It is a technique in which the separation of molecules is based on molecular structure and molecular composition. Two phases are involved in chromatography method. The one is stationary phase and the other is mobile phase.
The sample molecules show different interactions with the stationary phase.
The liquid is in the mobile phase which pumps through bed of particles while the particles are in the stationary phase. A mixture of the sample molecules is introduced into the liquid. The mixture of molecules is migrated by the mobile phase through the stationary phase. Thus the separation is based on the distribution of solute between mobile phase and the stationary phase.
The various types of chromatographic technique are used. These methods are based on the supports which use in separations such as silica gel, glass plates, volatile gases, paper and liquids etc. The different types of chromatographic techniques are given as below.
- Adsorption Chromatography
- Partition Chromatography
- Affinity Chromatography
- Gas Chromatography
- Liquid Chromatography
- Column Chromatography
- Paper Chromatography
- Flash Chromatography
- Reverse phase Chromatography
- Ion exchange Chromatography
This chromatography is especially used for analytical purpose. In the gas chromatography, the carrier gas is worked as mobile phase which have an inert gas. The stationary phase is a packed column. Generally gas-liquid
chromatography is used. It involves a vaporized sample is injected into the chromatographic column.
The sample is migrated through the column with the flow of carrier gas. The liquid stationary phase is adsorbed on the surface of the inert solid. The separation method can be affected by the polarity of stationary phase, temperature,
carrier gas flow, length of column, material amount etc.
The chemically inert gas is used as the carrier gas. Generally nitrogen, helium, argon, and carbon dioxide are used as carrier gas. It depends on the type of detector. The carrier gas system also has a molecular sieve for removing water and impurities. Sample injection port
The sample should not too large or to small to introduced onto the column. It may be reason for band broadening and resolution loss. Generally micro syringe is used for a proper injection of sample. The sample is injected through a rubber septum into a flash vaporizer port at the head of the column.
The sample size range is 10 to 20 micro liters for packed columns while the capillary columns need much less amount of sample (10-3 micro liter). The split or split less injector can be used as shown in figure. The sample is injected into glass liner of a heated chamber through the septum. The carrier gas enters the chamber. The sample vaporizes and the mixture of carrier gas, vaporized solvent and solutes are produced. A specific amount of this mixture enters onto the column.Columns
- Two columns packed and capillary (open tubular) are used. In the packed columns, a finely divided, inert, solid support material is coated with liquid (stationary phase). Generally they have length up to 1.5 - 10m and 2-4 mm internal diameter.
- The internal diameter of capillary columns is in tenths of a millimeter. Two types of capillary columns are; wall-coated
- open tubular (WCOT) and support-coated open tubular (SCOT). Capillary column are more used than packed columns.
- These have thin walls, flexible, good physical strength and have low reactivity than the glass capillary columns.
The column temperature depends on the boiling point of the sample. The temperature should be controlled in the range of tenths of a degree.
Detectors- Various types of detectors can be used in gas chromatography. Some are non-selective detector, specific detector, concentration dependent detector, mass flow dependent detector etc. the commonly used detector is flame ionization detector.
This is useful detector for organic compounds. The main features of the FID are high sensitivity, good linear range, and low noise. But it also have tendency to destroy the sample.
The detector is attached from the column. So the effluent enters in detector from the column and mix with gas of detector (hydrogen and air). The effluent gets ignited under the influence of flame ignition coil. The compound burns in the flame and produces ions and electrons. These ions and electrons can conduct electricity with the flame. A collector electrode is located above the flame. The current due to ionization of organic compounds is measured in recorder.
A piece of specialized paper is used paper chromatography. It is a planar system in which cellulose filter paper is act as stationary phase. The separation of compounds occurs on stationary phase. This is used for separation of amino acids, for detecting organic compounds, bio molecules, Hormones and drugs etc.
The substances are distributed between liquid phases. One phase is the water which present in pores of filter paper and other phase is mobile phase which moves on the paper. The separation of mixture is due to different attraction force towards stationary phase (water) and mobile phase (solvents).
Types of paper chromatography
The classification is based on the procedure of the development of chromatogram on the paper. So there are five types of chromatography.
- Ascending chromatography
- Descending chromatography
- Ascending- descending mode
- Radial mode
- Two dimensional chromatography
Mostly ascending type or radial type chromatography is used because they are easy to handle and not time consuming. They give chromatogram with fast speed.
- Selection of development: It depends on mixture, solvent, paper etc
- Selection of filter paper: it is based on size of pores of filter paper, quality of sample and method of separation.
- Preparation of sample: The sample is dissolved in suitable solvent which used as mobile phase.
- Sample spotting on the paper: Capillary tubes are used for spotting of sample on the paper.
- Chromatogram development: Sample spotted paper is immersing in the mobile phase which moves over the sample on the paper. This movement is based on the capillary action of paper.
- Drying of the paper and detection: After the development of chromatogram, the paper is dried with an air drier without touching on sample spots. The detecting solution is sprayed on the developed paper and dried to know about the sample chromatogram spots.
Liquid chromatography is an analytical technique of chromatography that is used for separating molecules. It is based on the different adsorption affinity of sample solution (solute) towards solvent. Due to the difference in ion exchange, partitioning or adsorption affinity, the mixture components easily separate from each other. These differences are used to determine the transit time of the solutes. This is the time of solutes in which they move through a column.
In a simple liquid chromatography, a column with a narrow bottom is used. In this column, the stationary phase is in equilibrium with a solvent. The interaction between stationary phases and the solutes can be, In the case of solids, ionic groups on a resin, liquids on an inert solid support, porous inert particles than the interactions are adsorption, ion-exchange, partitioning, and size-exclusion respectively. The mixture is filled from the top of the column with excess of solvent. The various components in the sample move with different rates due to different partition capacity between both the phases (the mobile liquid phase and the stationary phase). Thus the aliquots of the column effluent are collected as a function of time. Conventional LC is used in purification and separation of components and also in ultra trace separations. But for analytical separations of solutions for detection, the high-performance liquid chromatography instruments (HPLC) is used. It gives the faster results with high resolution.
In column chromatography method, the separation of a liquid or gaseous solution of the mixture is because of their flow through a packed tube filled with finely divided solid. This tube is coated with which an adsorbent liquid or can be a long capillary tube with coating of adsorbent liquid. The separation is based on rate of travel in the tube. Thus by determine the interaction of components, their separation and identification can be easily evaluated. This technique is especially used to purify the proteins.
- Stationary phase- The stationary phase is gel (silica or alumina gel), resin or metric. Three types of resin are Gel filtration, ion exchange and affinity. The reservoir is used to keep the constant pressure of column.
- Mobile Phase- The solvent is used as mobile phase. The commonly used solvents are hexane with ethyl acetate or toluene etc. water and methanol is not used as solvent because they are capable of dissolving silica gel. The choice of column depends upon the procedure of separation. By varying the diameter of columns, milligram to kilo gram quantities of materials can be separated.
- Column packing- Two methods are commonly used for column packing. The first is dry packing and the second is the slurry method. In dry packing, the micro scale column is used. It fills with a non polar solvent. Than alumina or silica powder is added and tapes the side of column with a pencil. The solid should move in the column. Try to avoid the cracks, air bubbles in the column. In the second dry pack method, the stationary phase is deposited in the column prior to the solvent. This is not a good method for packing.In the slurry method, first the solid stationary phase and small amount of non-polar solvent is mixed in a beaker for making a homogeneous mixture. Pour this mixture into the column carefully. It is a best method of column packing. After packing of column, the solvent is added from the top of the packing.
- Sample adding- The solution of sample mixture is made by using minimum amount of polar solvent. This solution is than added in the column form top side. A thin layer of white sand is also added to the top of the column which works as protective layer. Than again solvent is added. Thus the fractions of columns are made after some time. If the bands are colored than they move down with the solvent up to the end of column and collect them individually. But if they are color less than thin layer chromatography (TLC) is use for their detection. The spots of fractions are developed on TLC plate and identify all the components. The purification can be done by evaporating the solvent.
This is modified type of column chromatography. Column chromatography is a very time consuming method. But in the Flash Chromatography, pressure (up to 10 psi of air or nitrogen) is used to force the mobile phase through the column. This pressure increases the rate of the mobile phase and this gives the band with high speed. For getting good separation, fine alumina or silica (250 to 400 meshes) is used as stationary phase. It gives the high resolution results. The procedure is same as column chromatography.
In HPLC (High Performance Liquid Chromatography)
technique, high pressure pumps are used to increase the rate of movement of solvent through a packed column which is connected to detectors.
- Modern HPLC is especially used for separating proteins, lipids and nucleic acids in biochemistry. The aqueous mobile phases are required for their separation. Such as methanol or acetonitrile with water but they can easily dissolve silica or alumina stationary phases.
- Due to this reason, some non polar mobile phases are used which is known as reverse-phase. This is also called reverse-phase packing.
- These are made by bonding between hydrocarbon molecules (excess) and surface of silica gel particles.
- This makes the silica gel surface non polar. Thus the elution shows the opposite behavior towards alumina or silica column.
- So the packing and solvent behaves as non polar and polar respectively for the sample. The interaction of the non polar components of the solutes and the non polar stationary phase is the main cause of retention.
It’s the oldest type of chromatography. It consists of a mobile liquid or gaseous phase that is adsorbed on the surface of a stationary solid phase. There is equilibrium between both the phases. This is the cause of separation of different solutes.
The main principle of this method is that a thin film formed on the surface of a solid. The solid is supported by a liquid stationary phase. The solute is in equilibrium with both the phases.
This is the most specific type of chromatography. It is based on the specific interaction between two molecules. The one is solute molecule and a second molecule is immobilized on a stationary phase. For example, the immobilized molecule can be an antibody which interacts on the particular area of protein.
When a solute with mixture of proteins is passed by immobilized molecule, only the particular protein is reacted to this antibody. Thus the antibody binds to the stationary phase. After this interaction, the extraction of protein is completed by altering its ionic strength or pH. The packing material is called the affinity matrix. It should be inert. The “Agarose”
is used for this purpose.
This is useful in various fields. Some of the uses are described as below.
- It is commonly used technique for the separation of molecule. For example, it is used to remove pesticides and insecticides like DDT in the water and poly chlorinated biphenyls.
- It is widely used for testing the purification of drinking water.
- In pharmaceutical companies, it is used for producing pure materials for medicines and also for checking the contamination presence in medicines.
- It is used in pharmacy for detecting the chiral compounds (Enantiomers and optical isomers).
- In the food industry, this technique is very useful for analyzing and the separation of additives, proteins and amino acids etc. it used in forensic science for detecting the presence of drugs.