Kaplan MCAT OChem Ch. 12: Purification and Separation

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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

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