Separations and Purification

Question 1

basic definition of extraction

Answer 1

transfer of a dissolved compound from one solvent to another in which it is more soluble

Question 2

extraction process

Answer 2

• 2 solvents are immiscible
• shake
• aqueous and organic phase separate
• organic usually on top (density dependent)
• separators funnel
• usually we repeat several times with several aliquots of water
• can also isolate by use of rotovap

Question 3

wash process

Answer 3

• almost identical to extraction but opposite
• small amount of solvent used to extract and remove impurities

Question 4

filtration

Answer 4

• solid is residue and liquid that passes through filer is filtrate
• isolate solid
• gravity and vacuum

Question 5

recrystallization process

Answer 5

• dissolve product in minimal amount of hot solvent and allow to crystallize while it cools
• solvent chosen one in which product soluble only at high temp
• when solution cools, only desired product recrystallize out of solution excluding impurities

Question 6

simple distillation

Answer 6

• separate liquids boil below 150°C
• at least 25°C diff in bp
• low temp, maybe a stirrer in distilling flask
• lower bp liquid condensed as it travels though to receiving flask

Question 7

superheating

Answer 7

liquid heated to temp above bp without vaporization, gas bubbles within a liquid can’t overcome atmospheric pressure and surface tension

Question 8

vacuum distillation

Answer 8

• identical to simple but with vacuum to lower ambient pressure
• for liquid w bp over 150°C

Question 9

fractional distillation

Answer 9

• 2 liquids w bp difference lower than 25°C
• glass beads or steel wool packing
• vapor rises up fractional column

Question 10

TLC and paper chromatography

Answer 10

• TLC: silica gel or aluminum polar stationary phase
• paper: polar cellulose paper
• spot sample
• place in eluent
• calculate Rf after drying and observing under UV light

Question 11

retardation factor

Answer 11

Rf = distance spot moved/distance solvent front moved

Question 12

reverse-phase chromatography

Answer 12

stationary phase is nonpolar so polar molecules move up plate quickly

Question 13

column chromatography

Answer 13

column filled with silica or aluminum beads

gravity moves solvent and compounds down

can use gas pressure to speed it up

solvent drips out end of column

different fractions contain different compounds

solvent can be evaporated leaving behind compound of interest

Question 14

ion-exchange chromatography

Answer 14

beads coated with charged substances

for example DNA backbone neg charge or protein holds onto pos

salt gradient used to elute charged molecules that have stuck to the column

Question 15

size-exclusion chromatography

Answer 15

beads used in column contain tiny pores of varying sizes

small compounds enter and are slowed down

common approach to protein purification is to use ion-exchange and then size-exclusion column

Question 16

affinity chromatography

Answer 16

column w high affinity for protein (receptors or antibody)

common stationary phase is Ni

protein eluted used washing compound w free receptor to ultimately free protein from column

can be difficult to remove protein from eluent

Question 17

gas chromatography

Answer 17

eluent is gas

absorbent is crushed metal or polymer inside a 30 ft column

column is coiled and kept inside an oven to control its temperature

mixture is then injected into the column and vaporized

gaseous compounds travel at diff rates

injected compounds must be volatile

compounds registered by detector

mass spectrometry for molecular weight determination, ionization and fragmentation used

Question 18

HPLC

Answer 18

• eluent is liquid under pressure, travels through column of defined composition
• small sample injected into column and separation occurs as it flows through
• pass through detector and solvent collected at the end