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Orgo > Ch. 12: Separations and Purifications > Flashcards

Ch. 12: Separations and Purifications Flashcards

1
Q

what are the 4 methods that use solubility characteristics to separate compounds from a mixture?

A
  1. extraction
  2. wash
  3. filtration
  4. recrystallization
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2
Q

defn + func: extraction

A

one of the simplest ways to separate out a desired product

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

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3
Q

what is the fundamental concept that extraction is based on?

A

like dissolves like (polar substances will associate with other polar substances, nonpolar with nonpolar)

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4
Q

defn: immiscible

A

form two layers that do not mix, like water and oil

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5
Q

should the two solvents be immiscible when we perform extraction or not?

A

yes, they should be

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6
Q

process (6): extraction

A
  1. the two layers are temporarily mixed by shaking so that solute can pass from one solvent to the other
  2. the water (aqueous) and ether (organic) phases will separate on their own, given time to do so
  3. in order to isolate these two phases, we use a piece of equipment called a separatory funnel
  4. gravitational forces cause the denser layer to sink to the bottom of the funnel, where it can be removed by turning the stopcock at the bottom
  5. once we drain the aqueous layer from the separatory funnel, we repeat the extraction several times
  6. additional water is added to the separatory funnel, it is shaken and allowed to settle, and the aqueous layer is once again drained off (multiple extractions with fresh water are more effective for obtaining the most prodcut, rather than a single etraction with a larger volume of water)
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7
Q

align nonpolar and polar with aqueous phase/layer and organic phase/layer

A

nonpolar –> organic phase

polar –> aqueous phase

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8
Q

which layer is more common to be up top? what is the position of the layers determined by?

A

it is more common for the organic layer to be on top, but the opposite can also occur

the position of the layers is determined by their relative densities

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9
Q

func + name: rotovap

A

rotary evaporator

once the desired product has been isolated in the solvent, we can obtain the product alone by evaporating the solvent usually using the rotovap

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10
Q

defn + func: wash

A

a small amount of solvent is used to extract and remove impurities, rather than the compound of interest

another way to take advantage of solubility properties, the reverse of the extraction

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11
Q

defn: filtration

A

isolates a solid from a liquid

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12
Q

process (2): filtration

A
  1. pour a liquid-solid mixture onto a paper filter that allows only the solvent to pass through, like a coffee filter
  2. at the end of filtration, one is left with the solid (the residue) and the flask full of liquid that passed through the filter (filtrate)
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13
Q

how can filtration be modified?

A

depending on whether the substance of interest is the solid or is dissolved in the filtrate

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14
Q

defn + what is the product of interest: gravity filtration

A

the solvent’s own weight pulls it through the filter

more commonly used when the product of interest is in the filtrate

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15
Q

why is hot solvent often used in gravity filtration?

A

to keep the product dissolved in liquid

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16
Q

defn + process: recrystallization

A

a method for further purifying crystals in solution

dissolve or product in a minimum amount of hot solvent and let it recrystallize as it cools

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17
Q

what type of solvent should be chosen for recrystallization? why?

A

one in which the product is only soluble at high temperatures

so that when the solution cools, only the desired product will recrystallize out of the solution, excluding the impurities

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18
Q

why does distillation come in handy?

A

when the product itself is a liquid that is soluble in the solvent

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19
Q

defn + process (3): distillation

A

takes advantage of differences in the boiling point to separate two liquids by evaporation and condensation

  1. the liquid with the lower boiling point will vaporize first, and the vapors will rise up the distillation column to condense in a water-cooled condenser
  2. this condensate then drips down into a vessel
  3. the end product is called the distillate
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20
Q

why is the heating temperature kept low in distillation?

A

so that the liquid with the higher boiling point will not be able to boil and therefore will remain liquid in the initial container

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21
Q

defn + condition: simple distillation

A

the least complex version of distillation

should only be used to separate liquids that boil below 150 deg C and have at least a 25 deg C difference in boiling points

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22
Q

what do the restrictions placed on simple distillation allow for?

A

prevent the temperature from becoming so high that the compounds degrade and provide a large enough difference in boiling points that the second compound won’t accidentally boil off into the distillate

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23
Q

apparatus (3): simple distillation

A
  1. a distilling flask containing the combined liquid solution
  2. a distillation column consisting of a thermometer and a condenser
  3. a receiving flask to collect the distillate
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24
Q

what 3 additional pieces of equipment may be introduced into distillation to break surface tension and prevent superheating?

A
  1. boiling chip
  2. ebulliator
  3. magnetic strirer
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25
Q

when does superheating occur?

A

when a liquid is heated to a temperature above its boiling point without vaporization

occur when gas bubbles within a liquid are unable to overcome the combination of atmospheric pressure and surface tension

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26
Q

when do we use vacuum distillation?

A

whenever we want to distill a liquid with a boiling point over 150 deg C

27
Q

what happens when we use a vacuum in vacuum distillation?

A

we lower the ambient pressure which then decreases the temperature that the liquid must reach in order to have sufficient vapor pressure to boil

28
Q

what benefit does vacuum distillation provide?

A

it allows us to distill compounds with higher boiling points at lower temperatures so that we do not have to worry about degrading the product

29
Q

process (3): vacuum distillation

A
  1. the initial solution is placed in the heated distilling flask, where the components of the solution with the lowest boiling points will vaporize first
  2. the vapor then condenses in the water-cooled condenser
  3. this distillate drips into the receiving flask
30
Q

func: fractional distillation

A

to separate two liquids with similar boiling points (less than 25 deg C apart)

31
Q

process (4): fractional distillation

A
  1. a fractionation column connects the distillation flask to the condenser
  2. as the vapor rises up the column, it condenses on these surfaces and refluxes back down until rising heat causes it to evaporate again, only to condense again higher in the column
  3. each time the condensate evaporates, the vapor consists of a higher proportion of the compound with the lower boiling point
  4. by the time the top of the column is reached, only the desired product drips down the receiving flask
32
Q

defn: fractionation column

A

a column in which the surface area is increased by the inclusion of inert objects like glass beads or steel wool

33
Q

what is the universal concept behind chromatography?

A

the more similar a compound is to its surroundings (whether by polarity, charge, or other characteristics) the more it will stick to and move slowly through its surroundings

how strongly they adhere to the solid/stationary phase (or how easily they come off into the mobile phase)

34
Q

aka (2): solid medium

A

stationary phase
adsorbent

35
Q

what state of matter is the mobile phase usually?

A

a liquid (or a gas in gas chromatography)

36
Q

defn: elute

A

displace

37
Q

defn: partitioning + partitioning coefficient

A

depending on the characteristics of the substances in the sample and the polarity of the mobile phase, the mobile phase will adhere to the stationary phase with differing strengths, causing the different substances to migrate at different speeds

different compounds have different partitioning coefficients and will elute at different rates

38
Q

process (4): chromatography

A
  1. place the sample onto a solid medium
  2. then run the mobile phase through the stationary phase –> this will elute the sample and carry it through the stationary phase
  3. partitioning, representing an equilibrium between the two phases
  4. this results in separation within the stationary phase, allowing us to isolate each substance individually
39
Q

what is the most common property that we exploit in chromatography on the MCAT? give 2 examples

A

polarity!

examples
1. silica gel (super polar) is stationary phase in TLC
2. cellulose (polar) is stationary

40
Q

in practice, what do we measure instead of the speed at which compounds move through media? (2) + what types of chromatography are associated with each ansewr

A
  1. how far each substances travels in a given amount of time (TLC)
  2. how long it takes to elute (column or gas chromatography)
41
Q

what are the 4 main types of chromatography?

A
  1. thin-layer and paper chromatography
  2. column chromatography
  3. gas chromatography (gas-liquid chromatography)
  4. high-performance liquid chromatography (HPLC)
42
Q

diff: thin-layer (TLC) vs. paper chromatography

A

extremely similar techniques, only vary in stationary phase medium

thin-layer: a thin layer of silica gel or aluminum adherent to an inert carrier sheet

paper: paper composed of cellulose

43
Q

defn: spotting

A

when we apply a small, well-defined spot of sample directly onto the silica or paper plate

44
Q

what is used as a developing chamber in thin-layer and paper chromatography?

A

usually a beaker with a lid or a wide-mouth jar

45
Q

defn: eluent

A

a shallow pool of solvent

46
Q

process (5): thin-layer and paper chromatography

A
  1. place the sample that we want to separate directly onto the adsorbent itself (spotting)
  2. the plate is then developed (place the adsorbent upright in a developing chamber with eluent at the bottom of the jar)
  3. the spots of the sample must be above the solvent level or they will dissolve into the solvent pool rather than running up the plate
  4. when set up properly, the solvent will creep up the plate by capillary action, carrying the various compounds in the sample with it at varying rates
  5. when the solvent front nears the top of the plate, the plate is removed from the chamber and allowed to dry
47
Q

what are the components of TLC usually made of?

A
  1. often done with silica gel: polar and hydrophilic
  2. mobile phase: usually an organic solvent of weak to moderate polarity (so it doesn’t bind well to the gel)
48
Q

what is the impact of having an organic solvent of weak to moderate polarity as the mobile phase of TLC?

A

nonpolar compounds dissolve in the organic solvent and move quickly as the solvent moves up the plate

the polar molecules stick to the gel

the more nonpolar the sample is, the further up the plate it will move

49
Q

defn: reverse-phase chromatography

A

the opposite of TLC
stationary phase = nonpolar
- polar molecules move up the plate quickly
- nonpolar molecules stick more tightly to the stationary phase

50
Q

what are 2 ways to get around the fact that spots of individual compounds on TLC and paper chromatography are white, which makes them impossible to see?

A
  1. developed plate placed under UV light (will show any UV-sensitive compounds)
  2. iodine, phosphomolybdic acid, or vanillin can be used to stain the spots (will destroy the compounds s.t. they cannot be recovered)
51
Q

how are unknown compounds identified during TLC?

A

using the retardation factor (Rf) –> is relatively constant for a particular compound in a given solvent

52
Q

eqn: Rf (retardation factor)

A

Rf = distance spot moved/distance solvent front moved

53
Q

defn: preparative TLC

A

a larger scale of TLC as a means of purification

as the large plate develops, the larger spot of sample splits into bands of individual compounds, which can be scraped off and washed to yield pure compounds

54
Q

char + setup: column chromatography (5)

A
  1. uses similar principles as TLC
  2. uses an entire column filled with silica or aluminum beads as an adsorbent (allows for much greater separation than TLC or paper)
  3. uses gravity to move the solvent and compounds down the column
  4. the solvent drips out the end of the column and the different fractions that leave the column can be collected over time, with each fraction containing different compounds
  5. after collection: evaporate solvent, leaving behind the compounds of interest
55
Q

process summary (2): column chromatography

A
  1. the sample is added to the top of the column, and a solvent is poured over it
  2. the more similar the sample is to the mobile phase, the faster it elutes; the more similar it is to the stationary phase, the more slowly it will elute (if at all)
56
Q

what are 2 ways that you can speed up/aid column chromatography?

A
  1. force the solvent through the column using gas pressure (= flash column chromatography)
  2. solvent polarity can be changed to help elute the desired compound
57
Q

why is column chromatography particularly useful in biochem?

A

it can be used to separate and collect macromolecules such as proteins or nucleic acids

58
Q

process/set-up: ion-exchange chromatography (2)

A
  • the beads in the column are coated with charged substances s.t. they attract or bind compounds that have an opposite charge
  • after all other compounds have moved through the column, a salt gradient is used to elute the charged molecules that have stuck to the column
59
Q

process/setup: size-exclusion chromatography (3)

A
  • the beads in the column contain tiny pores of varying sizes
  • the pores allow small compounds to enter the beads, slowing them down
  • large compounds can’t fit, so they move around them and travel through the column faster

THIS MIGHT BE CONTERINTUITIVE

60
Q

process/set-up: affinity chromatography (6)

A
  1. a protein of interest is bound by creating a column with high affinity for that protein
  2. this is accomplished by coating the beads with a receptor that binds the protein OR a specific antibody to the protein s.t. the protein is retained in the column
  3. common stationary phase molecules: nickel, antibodies or antigens, enzyme substrate analogues
  4. once the protein is retained in the column, it can be eluted by washing the column with a free receptor (or target or antibody), which will compete with the bead-bound receptor and ultimately free the protein from the column
  5. eluents can be created with a varying pH or salinity level that disrupts the bonds between the ligand and the protein of interest
  6. drawback of the elution step: the recovered substance can be bound to the eluent, and might be difficult to remove
61
Q

process + aka + char: gas chromatography (7)

A

aka: vapor-phase chromatography

  1. the eluent is a gas (usually He or N) instead of liquid
  2. adsorbent: a crushed metal or polymer inside a 30-ft column
  3. the column is coiled and kept inside an oven to control the temp
  4. the mixture is then injected into the column and vaporized
  5. the gaseous compounds travel through the column at different rates bc they adhere to the adsorbent in the column to different degrees and will separate in space by the time they reach the end of the column
  6. the injected compounds must be volatile
  7. the compounds are registered by a detector, which records them as a peak on a chart
62
Q

defn: volatile

A

low melting-point, sublimable solids or vaporizable liquids

63
Q

func + process (3): mass spectrometry

A
  • for molecular weight determination

involves the ionization and fragmentation of compounds

the fragments are then run through a magnetic field, which separates them by a mass-to-charge ratio

the total molecular weight can be determined OR the relative concentrations of the different fragments can be calculated and compared against reference values to identify the compound

64
Q

setup/process + char: high-performance liquid chromatography (HPLC) (6)

A
  1. eluent = liquid –> travels through a column of a defined composition
  2. variety of stationary phases chosen depending on target molecule and the quantity of material that needs to be purified
  3. a small sample is injected into the column, separation occurs as it flows through
  4. the compounds pass through a detector and are collected as the solvent flows out of the end of the apparatus
  5. computerized –> sophisticated solvent gradients, temperature can be applied to the column to help resolve the various compounds in a sample
  6. uses liquid under pressure

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