12.3 Chromatography Flashcards
chromatography
uses physical and chemical properties to separate and identify compounds from a complex mixture
based on the concept that the more similar a compound is to its surroundings (by polarity, charge, etc.), the more it will stick to it
chromatography method
the sample is placed on the stationary phase (adsorbent)
we run the mobile phase through the stationary phase
the displaces (elutes) the sample and carries it through the stationary phase
components within the sample will adhere to the stationary phase with differing strengths, causing them to migrate at different speeds (partitioning)
thin-layer chromatography
uses a thin layer of silica gel or alumina (adherent to an inert carrier sheet) as a stationary phase
paper chromatography
uses paper (composed of cellulose) as the stationary phase
reverse phase chromatography
the stationary phase used is nonpolar
the mobile phase used is polar
(opposite of thin-layer and paper chromatography)
retardation factor (Rf)
relatively constant for a particular compound in a given solvent (an can be used to identify unknown compounds)
Rf = distance travelled by solute / distance of solvent front
preparative TLC
a technique where thin-layer chromatography is performed on a larger scale than usual in order to obtain pure compounds
the components can be scraped off and washed to yield pure compounds
eluent
the solvent (mobile phase)
adsorbent
the stationary phase
column chromatography
uses an entire column filled with silica or aluminum beads as an adsorbent
uses gravity as opposed to capillary action
flash column chromatography
forcing the solvent through the column using gas pressure to speed up the process
ion-exchange chromatography
the beads in the column are coated with charged substances so that they attract or bind compounds that have an opposite charge (ex. negatively charged backbone of DNA)
may retain the charged molecules completely
after all other compounds have moved through the column, a salt is used to elute the charged molecules that stuck to the column
size-exclusion chromatography
the beads used in the column contain tiny pores of varying sizes
tiny pores allow small compounds to enter the beads, slowing them down
large compounds can’t fit into the pores and travel through the column faster
affinity chromatography
a protein of interest is bound by creating a column with high affinity for that protein
ex. coating beads with a receptor that binds the protein to retain the protein in the column
once the protein is retained in the column, it can be eluted by washing the column with a free receptor which will compete with the bead-bound receptor and free the protein from the column
the protein may also be released from the receptor via varying pH or salinity levels that disrupt the bonds between protein and receptor
gas chromatography (vapour-phase chromatography)
the eluent is gas instead of liquid
adsorbent = crushed metal or polymer inside a long coiled column
the mixture is ejected into the column and vaporized
gaseous compounds adhere to the adsorbent to different degrees and travel through the column at different rates
compounds are registered by a detector, which records them as a peak on a chart as they exist the column
high performance liquid chromatography
similar to column chromatography: eluent is a liquid; travels through a column of defined composition
a small sample is injected into the column (via pressure), and separation occurs as it flows through
compounds pass through a detector and are collected as solvent flows through the apparatus
entire process is computerized
