Chromatography

Question 1

What is Chromatography?

Answer 1

Chromatography is the process of separation of components from a mixture of compounds

the partition (division/separation) of the compounds in the mixture between a stationary phase and a mobile phase

Question 2

How can chromatography be used?

Answer 2

qualitatively to assess purity - all move to the same distance = cannot tell how many analytes there are

preparatively to purify compounds - separates analytes

it can be used quantitatively with GC or HPLC with an internal standard - find out how many analytes there are

Question 3

What are the stationary and mobile phase?

Answer 3

the stationary phase is fixed in place - solid packing material
the mobile phase carries the compounds (analytes) along - liquid or gas

Question 4

Why are analytes (compounds) separated in chromatography?

Answer 4

analytes are separated due to their different distributions between stationary and mobile phases
this distribution is an equilibrium

Question 5

What is the partition coefficient?

Answer 5

the distribution of analytes through the 2 phases is an equilibrium
it is the equilibrium constant
it gives a numerical measure of the distribution of analytes in the mixture

A[stationary]/ A[mobile]

Question 6

What is TLC?

Answer 6

Thin Layer Chromatography

normal phase chromatography or reverse phase chromatography

Question 7

How does TLC work?

Answer 7

stationary phase = uses a solid backing material - silica gel or alumina (polar)
mobile phase = uses a mixture of organic solvents (non-polar)
unknown mixture is spotted onto the plate near the bottom (at least 1 cm away)
tank prepared with mobile phase
plate is placed in the tank
solvent moves up the plate due to capillary action
once the solvent level reaches the top, the analytes can be seen

Question 8

How can analytes be seen on the plate after movement?

Answer 8

it can be seen under UV light or chemical staining

Fluorescent light

Question 9

What is normal phase chromatography?

Answer 9

stationary phase is polar due to OH groups = silica gel or alumina
mobile phase is non-polar = mixture of organic solvents
used for non-polar separations

strong interactions between non-polar mobile phase and mixture
polar analytes will interact strongly with the stationary phase and be found near the bottom of the plate whereas non-polar analytes will move up faster

Question 10

What is reverse phase chromatography?

Answer 10

stationary phase is non-polar = C-18 modified silica gel
mobile phase is polar = water can be used, polar organic solvents
used for polar separations

strong interactions between polar mobile phase and mixture
non-polar analytes will interact strongly with the stationary phase and be found near the bottom of the plate whereas polar analytes will move up faster

Question 11

What is the Rf value?

Answer 11

distance moved by analytes/ distance moved by solvent

affected by polarity of the solvent
not affected by the size of the plate

Question 12

What is column chromatography?

Answer 12

can be normal phase or reverse phase

glass columns are be packed with different stationary phases (silica gel, alumina, sephadex, reverse-phase C-18 silica) - one at a time
add sample to the top
sample is absorbed onto the stationary phase
solvent is added and it washes down the column
separates the analytes into components

Question 13

What is column chromatography separation based on?

Answer 13

polarity - using normal or reverse phase chromatography

size - uses sephadex as a stationary phase

Question 14

How can column chromatography be analysed?

Answer 14

components collected (fractions/eluates) can be analysed via TLC to find out their contents and purity

TLC is usually used to determine whether separation has been successful

analysis with NMR, MS, UV and/or IR

Question 15

What is flash chromatography?

Answer 15

can be normal phase or reverse phase

Glass columns can be pressurised with air to speed up the movement through the column
Compounds can be detected by UV or ELSD (Evaporative light scattering detector)

Question 16

What are the advantages and disadvantages of flash chromatography?

Answer 16

advantage - speed of separation = faster

disadvantage - loss of resolution = poorer separation of components

Question 17

What is HPLC and UHPLC?

Answer 17

high performance liquid chromatography
ultra high performance liquid chromatography
can be normal phase or reverse phase

can use small columns - analytical
can use large columns - preparative or semi-preparative

Question 18

What is the difference between HPLC and UHPLC?

Answer 18

UHPLC can be run as HPLC but HPLC cannot be run as UHPLC

UHPLC can use smaller particles
-better resolution
better separation

Question 19

What is gas chromatography?

Answer 19

mobile phase must be an inert, dry and oxygen free gas - compounds could react with oxygen or water
stationary phase is
-packed column = packed with finely ground diatomaceous earth (porous rock) coated with a high boiling liquid (usually waxy polymer)
-capillary column - stationary phase lines the wall of the thin glass tube which supports the liquid phase

Question 20

How is gas chromatography used?

Answer 20

used to separate the components
must be passed through mass spectrometer, flame ionisation or thermal conductivity to identify components or give molecular ions/formula

Question 21

How does gas chromatography work?

Answer 21

gas is released and flow controller controls how quickly gas flows through
gas enter the column over with the sample that is injected in via in the injector port
column oven is heated
- lower bp compounds vaporise first and move alone mobile phase
- higher bp compounds remain in liquid phase longer = longer retention time (stationary phase is liquid)

some compounds interact differently with the stationary phase

• some are more soluble than others
• non-volatile compounds cannot be used = won’t vaporise
• large compounds cannot be used = takes too long ti vaporise/ needs higher temperature

Question 22

What is retention time in gas chromatography?

Answer 22

time from injecting the sample to detect of the analytes

Question 23

How can analytes be detected in gas chromatography?

thermal conductivity

Answer 23

thermal conductivity

• electrically heated source is maintained at constant power
• temperature of the source depends on the TC of surrounding gases
• the source is usually a thin wire made of platinum or gold.
• the resistance within the wire depends upon temperature, which is dependent upon the thermal conductivity of the gas
• measures change in carrier gas TC caused by presence of sample

Question 24

How can analytes be detected in gas chromatography?

flame ionisation

Answer 24

flame ionisation
-organic compounds burn and produce ions
- ions can be attracted to either the anode (negative) or cathode (positive) of the detector, producing a small current that can be picked up
-the more the ions, the larger the current and the larger the peak.
mass spectrometry

Question 25

What are the properties of each type of chromatography?
preparative 
quantitative 
qualitative
normal or reverse

Answer 25

all can be normal phase or reverse phase

qualitative - pure or not (all move the same distance)
quantitive - how many different analytes there are/concentration
preparative - isolate and purify
analytical - separates

TLC - qualitative only
Column/Flash - qualitative or quantitative, preparative
HPLC - qualitative or quantitative, preparative with preparative column
GC - qualitative or quantitative