Lab Techniques: Separations and Spectroscopy Flashcards
(36 cards)
distribution/partition coefficient
the ratio of the substance’s solubilities in the two solvents
outline the process of a liquid-liquid extraction
one substance is separated from a mixture of substances by adding a solvent in which the compound of interest is highly soluble. If the solution containing the compound of interest is shaken with a second solvent (completely immiscible with the first, and allowed to separate into distinct phases, the compound of interest will distribute itself between the phases based upon the solubility in each of the individual solvents.
what does solubility depend on?
the polarity of the solute and the polarity of the solvent
how do you extract basic compounds from organic mixtures?
treat with dilute acid (5-10% HCl) to protonate basic functional group and produce a positive ion. the cationic salts are soluble in aqueous solution and can be extracted using water
how do you extract carboxylic acid compounds from organic mixtures
treat with dilute weak base (5% NaHCO3) to produce anionic salts which can be extracted with water
when can you use dilute hydroxide solution (10%) to extract an acidic compound from an organic mixture?
when the compound of interest has a pH lower than hydroxide (pKa=16) and there are no other compounds that might also be extracted
thin-layer chromatography (TLC0
solid-liquid partitioning technique in which the mobile liquid phase ascends the polar stationary phase (thin layer of absorbant-silica that is coated on a supporting glass plate)
what determines the separation and rate of elution in TLC?
the more polar components travel slower (more interaction with stationary phase) and nonpolar components travel faster
column (flash) chromatography
uses a chromatography column filled with silica gel (polar stationary phase) and saturated with chosen organic solvent (mobile liquid phase), mixture of compounds is added to top and allowed to travel down column
what determines the separation and rate of elution in column chromatography?
the more polar compounds travels slower (more interaction with polar stationary phase), nonpolar compounds travel faster
when do you use TLC?
when samples are very small, when you want to determine the components present in a mixture
when do you use column chromatography?
when you want to isolate bulk compounds
when do you use ion exchange chromatography
when materials to be separated have varying charge states, often used in the separation of mixtures of proteins
ion exchange chromatography
column chromatography; mobile liquid phase containing analyte passed through column packed with a polymeric resin (solid stationary phase) containing either positive or negative charged molecules on the polymer surface
when is HPLC appropriate?
when speed and efficiency is important
high performance liquid chromatography (HPLC)
mobile phase (organic solvent) is pressurized by pump, sample is injected by syringe and mobile phase carries the sample to the column (containing stationary phase), sample is separated and detected when exiting column
what determines the elution rate in HPLC?
the differing affinities of various compounds for either a stationary phase or a mobile phase; more polar compounds will elute first if mobile phase is stationary; i.e. the compound polarity that is similar to mobile phase will elute faster
reverse phase HPLC
stationary phase is a silica gel bonded to a nonpolar group to create a nonpolar stationary phase, mobile phase is more polar than the stationary phase
how do you analyze charged compounds such as amino acids using HPLC?
stationary phase=ion exchange column, usually cation exchange, mobile phase=polar protic or acidic solvent that ensures solubility and suppresses the dissociation of the COOH group on the amino acid
what determines the elution order/difference in affinity to the column of amino acids in HPLC?
the various R groups and the corresponding intermolecular forces as a result
size exclusion chromatography
target materials are dissolved in solvent (mobile phase) and loaded into a column packed with chemically inert, porous polymer beads (stationary phase)
when do you use size exclusion chromatography?
when you want to separate bulk materials based on difference in molecular size, relatively fast elution, minimal loss of material on the column
what determines the rate of elution in size exclusion chromatography?
sizes of pores in bead allow permeation of small molecules but exclude larger ones. So, small molecules follow intraparticle path and are small, large particles follow interparticle path and are faster
when do you use affinity chromatography?
to purify proteins or nucleic acids from complex biochemical mixtures rather than a reaction mixture