MCAT Biology Ch12: Purification and Separation Flashcards
Extraction
transfer of a dissolved compound (the desired product) from starting solvent into a solvent in which the product is more soluble.
Extraction (details)
- idea of “like dissolves in like”
- leave impurities in first solvent
- two solvents are immiscible; if shaken, solute can pass from one solvent to the other
- use separatory funnel
- obtain compound by evaporating solvent, using rotary evaporator
separatory funnel
- piece of glassware that aids in separating two layers
- gravity force cause heavier one to sink (usually organic on top and aq. on bottom, although can be reverse, these depends on density)
- since small amount of extract remains behind, multiple extraction are necessary (multiple extractions instead of same amount in a single one)
wash
another way to take advantage of solubility properties is do reverse of extraction and removed unwanted impurities, washing the product of unwated impurities
filtration
- another simple filtration,
- isolates solid from liquid
- liquid-solid mix into paper filter, allowing solvent pass; solid on filter paper (residue) and flask full of liquid (filtrate)
two basic types of filtration
gravity and vacuum filtration
gravity filtraton
- solvent own weight pulls through filter
- substance of interest to be in SOLUTION, and impurities to remain undissolved
- prod. is dissolved
- usually w/ hot solvent
vacuum filtration
- liquid-solid mixture
- solvent forced through filter by vacuum, attached to vacuum flask
- faster
- large quantities of solid (desired product usually)
recrystallization
- purify solid product even further
- prod. in min. hot solvent –> recrystallize as cools
- solvent requirements: soluble only at high temps and polarity (if solubility info not known, intermediate polarity generally desirable), low enough FP
- mixed solvent sol case: first highly soluble, slowly add second in drops that less soluble –> ppt –> heat to redissolve the ppt –> cool –> isolated w/ vacuum filtration
sublimation
- purification (solid to gas) because impurities will not sublime easily
- produre vapors that condense on a chilled glass tube (cold finger)
cold finger
piece of glassware chilled by packing with dry ice or running cold water through it
- under vacuum because lower pressure (less likely pass liquid phase), reduces temp. for sublimation and danger of compound decomposing
- conditions dpeend on compound we’re purifying (diff. phase diagram)
centrifugation
- particles in solution settle (sediment) at diff. rates depending on mass, density and shape.
- heavy, dense particles at bottom, lighter top
- spin the solution via a centrifuge, in test tube, subjected to centrifugal tube
- separate large particles such as cells, organelles, and bio macromolecules
distillation
- separating two liquids that are soluble through diff. in BP to separate by vaporization and condensation
- lower BP vaporize first –> rise up and condense in water-cooled distillation column –> dripping down column into a vessel that catches the distillate
- temp is kept low so higher BP will never boil (remain in initial container)
types of dilstillation
simple, vacuum, and fractional
simple distillation
- most basic kind
- below 150 C, least 25 C diff
- apparatus consists of:
1. distilling flask - two liquids
2. distillation column - thermometer and a condenser
3. receiving flask - collect distillate
vacuum distillation
- BP over 150 C
- lowers pressure over surface of liquid –> dec temp –> lower BP –> boil liquid faster
fractional distillation
- similiar BP (less than 25 C)
- use fractioning column to connect the distillating flask to distillation column
- fractional column - any filled w/ inert objects (glass beads, steel wool) –> inc. SA –> vapor rise –> condense on the surface –> more heat –> reevap–> rise further –> recondense –> greater proportion of lower BP component to top of fractioning column our desired substance –> condense in distillation column –> receiving flask
chromatography
- more similar compound to surroundings –> stick –> slowing through surroundings
- sample on solid medium (stationary phase or adsorbent) –> run mobile phase (liquid or gas) through stationary phase –> displace (elute) sample and carry through stationary medium –> adhere to SP w/ diff strength –> diff. substances migrate at diff. speeds (partitioning) –> representing equilibrium between two phases (diff compound have diff eq) –> isolate
- depending on substance and polarity of mobile phase –> adhere to stationary phase
- many diff. stationary phase, most likely diff. is polarity (MCAT)
- analysis based on speed through media; either measure:
1. how far in given time
2. how long elute off column (column or gas chromotography)
stationary phase or adsorbent
solid medium in which sample is placed
mobile phase
usually liquid or gas which is run through stationary phase
thin-layer chromotography (TLC)
uses silica gel, highly polar substance as SP –> polar adhere –> elute slower
elute
displaces the sample
four types of chromotography
TLC, column chromatography, gas chromatography (GC) and high-pressure (or performanc) liquid chromatography (HPLC)
thin-layer chromatography
-adsorbent - piece of paper or thin layer of silica gel or alumina adhered to inert carrier sheet (glass or plastic)
=place mixture want to separate on adsorbent (spotting) –> TLC plate is developed –> make sure initial spots are above solvent –>solvent up plate via capillary action –> carrying diff. compounds varying rates –> solvent near top –> remove plate –> dry
-silicia gel is polar and hydrophobic while mobile phase usually an organic solvent (often mixture) of weak to mod polarity, doesn’t bind well, so nonpolar compounds hang w/ organic solvent and move quickly as solvent moves up plate, whereas molre polar molecules stuck on gel
- since spots are white:
1. place developed TLC plate under ultraviolet light
2. iodine, phosphomolybdic acid, vanillin for stain - *problem - stain destroys compound (by oxidation)
- distance compound travels/distance solvent travels = Rf value
- constant value of particular compound in given solvent –> identity of unknown compound.
-small scale, usually for identity
spotting
place mixture want to separate on adsorbent in TLCs, small, well-defined spot onto plate
developed
placing adsorbent upright in a developing chamber (usually beaker w/ lid or wide-mouthed jar) containing shallow pool of eluant (solvent) at bottom
eluant
solvent at bottom of developing chamber when TLC is developed
reverse-phase chromotography
stationary phase is very nonpolar, so polar molecules move up plate very quickly whereas nonpolar stick on palte
Preparative or prep TLC
- larger scale as means of purification
- large TLC plate that has big streak of mixture –> split streak of individual compounds –> scrape –> rinse w/ polar solvent –> pure compounds from silica
column chromatography
- column filled w/ silicia or alumina beads as adsorbent
- solvent and compounds move down by gravity –> sovlent drips out –> collect diff frac varying times –> each frac bands for diff compounds –> evaporate –> isolate compounds
- solvent polarity can be changed to help elute our compound
- can separate marcomolecules
flash column chromatography
solvent and compounds move down by gravity; to speed up, use N2 gas in column chromatography
ion exchange chromatography (column chromatography )
beads are charged substances, attracting opposite charges –> salt gradient elute charged molecules stuck on column
size-exclusion chromatography (column chromatography)
column has tiny pores allowing small molecules in, slowing them down, large molecules travel faster
- different MW
- can be used in protein purification (previous is ion exchange)
affinity chromatography (column chromatography)
- bind any substance of interest –> bind tightly to column tightly –> elute by washing w/ free receptor (or target or antibody) –> compete w/ bead-bound receptor –> free substance
- drawback is our inhibitor or recptor bound to our biological target – diff to remove
gas chromatography/vapor-phase chromatography
- separation
- elutant is gas; adsorbent is 30 foot column that’s coiled and in oven to control temperature –> mixture injected and vaporized –> travel at diff rates since adhere diff and separate by time –
- requirement of gas is that is be volatile, low MP, subllimate solids and liquids
- injected pure molecules analyzed by computer –> detector –> mass spec –> peak –> for MW
HPLC
- eluant is liquid
- variety of column whose stationary phase depending on target molecule and size depending on material needed to purify
- lower pressure
- small sample injected in column and separation occurs as it flows through –> detector and collected as solvent flows out the end of apparatus.
electrophoresis
- separate a mixture of compound that carry a charge
- usually macromolecules to an electric field, move according to size and charge
- neg charge to anode, pos charged to cathode.
migration velocity
-for electrophoresis; frictional force coefficient, depends on mass and shape of migrating molecues
- more charged –> strong field and faster
- bigger and convoluted –> slower
agarose gel electrophoresis
- separate pieces of nucleic acid
- medium used is agarose
- since every piece NA is neg charged –> separated based on size and shape alone
- stain gels w/ ethidium bromide –> bind to NA –> fluorescence
- get out compound (prep) by cutting desired band out of gel and eluting out the nucleic acid
faster: inc voltage (inc. E) and use lower percentage of gel (dec. f)
agarose
medium used in agarose gel electrophoresis
SDS-Page
- separates proteins on mass alone
- poly gel
- disrupts all noncovalent interxns
- binds proteins –> creates large chains w/ net neg. charges –> neutralizing protein’s original charge –> proteins move through gel (depending on f, depending on mass) –> separation, stains protein bands
isoelectric focusing
-acidic and basic prop. of amino acids
isoelectric point, pI
- each protein characterized this way, in which is the pH at which its net charge (sum of all the charges on all of its amino acids) is zero
- if mix of protein placed in electric field that exists across gel w/ pH gradient, proteins move until pH equal pI –> net charge zero and stop moving
if pI = 9 put in gel of pH = 7 –> more protons around protein –> attach basic sites on protein –> net pos charge –> toward neg. charged cathode –> basic siide –> fewer protons in gel –> pH inc –> near pH of 9 –> protons creating pos. charge will dissociate –> neutral protein
- acid w/ protons –> pos charge –> anode is positively charged
- basic w/ neg OH –> neg. charged cathode
