5C Seperation and Purification Flashcards
Extraction
Separates two compounds based on polarity. Combines two immiscible solvents, one of which easily dissolves compound of interest.
- Nonpolar layer = organic layer
- Polar layer = aqueous layer
- Isolate organic and aqueous layer based on densities usually using relative densities. Aqueous layer is usually more dense than organic, so it is drained first.
- Compounds with H-bonding like alcohols or acids move easily into aqueous layer (polar)
- Compounds with only dipole-dipole less likely
- Only Van der Waals, least likely to move into aqueous
Wash
Reverse of extraction in order to remove unwanted impurities. A small amp of solute is used to extract and remove impurities, rather than compound of interest.
Filtration and types
isolates a solid (residue) from a liquid (filtrate). Pour liquid-solid mix onto paper filter, allowing solvent to pass
- Gravity filtration: use when product of interest is in filtrate (liquid). Hot solvent used to keep product dissolved in liquid
- Vacuum filtration: used when solid (residue) is desired. Solvent is forces through a filter by a vacuum connected to flask
Recrystallization
used to remove impurities. Product is dissolved in minimum amount of solvent. If impurities are more soluble , the crystals will reform while the flask cools, eluding the impurities
Distillation and types
separates liquids according to differences in their boiling points. Liquid with LOWEST BP vaporizes first and is collected as the distillate. An isomer that has higher intermolecular bonding (exist between molecules), has a higher BP compared to an isomer that experiences more intramolecular bonding (forces that hold atoms within mol) has lower BP.
- Simple Distillation: BP < 150 C and are at least 25 C apart
- Vacuum Dist: BP > 150 C. Vacuum lowers air pressure, which decreases temp liquid must reach in order to boil
- Fraction distillation: BP < 25 C because it allows more refined sep of liquids by BP. A fractionated column has increased surface area by inclusion of glass beads or steel wool. Vapor consists of a higher proportion of compound with LOWER BP and condenses in receiving flask.
Column Chromatography
Sep two molecules from a mixture. Usually, stationary phase: polar molecule and mobile phase typically non polar. It uses beads of a polar compound (stationary phase) with a non polar solvent (mobile). Utilizes polarity, size, or affinity to separate compounds based on physical properties
Liquid Chromatography
silica = stationary phase and nonpolar liquid used as mobile phase
High-performance/pressure liquid chromatography
type of liquid chromatography that uses high pressure to pass solvent through a more finely-ground stationary phase (smaller absorbent particles), which increase the interactions between the molecular and the stationary phase (relies on pressure instead of gravity to pass mobile phase through column) This gives HPLC higher resolving power (ability to distinguish between compounds). It is used if sample sizes small or forces such as capillary action affect results
Gas chromatography
Vaporizes l liquid before separating. Molecules are separated based on polarity and polling point according to how well they adhere to adsorbent in column.
- Stationary: crushed metal or polymer. Polarity matches solute
- Mobile: non-reactive gas
- May be combined with mass spec, which ionizes and fragments molecules and then passes fragments through magnetic field to determine weight of structure
Paper and thin-layer chromatography
Stationary (adsorbent): polar e.g., silica, alumina, or paper
Mobile (eluent): non polar solvent, which climbs card through capillary action
-Rf values calculated = dis spot moved/dist solvent front moves
Reverse-phase TLC chrom
Uses non polar card with polar solvent
Separation of enantiomers (Racemic mix)
Reacting two enantiomers (+, -_ with a single enantiomer of another compound leads to two diastereomers. E.g., If you had two enantiomers that contain one chiral carbon (+) and (-) and react with a (+) enantiomer of another compound, two diasteromers would result (+,+) and (-, +). Diasteromers have diff physical properties and can be separated by crystallization, filtration, distillation, and others. Once separated, diasteromers can be reacted to form enantiomers
Gel electrophoresis
sep macromolecules. For its and small molecular, the gel is Polyacrylamide. For larger mols (<500 bp), Gel is agarose. Neg charged molecules travel toward anode (+) at bottom. Large molecules move slower. Coomaissie blue stain can be used for visualization
Thin-layer chromosome with UV light
TLC can be useful to monitor reactions and UV light can be used to visualize e results from TLC If compounds from reaction mix can absorb uv light. UV light carries a large art of energy that can excite electrons of UV chromophores (C=O, nitrous groups, alkyl halides, and conjugated systems)
Native-PAGE
A polyacrylamide gel electrophoresis method for proteins using NON-DENATURING conditions. Pts keep native charge and structure so separation based on CHARGE AND SIZE
