Lab Techniques: Separations and Spectroscopy

Question 1

distribution/partition coefficient

Answer 1

the ratio of the substance’s solubilities in the two solvents

Question 2

outline the process of a liquid-liquid extraction

Answer 2

one substance is separated from a mixture of substances by adding a solvent in which the compound of interest is highly soluble. If the solution containing the compound of interest is shaken with a second solvent (completely immiscible with the first, and allowed to separate into distinct phases, the compound of interest will distribute itself between the phases based upon the solubility in each of the individual solvents.

Question 3

what does solubility depend on?

Answer 3

the polarity of the solute and the polarity of the solvent

Question 4

how do you extract basic compounds from organic mixtures?

Answer 4

treat with dilute acid (5-10% HCl) to protonate basic functional group and produce a positive ion. the cationic salts are soluble in aqueous solution and can be extracted using water

Question 5

how do you extract carboxylic acid compounds from organic mixtures

Answer 5

treat with dilute weak base (5% NaHCO3) to produce anionic salts which can be extracted with water

Question 6

when can you use dilute hydroxide solution (10%) to extract an acidic compound from an organic mixture?

Answer 6

when the compound of interest has a pH lower than hydroxide (pKa=16) and there are no other compounds that might also be extracted

Question 7

thin-layer chromatography (TLC0

Answer 7

solid-liquid partitioning technique in which the mobile liquid phase ascends the polar stationary phase (thin layer of absorbant-silica that is coated on a supporting glass plate)

Question 8

what determines the separation and rate of elution in TLC?

Answer 8

the more polar components travel slower (more interaction with stationary phase) and nonpolar components travel faster

Question 9

column (flash) chromatography

Answer 9

uses a chromatography column filled with silica gel (polar stationary phase) and saturated with chosen organic solvent (mobile liquid phase), mixture of compounds is added to top and allowed to travel down column

Question 10

what determines the separation and rate of elution in column chromatography?

Answer 10

the more polar compounds travels slower (more interaction with polar stationary phase), nonpolar compounds travel faster

Question 11

when do you use TLC?

Answer 11

when samples are very small, when you want to determine the components present in a mixture

Question 12

when do you use column chromatography?

Answer 12

when you want to isolate bulk compounds

Question 13

when do you use ion exchange chromatography

Answer 13

when materials to be separated have varying charge states, often used in the separation of mixtures of proteins

Question 14

ion exchange chromatography

Answer 14

column chromatography; mobile liquid phase containing analyte passed through column packed with a polymeric resin (solid stationary phase) containing either positive or negative charged molecules on the polymer surface

Question 15

when is HPLC appropriate?

Answer 15

when speed and efficiency is important

Question 16

high performance liquid chromatography (HPLC)

Answer 16

mobile phase (organic solvent) is pressurized by pump, sample is injected by syringe and mobile phase carries the sample to the column (containing stationary phase), sample is separated and detected when exiting column

Question 17

what determines the elution rate in HPLC?

Answer 17

the differing affinities of various compounds for either a stationary phase or a mobile phase; more polar compounds will elute first if mobile phase is stationary; i.e. the compound polarity that is similar to mobile phase will elute faster

Question 18

reverse phase HPLC

Answer 18

stationary phase is a silica gel bonded to a nonpolar group to create a nonpolar stationary phase, mobile phase is more polar than the stationary phase

Question 19

how do you analyze charged compounds such as amino acids using HPLC?

Answer 19

stationary phase=ion exchange column, usually cation exchange, mobile phase=polar protic or acidic solvent that ensures solubility and suppresses the dissociation of the COOH group on the amino acid

Question 20

what determines the elution order/difference in affinity to the column of amino acids in HPLC?

Answer 20

the various R groups and the corresponding intermolecular forces as a result

Question 21

size exclusion chromatography

Answer 21

target materials are dissolved in solvent (mobile phase) and loaded into a column packed with chemically inert, porous polymer beads (stationary phase)

Question 22

when do you use size exclusion chromatography?

Answer 22

when you want to separate bulk materials based on difference in molecular size, relatively fast elution, minimal loss of material on the column

Question 23

what determines the rate of elution in size exclusion chromatography?

Answer 23

sizes of pores in bead allow permeation of small molecules but exclude larger ones. So, small molecules follow intraparticle path and are small, large particles follow interparticle path and are faster

Question 24

when do you use affinity chromatography?

Answer 24

to purify proteins or nucleic acids from complex biochemical mixtures rather than a reaction mixture

Question 25

what is affinity chromatography based on?

Answer 25

the highly specific interactions between macromolecules

Question 26

affinity chromatography

Answer 26

target molecule is isolated using specific antibody, antibody-protein isolated by microbe-derived proteins (AGL) which is covalently linked to a solid support-stationary phase (can also use solid resin) which binds to protein of interest. After centrifuging sample and decanting the supernatant, target can be isolated

Question 27

affinity tag

Answer 27

used when no commercial antibody available, affinity tagged proteins have an affinity to bind to specific ions

Question 28

gas chromatography

Answer 28

a form of column chromatography in which the partitioning of the compounds to be separated takes place between a mobile gas phase and a stationary liquid phase

Question 29

how does gas chromatography work?

Answer 29

sample is vaporized by heater and carried with an inert gas stream (helium-mobile gas phase), moved into a column of particles coated with liquid absorbant (stationary liquid phase), components interact differently with absorbant based on their relative volatilities. Many gas-liquid partitioning processes later, the individual components are separated. After exiting the column, it is burned to allow detection of resulting ions

Question 30

what determines elution rate in GC?

Answer 30

different volatilities; more volatile moves faster (less interaction with stationary phase), less volatile gases move slower (more interaction with stationary phase)

Question 31

what are two factors that greatly influence melting and boiling point

Answer 31

1) branching-more branches=less surface area for intermolecular forces
2) molecular weight (size)=larger molecules have more surface area to interact and have greater intermolecular forces

Question 32

what is distilarion

Answer 32

the process of raising the temperature of a liquid until it can overcome the intermolecular forces that hold it together in the liquid phase. The vapor is then condensed back to the liquid phase and is subsequently collected in another container

Question 33

when is distillation appropriate

Answer 33

when large amounts of material need to be separated

Question 34

when is simple distillation performed?

Answer 34

performed when trace impurities need to be removed from a relatively pure compound or when a mixture of compounds with significantly different boiling points need to be separated

Question 35

when is fractional distillation performed?

Answer 35

when the difference in boiling points of the components in the liquid mixture is not large

Question 36

how does fractional distillation work?

Answer 36

fractional distillation column packed with appropriate material (ex. glass beads), allows liquid to be subjected to multiple vaporization-condensation cycles, as the cycle progresses, the composition of vapour gradually becomes enriched in the lower boiling component which can be condensed and collected