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119 terms

Kaplan MCAT OChem Ch. 12: Purification and Separation

STUDY
PLAY
types of basic techniques
extraction

filtration

recrystallization

sublimation

centrifugation
extraction
separate desired product, transfer of dissolved compound (desired) from starting into solvent in which product is more soluble => leave most impurities behind the first solvent
extraction
two solvents are immiscible
extraction
water (aq) and ether (organic) separate => separatory funnel => gravitational forces cause heavier layer to sink => bottom layer can be removed => small amount will remain dissolved in ether => repeat extraction (multiple)
wash
advantage of solubility prop => reverse of extraction => removed unwanted impurities
extraction
prop of acids and bases:

acid disso => anion formed will be more soluble in aq. layer, so adding base will help extract an acid
filtration
another simple purification tech which isolates solid from liquid
filtratioin
liquid-solid mix on paper filter => solvent passes through => residue (solid) on filter paper and a flask full of filtrate (liquid)
gravity filtration
solvent's own weight pulls it through filter => pore become clogged w/ solid => slowing rate of filtration
gravity filtration
substance of interest in solution and impurities to remain undissolved

prod remains dissolved, usually carried out w/ hot solvent
vacuum filtration
separate liquid/solid mix
two types of filtration
vacuum and gravity
vacuum filtration
solvent forced through filter by vacuum on other side

need specific flask w/ valve that attaches vacuum (Buchner/vacuum flask)
vacuum filtration
much faster method, so used to isolate large quantities of solid, particularly when solid is the desired product
recrystalliztion
purify our solid product even further
recrystalliztion
dissolve our product in min of hot solvent => recrystallize as cools
recrystalliztion
1. make sure soluble at high temps ONLY
2. polarity of solvent
3. solvent have low enough freezng point
recrystalliztion
if solubility info not available, solvent w/ intermediate polarity is generally desirable for this
recrystallization
mixed solvent system may be used:
1. in a solvent that's highly soluble => slowly add less soluble solvent by drops => solid ppt =>heat => redissolve ppt => slowly cool for crystal formation, which can be isolated w/ vacuum filtration
recrystallization
impurities should be equally soluble at various temp
recrystalllization
results in crystal lattice
sublimation
purification because impurities in most rxns will not sublime easily
cold finger
produce vapors that condense on this chilled glass

piece of glass chilled by packing w/ dry ice or running cold water
sublimation
most performed under vacuum , since at low pressures, compounds less likely pass through liquid phase

low pressure also reduces temp
sublimation
danger is that compound will decompose, so optimal condition depends on compound we're purifying
to make solid sublime
1. raise temp at low enough pressure
2. lower pressure at very cold temp
centrifugation
separate particles of sediment at diff rates depending on mass, density and shape
centrifugation
heavy, dense particles will settle at bottom and lighter particles at top by hyperspeed spinning of sol via this device
centrifugation
subject to centrifugal forces
centrifugation examples.
1. sep large particles
2.in blood to separate cells from plasma
3. in cell debris to separate out organelles
4. sep big DNA molecules from smaller ones => get large pure DNA
types of distillation
simple
vacuum
fractional
distillation
separate two soluble liquids in diff. of BP by vaporization and condensation
distillation
liquid w/ lower BP vap. first => vap rise up => condense in water-cooled distillation column, drippping down column => vessel catches distillate
distillation
temp kept low so liquid w/ higher BP will never boil and remain liquid
simple distillation
most basic kind of distillation
simple distillation
separate liquids that below 150 C and have at least a 25 C diff
simple distillation appartus
1. distillating flask containing two liquids
2. distillation column w/ thermometer
3. condenser
4. receiving flask to collect
vacuum distillation
BP over 150C
vacuum distillation
lowering the pressure (atm) over surface of liquid => dec. temp liquid needs to reach BP
vacuum distillation
don't have to worry about degrading compound w/ excessively high heat and get liquid to boil faster
fractional distillation
similar BP (less than 25 C) use this

more precise
fractional distillation
use this column to connect distillating flask to distillation column
fractional distillation
as VP rises => condense on surfaces => more heat rise => condensation (lower BP sol) will re evap an rise further => recondense even higher => at top of column is desired substance -> condenses at distillation column => receiving flask
fractional column
any column filled w/ inert object => inc. SA of column
chromatography
thin-layer

column

gas chromatography

HPLC
types of column chromatography
ion exchange

size-exclusion

affinity
chromatography
more similar compound w/ surroundings => more it will stick and move slowly through its surroundings
chromatography
sample on stationary phase (adsorbent) => run mobile phase through stationary phase => adhere to stationary phase w/ diff. strengths => diff. substance to migrate at diff. speeds => each compound separating w/in stationary phase => isolate
stationary phase or adsorbent
solid medium that mobile phase runs on which elutes sample
mobile phase
usually liquid (gas in gas one)
elute
displaces
partitioning
adhere to stationary phase w/ diff. strengths => diff. substance to migrate at diff. speeds
stationary phase or adsorbent
plethora of diff. media w/ diff. properties that allow to separate compound

property most likely see is polarity
TLC
uses silicia gel, highly polar substance => adhere to gel => move (elute) slowly
column chromatography
size and charge and how quickly how compound moves through stationary phase
chromatography
analysis based on speed:

1. measure how far substance travels in a given amount of time (TLC)
2. time how long it takes substance to elute off column (gas)
TLC
adsorbent is either a piece of paper or a thin layer of silica gel or alumina adhered to an inert carriet sheeet (Glass or plastic)
TLC
to mix on adsorbent => developed in containing elutant => make sure initial sports on plate above solvent => solvent goes up via capillary action carry diff. compounds w/ it at varying rates => solvent front near top => plate removed => dry
spotting (TLC)
small, well defined spot
developed (TLC)
adsorbent upright in develop chamber (usually a beaker w/ lid or wide-mouthed jar)
elutant (TLC)
in beaker, shallow pool at the bottom
silicia gel
silcia gel usually polar and hydrphilic

mobile phase - an organic solvent that's weak to moderate, so doesn't bind well to gel
reverse-phase chromatography
opposite of TLC in terms of organic/inorganic stationary and mobile phase
TLC
spots usually white => developed TLC plate under UV light

alternate to white spots are iodine, phosphomolybdic acid, vanillin to stain spots (problem is that it destroys the compound by oxidation)
Rf value (TLC)
distanace that the compound travels/distance that solvent travels
Rf value (TLC)
relatively constant value for a particular compound in a given solvent => used to identify unknown compound
TLC
on small scale:(qual. ID)

on large scale: means of purification
Preparative or prep TLC
uses large TLC plate that has big streak => split into bands of indie compounds => scrape bands => rinse w/ polar solvent => recover pure compound from silica
column chromatography
column filled w/ silica or alumina bead as adsorbent => allow for greater separation => solvent and compounds move down column by gravity => solvent drips => collect diff fractions at varying times (each band has diff compound) => evaporate solvent => isolate compound
flash column chromatography
to speed of column chromatography. we force solvent through column w/ N2
column chromatography
solvent polarity can easily be changed to help elute our compound
ion exchange chromatography
beads in column are charge => attract/bind to compounds that have opposite charge => salt gradient is used to elute the charged molecules that have struck to column
size exclusion chromatography
column has tiny pores of vary sizes => small molecules go through => slowing them down while large molecules are faster
size exclusion chromatography
diff MW molecules may be fractioned
protein purification
uses an ion exchange column followed by a size-exclusion column
affinity chromatography
can bind any substance of interest
affinity chromatography
to purify sub A => bind tightly to column => elute A by washing w/ free receptor (target or antibody) which will compete w/ bead-bound receptor and free sub A

drawback: inhibitor/receptor bound to target, so difficult to remove
gas chromatography (GC)
aka vapor pahse chromatography (VPC)
gas chromatography
elutant is gas (He or N)

adsorbent: inside a 30-column that's coiled and kept inside an oven at controlled temp
gas chromatography
mix injected in column => vaporized => gaseous compounds travel through at diff rates since adhere to adsorbent at diff degrees
gas chromatography
requirement for compounds injected is that that be volatile, low MP, sublimable solids or liqiuds
gas chromatography
type of chromatography that are analyzed by computer where compounds are go to detector => peak chart => sometimes pure compounds => mass spec
chromatography
separates compounds based on how strong adhere to stationary/solid or how easily comes off mobile phase
HPLC
either high pressure of high performance liquid chromatography
HPLC
eluant is liquid => travel through column of a defined composition
HPLC
variety of columns whose stationary phase is chosen depending on target molecule
HPLC
size depends on quantity of material needed to be purified
HPLC
simliar to GC, expect eluent is liquid
HPLC
using low pressure now is chromatography
HPLC
small sample into column => separation occurs as flows through => detector => collected as solvent flows out end of apparatus (computer)
HPLC
sophisticated solvent gradients can be applied to column => help resolve various components in our mixture
retention time
in chromatography, how long it took for each to travel through column
types of electrophoresis
agarose gel electrophoresis

SDS-PAGE

ISOELECTRIC FOCUSING
electrophoresis
separate a mixture of compounds that carry a charge
electrophoresis
subjects compounds to electric field which moves them according to their charge and size
electrophoresis
neg charge => anode (anion -> anode)
pos charge => cathode (cation -> cathode)
migration velocity
v = Ez/f

E = electric field
z = net charge on molecule
f = fricitional coefficient, depending on mass and shape of moving molecules
electrophoresis
more charged => stronger electric field => faster

bigger/convulted => slower
nonspon electrolytic cell
used for electrophoresis has neg. cathode and pos. anode.
agarose gel electrophoresis
types of electrophoresis separates nucleic acids (DNA and RNA)
agarose gel electrophoresis
medium used is agarose
agarose gel electrophoresis
since every piece is neg charged, separated based on size and shape alone
agarose gel electrophoresis
ethidium bromide=> binds to nucleic acids => results under fluorescence
agarose gel electrophoresis
can also obtain compound (prep) by cutting desired band out of this and eluting out NA
to make gel run faster
inc. voltage => inc electric field

use lower % of agarose or acrylamide => dec. fric. coeff
agarose
nontoxic and easy to manipulate gel (unlike SDS PAGE)
SDS PAGE
separates proteins on the basis of mass alone
SDS PAGE
typically denatures protein (can use "native gels" to avoid this)
SDS PAGE
polyacryl gel used
SDS PAGE
this disrupts all noncovalent interactions

binds proteins => creates large chains w/ net neg. charges => neutralizing protein's original charge=> move => separate => stain bands
SDS PAGE
only variable affecting velocity if "f", which depends on mass
SDS PAGE and agarose gel
both separate molecules based on size
isoelectric focusing
acidic and basic prop of amino acids
isoelectric focusing
mix of proteins => electric field across gel w/ pH grad. -> proteins movie until reach point that which pH = pI -> so pH is zero (protein stops moving)
isoelectric focusing example
pI (9) place in pH (7) -> charge exists (here, more protons) => attach to basic sites of protein => net pos. charge on molecule => carry proteins toward neg. charged cathode => fewer protons in gel (pH incs) -> protons creating pos charge disso => protein becomes neutral
pH < pI (isoelectric focusing )
=> proteins protonated and pos charge => towards cathode
pH > pI (isoelectric focusing )
=> deprotonate and neg charged => migrate towards anode.
isoelectric point, pI
each protein may be characterized by this, which is the pH at which its net charge is zero
distillation
separates liquid based on BP, which in turn depend on intermolecular forces