Question

Explain in detail the following techniques giving their principle, different types, and applications:

a.Ion Exchange Chromatography

b.ColumnChromatography(Size-Exclusion and Affinity)

c.Thin Layer Chromatography

Answer 1

a) Ion- exchange chromatography

Ion exchange chromatography is a technique used to separate molecules according to their charge.

Principle- The principle of separation is the by reversible exchange of ions between the target ions present in the sample solution to the ions present on ion exchangers.

Types- Differentiated on the basis of types of exchangers i.e., cationic and anionic exchangers can be used.

1. Cationic exchangers possess negatively charged group, and these will attract positively charged cations. These exchangers are also called “Acidic ion exchange” materials.
2. Anionic exchangers have positively charged groups that will attract negatively charged anions. These are also called “Basic ion exchange” materials.

Applications-  Ion exchange chromatography has many uses including:

• separation of proteins from foods, for example, to investigate the effects of individual food components on health – this type of analysis is used in nutrition research
• separation of high value proteins from substances
• drinking water analysis for pollution and other constituents
• determination of water chemistries in aquatic ecosystems
• determination of sugar and salt content in foods.

b) Column chromatography

Size exclusion column chromatography

Principle- A column of gel particles or porous glass granules is in equilibrium with a suitable solvent for the molecules to be separated. Large molecules which are completely excluded from the pores will pass through the interstitial spaces, while smaller molecules will be distributed between the solvent inside and outside the molecular sieve and will then pass through the column at a lower rate.

Types- There are two basic types of size exclusion chromatography.

1. Gel permeation chromatography (GPC), which uses a hydrophobic column packing material and a non-aqueous mobile phase (organic solvent) to measure the molecular weight distribution of synthetic polymers.
2. Gel filtration chromatography (GFC), which uses a hydrophilic packing material and an aqueous mobile phase to separate, fractionate, or measure the molecular weight distribution of molecules soluble in water, such as polysaccharides and proteins.

Applications-

• separation of large molecules of the biological origin from inorganic and ionizable species. This is termed as desalting.
• purification of biological macromolecules.
• Molecular weight determination

Affinity column chromatography

Principle- It utilizes the reversible biological interaction or molecular recognition called affinity which refers to the attracting forced exerted in different degrees between atoms which cause them to remain in combination.

Types- Affinity chromatography can be broadly divided into two method types:

• The first method uses a naturally occurring structure or sequence of amino acids on the protein as the binding site. Examples include the affinity of Affi-Gel Blue support binding for albumin’s bilirubin-binding site and the binding of protein A in the Affi-Gel and Affi-Prep protein A supports to the Fc region of IgG.
• The second method involves binding to a special amino acid sequence engineered into the protein of interest, commonly referred to as a "tag". Two of the most commonly used protein tags are the polyhistidine tag, which binds to certain metal-containing complexes and the glutathione s-transferase (GST) sequence, which binds to glutathione.

Applications-

• Separation of mixture of compounds.
• Removal of impurities or in purification process.
• In enzyme assays
• Detection of substrates
• Investigation of binding sites of enzymes
• In in vitro antigen-antibody reactions
• Detection of Single Nuceotide polymorphisms and mutations in nucleic acids

c) Thin layer chromatography

Principle- TCL is based on the principle of separation through adsorption type. The separation relies on the relative empathy of compounds towards the mobile phase and stationary phase.

Applications-

• Thin layer chromatography can be used to identify natural products like essential oils or volatile oil, fixed oil, glycosides, waxes, alkaloids, etc
• It is widely used in separating multicomponent pharmaceutical formulations.
• It is used to purify of any sample and direct comparison is done between the sample and the authentic sample
• It is used in the food industry, to separate and identify colours, sweetening agent, and preservatives
• It is used in the cosmetic industry.
• It is used to study if a reaction is complete.

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