Chromatography

Question 1

What is chromatography

Answer 1

An analytical technique that separates components in a mixture between a mobile phase and a stationary phase

Question 2

what are the types of chromatography

Answer 2

• thin layer chromatography ( TLC) - a plate is coated with a solid and a solvent moves up the plate
• Column chromatography (CC) - a column is packed with a solid and a solvent moves down the column
• gas chromatography ( GC) - a column is packed with a solid or with a solid coated by a liquid , a gas is passed through the column under pressure at high temperature

Question 3

what is the mobile phase

Answer 3

may be a liquid or a gas
the part where the molecules can move through

Question 4

what is the stationary phase

Answer 4

may be a solid or either a like or solid on a solid support
This is the part where molecules can’t move

Question 5

in gas liquid chromatography ( GC) what is the mobile phase

Answer 5

An inert gas such as nitrogen , helium , argon

Question 6

in gas liquid chromatography what is the stationary phase

Answer 6

a liquid on an inert solid

Question 7

what does the separation by column chromatography depends on

Answer 7

the balance between solubility in the moving phase and retention in the stationary phase

Question 8

what is a solid stationary phase separated by

Answer 8

adsorption

Question 9

what is a liquid stationary phase separated by

Answer 9

relative solubility

Question 10

what happens if the stationary phase was polar and the mobile phase was non polar

Answer 10

non polar compounds would pass through the column more quickly than polar compounds as they would have a greater solubility in the non polar mobile phase

Question 11

what’s the method for TLC

Answer 11

• wear gloves , draw a pencil line 1cm above the bottom of a TLC plate and mark spots for each sample , equally spaced along the line
• Use a capillary tube to add a tiny drop of each solution to a different spot and allow the plate to air dry
• add solvent to a chamber or large beaker with a lid so that it’s no more than 1cm in depth
• place the TLC plate into the chamber , making sure that the level of the solvent is below the pencil line . Replace the lid to get a tight seal .
• When the level of the solvent reaches about 1cm from the top of the plate , remove the plate and mark the solvent with a pencil . Allow the plate to dry in the fume cupboard .
• Place the plate under a UV lamp in order to see the spots
  Draw around them lightly in pencil
• calculate Rf values of the observed spots

Question 12

why do we use a pencil line for TLC

Answer 12

Will not dissolve in the solvent

Question 13

why do we use a tiny drop of each solution in TLC

Answer 13

too big a drop will cause different spots to merge

Question 14

why don’t we use too deep of a solvent in TLC

Answer 14

If the solvent is too deep it will dissolve the sample spots from the plate

Question 15

Why do we use a lid in TLC

Answer 15

to prevent evaporation of toxic solvent

Question 16

why do we dry in a fume cupboard in TLC

Answer 16

as the solvent is toxic

Question 17

why do we use a UV lamp in TLC

Answer 17

used if the spots are colourless and not visible

Question 18

what are some preventions of risks in TLX

Answer 18

Wear plastic gloves to prevent contamination from the hands to the plate

Question 19

how do we calculate the rf value

Answer 19

distance moved by amino acid / distance moved by the solvent

Question 20

what’s the method for column chromatography

Answer 20

• a glass tube is filled with the stationary phase usually silica or alumina in powder form to increase the surface area
• a filter or plug is used to retain the solid in the tube
• solvent is added to cover all the powder
• the mixture to be analysed is dissolved in a minimum of a solvent and added to the column
• a solvent or mixture of solvent is then run through the column
• the time for each component in the mixture to reach the end of the column is recorded ( retention time )

Question 21

what is the mobile phase in column chromatography

Answer 21

solvent
( eluent )

Question 22

what is the stationary phase in column chromatography

Answer 22

aluminium oxide / silicon oxide in powder form to increase surface area

Question 23

what is HPLC and what does it stand for

Answer 23

High performance liquid chromatography
- it’s a type of column chromatography used in industry

Question 24

what’s the mobile phase for HPLC

Answer 24

a liquid

Question 25

what’s the ststionary phase for HPLC

Answer 25

solid silica

Question 26

what is gas liquid chromatography used for

Answer 26

to separate mixtures of volatile liquids

Question 27

what is the retention time in GC

Answer 27

the time taken for a particular compound to travel from the injection of the sample to where it leaves the column to the detector . It can be used to identify a substance

Question 28

what happens if some compounds have similar retention times

Answer 28

Will not be distinguished

Question 29

what does GC tell us

Answer 29

•how many components there are in the mixture by the number of peaks . • the abundance of each peak - the area under each peak will be proportional to the abundance of that component

Question 30

what is gas chromatography mass spec used in (GC-MS)

Answer 30

analysis , in forensics , environmental analysis , airport security and space probes

Question 31

EQ : suggest why it was necessary to use two different solvents ( TLC) (1)

Answer 31

Some of the amino acids did not dissolve / separate with the first / either solvent • Some amino acids have the same Rf values or have the same affinity with first / either solvent

Question 32

suggest a suitable reagent for the hydrolysis of a protein (1)

Answer 32

conc HCl

Question 33

EQ : suggest how the positions of the amino acids on the TLC plate were located (1)

Answer 33

Using ninhydrin / UV lamp

Question 34

EQ : state in general terms what determines the distance travelled by a spot in TLC (1)

Answer 34

The affinity for the mobile phase and the stationary phase

Question 35

EQ :To obtain the chromatogram , the TLC plate was held by the edges and placed in the solvent in the beaker in the fume cupboard . The lid was then replaced on the beaker Give one other practical requirement when placing the plate in the beaker (1)

Answer 35

solvent depth must be below start line

Question 37

What do you measure for stationary phase

Answer 37

Affinity for stationary phase

Question 38

what do you measure for moving / mobile phase

Answer 38

Solubility in moving phase

Question 39

EQ : suggest why propanone was present in the samples of the eluent collected first ( those with shorter retention times , ) whereas samples containing propan-2-ol were collected later

Answer 39

propanone is less polar than propan-2-ol Propan-2-ol has a greater affinity for the stationary phase OR propanone is more soluble in the moving phase

Question 40

EQ : Outline briefly why chromatography is able to separate a mixture of compounds . Practical details are not required (3)

Answer 40

- solvent is mobile - stationary phase - Separation depends on balance between solubility or affinity of compounds in each phase OR different rf values

Question 41

EQ : outline how TLC can be used to separate the amino acids present in the mixture . Explain which property of the amino acids allow them to be separated in this way (4)

Answer 41

- A spot of the amino acid mixture is placed on a thin-layer chromatography plate -The bottom of the plate (but not the spot) is then dipped into a solvent - As the solvent moves up the plate, the amino acids dissolve and move with it - Different amino acids have different solubilities in the solvent/polarities/attractions to the stationary phase, and so move at different rates, separating out on the plate