins in forensic samples, monitor the pharmacokinetics of drugs in clinical trials, study the metabolic pathways of drugs in pharmacology, etc.
• Estimation of drugs in formulation or crude extracts: Column chromatography can be used to quantify the amount of a drug or a substance that is present in a formulation or a crude extract. For example, column chromatography can be used to determine the purity and potency of pharmaceutical products, measure the concentration of active ingredients in herbal medicines, assess the quality and stability of food products, etc.

Column chromatography is a widely used technique for the separation and purification of compounds. However, like any other technique, it has its advantages and limitations. Some of the advantages of column chromatography are:

• It can separate any type of mixture, whether it is solid or liquid, organic or inorganic, polar or non-polar.
• It can handle any quantity of the mixture, from micrograms to kilograms, depending on the size and capacity of the column.
• It offers a wider choice of mobile phase, which can be adjusted to optimize the separation and elution of the compounds.
• It can be used for preparative purposes, where the separated compounds can be collected and reused for further analysis or synthesis.
• It can be automated, where the column operation, detection, and fraction collection can be controlled by a computer system.

Some of the limitations of column chromatography are:

• It is a time-consuming method, as it requires careful preparation of the column, sample, and mobile phase, as well as monitoring of the elution process.
• It consumes large amounts of solvents, which may be expensive, toxic, or hazardous to the environment.
• It may require complex and costly instrumentation, especially for high-performance liquid chromatography (HPLC) or gas chromatography (GC), which involve high pressure, temperature, and detection systems.
• It may suffer from some problems such as column deterioration, adsorbent degradation, peak broadening, tailing, or overlapping.

Therefore, column chromatography is a powerful and versatile technique for separation and purification of compounds, but it also has some drawbacks that need to be considered and overcome.

We are Compiling this Section. Thanks for your understanding.