Chromatography is a vital biophysical process that permits it to separate, identify and separation of the constituents of a mixture to enable the quantitative as well as qualitative evaluation. This is a separation process where a mobile component that is carrying a mixture is made to move when in contact with a stationary phase.
There are many different types of chromatography that differ in the stationary and mobile method used. Column chromatography is an approach where the compounds which are separated inserted on surface of the column containing an adsorbent. Then, they are moved through the column at various rates that are based on the affinity level of the substance for the adsorbent , as well as the solvent or mixture and are then removed from the column as they leave the column at various time intervals.
It is a solid and liquid technique where both the stationary and mobile phases are solid and the mobile phase is gas or liquid. It was invented by American scientist D.T Day in 1900 . M.S. Tswett was the Polish botanist, in the year 1906 employed adsorption columns during his research into plant pigments.
Forms of Column Chromatography
There are two types of column chromatography.
- Liquid chromatography (LC)
- Gas chromatography (GC)
The most popular forms of column chromatography include:
- Adsorption chromatography
- Partition chromatography
- Ion exchange chromatography
- Gel chromatography
Principle of Column Chromatography
In column chromatography, the stationary phase is placed in the form of a metal or glass column. The mixture of analytes then applied , and that mobile phase often known as the eluent, flows through the column through using a pumping device or gas pressure applied. The stationary phase can be coated on small, discrete particle size (the matrix) and then packed into the column , or applied as a thin layer to the wall inside within the column. As the eluent is pumped through the column, the analytes are separated by their distribution coefficients, and then emerge independently in the eluate after it is removed from the column.
Instrumentation of Column Chromatography
The typical column-chromatographic process that uses a liquid or gas mobile phase is made up of the following elements:
- Stationary phase: selected to be the most suitable one for the analytes that need to be separated.
- Column: In liquid chromatography, these typically are 25- 50 cm long with a 4mm in diameter. They are made from stainless steel. In gas chromatography they’re three to four meters long with a 4mm internal diameter , and are constructed out of stainless steel or glass. They could be the standard type that is that is filled with stationary phase, or the microbore type where the stationary component is encased directly on the inner walls of the column.
- Mobile phase and delivery system: Selected to supplement the stationary phase and , consequently, to differentiate between samples and deliver an unchanging flow through the column.
- Injector system: To inject tests up to the surface of the column, in a reproducible way.
- Detector and chart recorder: To provide an ongoing report of analytes in the eluate , as it is released in the column. Detection is typically determined by measuring an physical parameter, such as absorption of visible or ultraviolet light or fluorescence. A peak in the chart recorder is a representation of every analyte that is separated.
- Fraction collector: To collect the analytes that are separated for future biochemical research.
What is Elution?
Elution is a process that involves the removal of a substance’s ions through an exchange with another. The chromatographic method of extraction of an adsorbed substance out of a solid adhering material by using the solvent. The solvent is the eluent or mobile phase that flows throughout the column. If the nature of the eluent is in line with the chemical polarity of the molecules within the sample, they desorb from the adsorbent, and then dissolve in the eluent.
The percentage that is mobile that moves the sample components is referred to as the eluent. The mixture of solvent and solute that comes out of the column is referred to as the eluate. The eluate consists part of the liquid phase as well as analytes. A substance that is used to separate and move components of a mix through the chromatograph’s column. The liquid chromatography solvent is a liquid solvent , whereas in gas chromatography , it is the gas that is used as a carrier.
Steps in Column Chromatography
A. Preparation of the Column
The column is mostly an unpacked glass tube with the right stationary phase. Glass wool/cotton wool or an asbestos pad are located at the bottom of the column prior to packaging the stationary phase. After packing, a paper disc is placed on top to ensure it is sure that the stationary phase isn’t affected when introducing the mobile or sample phase. There are two ways of column preparation and they are:
- Dry packing / dry filling: In this method, the amount of adsorbent is added into a fine, dry powder into the column. The liquid is then allowed to move through the column until equilibrium is reached.
- Wet packing / wet filling: In this method, the solution of the absorbent along with an mobile layer is made and then filled in the column. This is considered to be the most efficient method of packing.
- Prior to using the column needs to be cleaned properly and dried.
- The column must also be free of impurities and filled evenly with the stationary phase.
B. Introduction of the Sample
The sample that is normally comprised of a variety of components is dissolved using a minimal amount of that mobile phase. The whole sample is introduced to the column at the same time and is then adsorbed onto the top of the column. In this area, each samples can be separated through an elution process.
The elution technique allows the various components are separated and separated from the entire column. This can be accomplished by two methods:
- Isocratic elution technique: Same chemical composition of the solvent or a solvent with identical polarity is utilized throughout the separation process. Eg. Use of chloroform alone.
- Gradient elution technique: Solvents that are slowly increasing polarity or strength for elution are utilized in the process of separation. E.g. initially benzene, then chloroform, then ethyl acetate then chloroform
D. Detection of Components
If the compounds that are separated from the column chromatography process have a color, then the process of the separation process can be observed visually. The compounds that are to be separated by column chromatography have no color. In this situation small amounts of liquid are collected sequentially in tubes that are labelled with the composition and chemical makeup of every of the fractions is examined using TLC.
Column Chromatography Experiment
- The stationary phase is by the use of solvent. The upper levels of the mobile phase as well as stationary phase needs to be in the same range. The mobile phase, also known as the eluent can be a solvent or a mixture of solvents. In the initial step, the mixture of compound that has to be separated is added to at the bottom of the column, without disrupting the top layer. The tap is switched on and the process of adsorption on the silica’s surface commences.
- In order to not disturb the stationary phase, the solvent solution is slowly added by gently rubbing the edges of the column. It is then added through the procedure as per requirements.
- The tap is switched on to start the motion of compounds within the mixture. The mechanism of movement is determined by the polarity of the molecules in the sample. Non-polar molecules are moving at a faster rate as compared to the more polar components.
- If, for instance, a compound mixture is composed of three distinct compounds, namely blue, red and green, then their order, based on their polarity will be like this: red>blue>green
- Since the polarity of the green compound is lower than the polarity of the green compound, it will move first. When it reaches the bottom of the column, it’s taken away in an unclean test tube. Following this, the red compound is gathered and the blue compound is removed. All of them are taken into distinct test tubes.
Types of Column Chromatography:
- Adsorption column chromatography – Adsorption Chromatography is a method of separation in which the constituents of the mix are adsorbed onto the surface of the adsorbent.
- Partition column chromatography – The stationary phase and mobile phase are both liquids in partition chromatography.
- Gel column chromatography – This method of chromatography separation occurs by means of a column filled with gel. It is stationary because it contains the solvent that is kept within the gaps of a solvent.
- Ion exchange column chromatography – A method of chromatography where the stationary phase is always an ion exchange resin.
Factors Affecting Column Efficiency
- Sizes and dimensions for the column
- Size of the particle of the Adsorbent
- The nature of the solvent
- The temperature of the column
Applications of Column Chromatography
Column chromatography is among the most efficient methods to cleaning and separation of liquids and solids. Its main application is:
- Separation of mixtures of compound.
- Purification or removal of impurities process.
- Isolation of active components.
- The isolation of metabolites from biological fluids.
- Estimation of the drugs in their formulations or crude extracts.
Advantages of Column Chromatography
- All kinds of mixtures is separated by column chromatography.
- The mixture can be separated.
- A wider range of mobile phases.
- In the preparative form the sample may be separated and used again.
- Automation is achievable.
Limitations of Column Chromatography
- A time-consuming process.
- A greater quantity of solvents is required , which could be costly.
- Automation makes the procedure more complex and expensive.