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#chromatography techniques

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Chromatography

Ion-exchange separates by:

Ion exchange chromatography exploits differences in net electric charge of analytes. Stationary phase functionalized with charged groups interacts electrostatically with oppositely charged solutes. Elution is controlled by pH influencing ionization and ionic strength providing competing counter-ions. Proteins with different pI values exhibit distinct charge at a given pH, causing differential affinity to matrix. Hydrophobicity governs reversed-phase and HIC, mass governs size exclusion, color is irrelevant. Selectivity can be modulated by gradient elution. This mechanism is central to purifica

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Chromatography

Cation exchange chromatography uses:

Ion exchange chromatography separates ions or charged biomolecules using oppositely charged matrices. A cation exchanger must bind positively charged analytes, therefore matrix carries negatively charged functional groups such as carboxymethyl or sulfopropyl residues. These anionic groups electrostatically attract cations from mobile phase while repelling anions. Elution is achieved by increasing salt concentration or changing pH to compete for binding. Conversely, anion exchangers bear positive groups like diethylaminoethyl. Selecting negatively charged matrix for cation separation is crucial

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details