MCAT Biology Ch12: Purification and Separation

Question 1

Extraction

Answer 1

transfer of a dissolved compound (the desired product) from starting solvent into a solvent in which the product is more soluble.

Question 2

Extraction (details)

Answer 2

• 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

Question 3

separatory funnel

Answer 3

• 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)

Question 4

wash

Answer 4

another way to take advantage of solubility properties is do reverse of extraction and removed unwanted impurities, washing the product of unwated impurities

Question 5

filtration

Answer 5

• 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)

Question 6

two basic types of filtration

Answer 6

gravity and vacuum filtration

Question 7

gravity filtraton

Answer 7

• 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

Question 8

vacuum filtration

Answer 8

• liquid-solid mixture
• solvent forced through filter by vacuum, attached to vacuum flask
• faster
• large quantities of solid (desired product usually)

Question 9

recrystallization

Answer 9

• 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

Question 10

sublimation

Answer 10

• purification (solid to gas) because impurities will not sublime easily
• produre vapors that condense on a chilled glass tube (cold finger)

Question 11

cold finger

Answer 11

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)

Question 12

centrifugation

Answer 12

• 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

Question 13

distillation

Answer 13

• 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)

Question 14

types of dilstillation

Answer 14

simple, vacuum, and fractional

Question 15

simple distillation

Answer 15

• 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

Question 16

vacuum distillation

Answer 16

• BP over 150 C

- lowers pressure over surface of liquid –> dec temp –> lower BP –> boil liquid faster

Question 17

fractional distillation

Answer 17

• 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

Question 18

chromatography

Answer 18

• 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)

Question 19

stationary phase or adsorbent

Answer 19

solid medium in which sample is placed

Question 20

mobile phase

Answer 20

usually liquid or gas which is run through stationary phase

Question 21

thin-layer chromotography (TLC)

Answer 21

uses silica gel, highly polar substance as SP –> polar adhere –> elute slower

Question 22

elute

Answer 22

displaces the sample

Question 23

four types of chromotography

Answer 23

TLC, column chromatography, gas chromatography (GC) and high-pressure (or performanc) liquid chromatography (HPLC)

Question 24

thin-layer chromatography

Answer 24

-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

Question 25

spotting

Answer 25

place mixture want to separate on adsorbent in TLCs, small, well-defined spot onto plate

Question 26

developed

Answer 26

placing adsorbent upright in a developing chamber (usually beaker w/ lid or wide-mouthed jar) containing shallow pool of eluant (solvent) at bottom

Question 27

eluant

Answer 27

solvent at bottom of developing chamber when TLC is developed

Question 28

reverse-phase chromotography

Answer 28

stationary phase is very nonpolar, so polar molecules move up plate very quickly whereas nonpolar stick on palte

Question 29

Preparative or prep TLC

Answer 29

- 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

Question 30

column chromatography

Answer 30

- 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

Question 31

flash column chromatography

Answer 31

solvent and compounds move down by gravity; to speed up, use N2 gas in column chromatography

Question 32

ion exchange chromatography (column chromatography )

Answer 32

beads are charged substances, attracting opposite charges --> salt gradient elute charged molecules stuck on column

Question 33

size-exclusion chromatography (column chromatography)

Answer 33

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)

Question 34

affinity chromatography (column chromatography)

Answer 34

- 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

Question 35

gas chromatography/vapor-phase chromatography

Answer 35

- 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

Question 36

HPLC

Answer 36

- 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.

Question 37

electrophoresis

Answer 37

- 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.

Question 38

migration velocity

Answer 38

-for electrophoresis; frictional force coefficient, depends on mass and shape of migrating molecues - more charged --> strong field and faster - bigger and convoluted --> slower

Question 39

agarose gel electrophoresis

Answer 39

- 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)

Question 40

agarose

Answer 40

medium used in agarose gel electrophoresis

Question 41

SDS-Page

Answer 41

- 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

Question 42

isoelectric focusing

Answer 42

-acidic and basic prop. of amino acids

Question 43

isoelectric point, pI

Answer 43

- 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