Liquid Chromatography

Chromatography is an essential analytical technique used for separating, identifying, and purifying mixture components for quantitative and qualitative analysis. Chromatography is based on the principle that-

Each element migrates at a different rate through the stationary phase under the influence of the mobile phase. The stationary phase is a porous solid like silica or alumina, whereas the mobile phase is solvent or gas.

Table of Content

• Components of Chromatography
• Liquid Chromatography
• Types of Liquid Chromatography
• Reversed-Phase Chromatography
• Normal Phase Chromatography
• Ion Exchange Chromatography
• Size Exclusion Chromatography
• R_f Value
• Advantages of Liquid Chromatography
• Applications of Liquid Chromatography
• Frequently Asked Questions – FAQs

Components of Chromatography

Based on the above approach, we can classify chromatography into three components.

• Stationary Phase: It is the phase in which the mobile phase passes. It is generally a solid phase or is a layer of liquid adsorbed on the concrete surface.
• Mobile Phase: It is either a liquid or gaseous phase chromatography system.
• Separated molecules.

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.
• Thus, components are separated by utilising these differences.
• 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.

Types of Liquid Chromatography

We can classify liquid chromatography into four types based on the components of chromatography:

• Reversed-Phase Chromatography
• Normal Phase Chromatography
• Ion Exchange Chromatography
• Size Exclusion Chromatography

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.

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.
• Ion Exchange Chromatography is generally used to separate compounds differing in functional groups.
• Ion Exchange Chromatography is also used to separate isomers.

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.

1. Cation Exchange Chromatography
2. 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 has the highest matrix tolerance and predictable elution patterns.
• A major limitation of ion-exchange chromatography is that it is limited to the ionisable group.

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.

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.

For example,

R_f value of substances A, B, and C will be as given under

R_f value of A= a/X

R_f value of B= b/X

R_f value of C=c/X

Advantages of Liquid Chromatography

• Liquid Chromatography is a cheap separatory technique to separate mixtures.
• The flow of the mobile phase, detection of separation bands and collection of each component is done manually.
• Glass wear used for liquid chromatography is readily available and inexpensive too.

Applications of Liquid Chromatography

• Liquid chromatography is used for testing ink samples.
• It is used in environmental analysis and cleanliness testing.
• It is used in food analysis and quality control
• It is used in the pharmaceutical industries and chemical industries.
• It is used in forensic science and hospitals.