Lecture 6 Flashcards
(19 cards)
What is the etymological origin of “chromatography,” and what is its primary purpose?
Derived from Greek “Chroma” (colour). It identifies unknown compounds and separates mixtures.
Name four key application areas of chromatography.
Nutraceuticals, Forensics, Research, Pharmaceutical industry.
List four general types of chromatography.
Paper, Thin layer, Column, HPLC, Gas chromatography.
In Thin Layer Chromatography, what are the stationary phase and mobile phase?
Stationary phase: Chromatography paper; Mobile phase: Water.
What factors determine how fast a dye travels up the paper in chromatography?
Solubility of the dye in water and its interaction with the paper.
What is an Rf value and how is it calculated?
Retention factor (Rf) helps identify components. Rf = (Distance from start to middle of spot) / (Distance from start to finish point of water).
How are biomolecules generally purified using modern chromatography techniques?
By separating them according to differences in their specific properties.
Name the four main types of chromatography for purifying biomolecules.
Gel filtration (Size Exclusion), Ion exchange, Hydrophobic interaction (Reversed phase), Affinity chromatography.
What is the primary separation principle in Gel Filtration Chromatography?
Separation based on differences in molecular size as molecules pass through a gel filtration medium.
What is a significant advantage of gel filtration regarding buffer composition?
Buffer composition does not directly affect resolution as molecules do not bind to the medium, allowing flexible conditions for downstream uses.
Why is gel filtration well-suited for sensitive biomolecules?
Because it handles biomolecules sensitive to changes in pH, metal ion concentration, co-factors, and harsh environmental conditions.
What is the basis for separation in Ion Exchange Chromatography (IEX)?
Differences in the net surface charge of molecules.
Why is the net surface charge of proteins highly pH-dependent?
Proteins are amphoteric; their charged groups have different pKa values, causing their net charge to change with the surrounding pH.
How does Hydrophobic Interaction Chromatography (HIC) separate proteins?
Based on differences in their surface hydrophobicity, using a reversible interaction with a hydrophobic column surface.
How do salts facilitate binding in HIC, and how are bound components eluted?
High salt concentrations enhance hydrophobic interactions for binding. Lowering the salt concentration gradually elutes bound components in order of hydrophobicity.
What is the principle behind Affinity Chromatography (AC)?
It separates biomolecules based on a reversible, specific interaction between a biomolecule and a unique ligand coupled to the chromatography matrix.
Why is AC considered excellent for purification?
Due to its high selectivity (high resolution) and high capacity, allowing for several thousand-fold purification with high recovery of active material.
Name three main elution methods in Affinity Chromatography.
Ionic-strength elution, pH elution, and Competitive elution.
What type of elution agent should generally be avoided in AC, and why?
Chaotropic agents (e.g., guanidine hydrochloride, urea) should be avoided whenever possible, as they are likely to denature the eluted protein.
