Chromotography L8 Flashcards
What role do functional groups play in chromatography?
They determine interaction of analyte(s) with stationary/mobile phases
Functional groups are sub-structures within molecules that influence their molecular properties.
Define non-polar groups in organic molecules.
Groups with evenly distributed electron density, primarily hydrocarbons
Non-polar groups participate in dispersion and hydrophobic interactions.
What defines polar groups in molecules?
Uneven distribution of electron density creating positive and negative charges
Polar molecules contain at least one heteroatom (O, N, P, S) and participate in dipole-dipole interactions.
What are the requirements for hydrogen bonding?
Electron-deficient hydrogen atom and a highly electronegative atom with an electron pair
Examples include O-H, N-H, and F-H bonds.
What do ionic functional groups do?
They donate or accept protons
Ionic groups can be charged or non-ionized depending on pH.
List some physical and chemical properties that allow separation of molecules.
- Solubility
- Ionic charge
- Molecular size
- Adsorption properties
- Binding specificity for other molecules
What is column chromatography?
A technique where a mixture is dissolved in a solvent and passed through a stationary phase for separation
Pure substances are collected in fractions based on their interactions.
How does High Performance Liquid Chromatography (HPLC) improve separation?
By using a pump to pass the mobile phase through the column for faster and higher resolution separations
HPLC is primarily an analytical technique.
What is the difference between normal phase and reversed phase liquid chromatography?
Normal phase has a more polar stationary phase, while reversed phase has a less polar stationary phase
Examples include toluene/silica for normal phase and water-methanol/C18 for reversed phase.
What is gel filtration also known as?
Size exclusion chromatography
It separates molecules based on size using a porous gel.
What principle does dialysis operate on?
Separation of molecules according to size through semipermeable membranes
It allows solvents and small molecules to diffuse while blocking larger molecules.
What influences hydrophobic interaction chromatography (HIC)?
Presence of certain salts in the running buffer
High salt concentration enhances interactions, while low salt concentration weakens them.
Describe reversed phase chromatography.
Separates molecules based on hydrophobicity with a more hydrophobic stationary phase
Non-polar solvents are used to reverse the hydrophobic effect.
What is the principle behind ion exchange chromatography?
Separation based on the affinity of molecules to the ion exchanger
Proteins bind to either cation or anion exchangers depending on their charge.
What types of biological interactions are used in affinity chromatography?
- Enzyme/substrate analogue
- Antibody/antigen
- Lectin/polysaccharide
- Nucleic acid/complementary base sequence
- Hormone/receptor
- Glutathione/GST fusion proteins
- Metal ions/poly (His) fusion proteins
