Separations and Purifications Flashcards
Chromatography
Separating mixtures by passing through medium. Literally means “ measurement of colors.”
Components:
- Analyte: mixture one wants to analyze or purify.
- Apparatus: setup
- Mobile phase: liquid or gas that carries the analyte through the apparatus.
- Stationary Phase: solid or liquid that analyte and mobile phase move through.
Paper chromatography
Liquid-liquid technique based on differing rates of migration across paper.
Stationary phase (water) is polar
Mobile phase is less polar than water
Separation based on relative solubilities (polarities)
- highly soluble molecules migrate faster
Polar molecules have higher affinity for stationary phase, leaving lower marks
Thin layer chromatography
liquid-solid chromatography technique where of differently adsorbed based on solubility/ polarity
Column Chromatography
Eluent: the mobile phase
Eluate: solvent + analyte that exists in the column
Elution: extracting a substance by washing it with solvent.
Liquid Column Chromatography
Separates based on polarity onto a solid adsorbent container in a column.
Molecules that interact more with the stationary phase move slowly and move quickly with the mobile phase.
Type: High Performance Liquid Chromatography (HPLC)
- uses pressure instead of gravity
Gas Chromatography
Gas-liquid chromatography technique that separates vaporized compounds
Compounds with low boiling points will have shorter retention times.
Large molecules will have longer retention times
Size-Exclusion Chromatography
Column Chromatography separates compounds based on size and shape
Stationary phase: beads with pores
Small molecules get trapped in pores and larger molecules exit the column first.
Ion exchange chromatography
Technique separated compounds based on charge
Affinity Chromatography
Separates compounds based on binding interactions
Gel Electrophoresis
Prepatory or analytical. Separates macromolecules—Nucleic acids and proteins based primarily on size (also shape/charge)
- small molecules travel quicker
Three forces influence :
- Electrostatic force
- Friction
- Electrophoretic Retardation
Isoelectric Focusing (IEF)
An Electrophoresis technique that separates proteins or peptides based on Isoelectric points. Preparatory or analytical
Factors affecting pI values
- based on amino acids that comprise it
- pH decreases as temperature rises.
Southern Blot
Used where researchers want to find specific sequences of DNA. Can be used
in the diagnosis of disease caused by mutation.
