Liquid chromatography separation technique .pptx

separation technique-Liquid chromatography (LC) is a technique that separates molecules in a liquid sample based on how strongly they stick to a stationary phase. It can be used to identify, quantify, and separate components in a mixture. A liquid sample is injected into a stream of solvent (mobile phase) that flows through a column packed with a stationary phase. The sample components separate as they flow through the column.

1. VALUE ADDED COURSE LIQUID CHROMATOGRAPHY

2. What is Liquid Chromatography? Liquid chromatography (LC) is an analytical technique in which the mobile phase is a liquid. It is carried out either in a column or a plane . The sample with the mobile phase is passed through a column or plane , accompanied by the stationary phase. Due to differences in adsorption, size, partitioning and ion exchange, different solutes will interact with the stationary phase to a different extent . Liquid Chromatography is an effective technique to separate mixtures. It is especially effective when the mixture is coloured . Even if the mixture is not coloured , we can visualise it using several visualisation methods like irradiating it with ultraviolet light.

3. Types of Liquid Chromatography Reversed-Phase Chromatography Normal Phase Chromatography Ion Exchange Chromatography Size Exclusion Chromatography

4. Reversed-Phase Chromatography Reversed-Phase Chromatography retains a non-polar stationary phase and polar mobile phase . Hydrophilic molecules in the polar mobile phase are transported to the column and eluted , whereas hydrophobic molecules of the polar mobile phase are absorbed in the stationary phase . Organic solvents, aqueous buffers, and a mixture of water are used to elute compounds in a reversed-phase column. In it, the alkyl chain is covalently bonded to stationary phase particles. Charged analytes can be separated through reverse-phase chromatography by ion pairing.

5. Normal Phase Chromatography Normal Phase Chromatography retains a polar stationary phase and a non-polar mobile phase. Silica and organic compounds with cyano and amino groups are commonly used in the stationary phase. Organic solvents like hexane or heptane mixed with polar solvents like chloroform, isopropanol and ethyl acetate are commonly used polar stationary solvents . Normal Phase Chromatography is generally used to separate chiral compounds, cis -trans isomers, geometric isomers and water-sensitive compounds. The less polar compounds are eluted first in normal phase chromatography, whereas more polar ones last.

6. Ion Exchange Chromatography Ion Exchange Chromatography retains the ionic stationary phase and aqueous buffer as the mobile phase . Ion exchanger Chromatography separates ions and polar compounds based on their affinity to the ion exchanger. It can be sub- categorised into two types, ie . Cation Exchange Chromatography Anion Exchange Chromatography A cation exchange chromatography retains positively charged cations , while an anion exchange chromatography retains negatively charged anions. Ion Exchange Chromatography is generally used to separate organic and inorganic ions from an aqueous solution. Ion Exchange Chromatography is generally used to separate compounds differing in functional groups. Ion Exchange Chromatography is also used to separate isomers . A major limitation of ion-exchange chromatography is that it is limited to the ionisable group.

7. Size Exclusion Chromatography Size Exclusion Chromatography is also known as Gel Filtration Chromatography. In Size Exclusion Chromatography, molecules are separated, based on their size and the pore size of the stationary phase. In some cases, molecules are also separated based on their molecular mass. In Size Exclusion Chromatography, more giant molecules are eluted first, whereas smaller molecules are last. Size Exclusion Chromatography is used to separate industrial polymers and proteins.

8. R f  Value R f  stands for Retention factor. It is the ratio of distance travelled by the analyte to that of the solvent front. It is the characteristic identification value of analytes . Compounds can be analysed and identified based on their R f   value. R f   = Distance travelled by analyte / Distance travelled by solvent .

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