separation techniques - chromatography

Question 1

what is chromatography?

Answer 1

involved partitioning of an analyte between 2 phases - mobile and stationary phase

Question 2

TLC meaning

Answer 2

thin layer chromatography

Question 3

what apparatus is needed for TLC?

Answer 3

TLC plate - made with a thin layer of absorbant coating e.g. SiO2, this is the stationary phase
a shallow pool of solvent which acts as the mobile phase

Question 4

how does TLC work?

Answer 4

sample is dotted onto baseline and plate is immersed into shallow pool of solvent, solvent will travel up the plate
as components/samples interact differently with stationary and mobile phases, they move at different speeds over the stationary phase therefore separation occurs

Question 5

how does column chromatography work?

Answer 5

sample is placed on top of packed column, filled with solvent, which then flows down column
components move down column at different rates forming bands
an example of this is gas chromatography

Question 6

what are the mobile phases in gas, liquid and supercritical chromatography?

Answer 6

mobile phase in gas chromatography = gas
mobile phase in liquid chromatography/TLC = liquid
mobile phase in supercritical chromatography = supercritical fluid

Question 7

how is separation in chromatography determined?

Answer 7

Kp = [Astationary]/[Amobile] - just like phase extractions
also for TLC Rf = distance travelled by component/distance travelled by solvent

Question 8

other than interactions stationary phase/column, what else influences separation of samples in gas chromatography?

Answer 8

boiling point

Question 9

what is the purpose of a flow controller in a gas chromatographer?

Answer 9

allows flow rate of gas to be controlled, lower flow rate = slower measurement process and greater separation
- flow rate can impact the value of the partition coefficient between column and analyte, so controlling it precisely is important for reproducability

Question 10

give 2 necessary properties of the carrier gas/solvent in column chromatography

Answer 10

• it must be inert - this means it will not interfere with results by reacting with gas being examined
• must have a very high purity

Question 11

give 3 examples of common inert carrier gases

Answer 11

H2, N2 and He

Question 12

which carrier gases are preferred for gas chromatography + why?

Answer 12

H is very explosive + hard to store
He is preferred for safety reasons but it is very expensive

Question 13

how does boiling point affect separation of analytes in gas chromatography?

Answer 13

the lower the boiling point the faster the gas will pass through the column

Question 14

what properties are necessary for a sample/analyte to be used for gas chromatography?

Answer 14

• must be thermally stable, cannot decompose at high temps which are necessary for the process
• must have an appreciable vapour pressure at column temp, as it must be able to turn into a gas

Question 15

what kinds of compounds can be used for gas chromatography?

Answer 15

all ‘permanent gases’ (= gas at room temp) as well as most non-ionised small/medium organic molecules

Question 16

give 2 compounds gas chromatography does not work for

Answer 16

macromolecules e.g. polymers/salts
non-volatile compounds

Question 17

what is the purpose of an injector in a gas chromatographer?

Answer 17

injector must deliver precisely a small amount of the material onto the column
- necessary as the capillary column is very thin and only a very small amount is needed

Question 18

under what temperature is gas chromatography typically done?

Answer 18

250 C - ensures all material is in vapour phase

Question 19

state 1 type of injector often used in gas chromatography

Answer 19

split-splitless injector

Question 20

how does a split/splitless injector work?

Answer 20

it injects a small fraction of sample into the column, with the rest being sent to exhaust, once the sample is injected into the column it vapourises
- this works because the injector has an outlet valve kept open, and depending on the flow rate of carrier gas most of the sample is ejected

Question 21

how is split ratio calculated?

Answer 21

split ratio = (split outlet flow rate + column outlet flow)/ column outlet flow

Question 22

how does concentration of sample affect split ratio?

Answer 22

more concentrated samples are run with a higher split ratio, weaker samples are run with a low split ratio or splitless (so all sample goes in column)

Question 23

what are the advantages of using split/splitless injectors?

Answer 23

small + precise amount of sample injected, this can aid in separation + reproducibility, and can be used for capillary columns that have a small capacity

Question 24

what are the disadvantages of using split/splitless injectors?

Answer 24

trace analysis is limited as such a little fraction is detected (this can change ratio), this can discriminate against heavier/less volatile samples as what enters the column might not be a true representation of the sample (internal standard method is used to avoid this dsicrimination)

Question 25

what are the 2 types of columns

Answer 25

pack columns and open tubular columns (capillary columns)

Question 26

why are open tubular columns beneficial?

Answer 26

offer high resolution, good sensitivity, reduced time and greater chemical inertness

Question 27

what is the difference between packed columns and capillary columns?

Answer 27

capillary columns are much longer, thinner and have lower flow rates and sample capacities

Question 28

what are the types of capillary columns?

Answer 28

WCOT (wall coated open tubular) - stationary phase located directly onto inner wall of the column PLOT (porous layer open tubular) - inner wall of column is extended by addition of a porous layer e.g. SiO2coated open tubular) - stationary phase applied into a solid support coated on the internal wall of column

Question 29

other than silica, SiO2, what is one other common stationary phase in TLC and gas chromatography?

Answer 29

alumina, Al2O3

Question 30

can compounds that react with the stationary phase be used?

Answer 30

no, this will deactivate the column

Question 31

what affects whether or not compounds interact with stationary phase/column?

Answer 31

if samples have high affinities to the column, they interact, and vice versa less interaction means the sample will come out faster

Question 32

what kind of compound will non polar stationary phases interact with and how?

Answer 32

it will interact with other non polar compounds via dispersion forces here separation depends more on volatility of analytes following trend in b.p rather than interactions

Question 33

what kind of compound will polar stationary phases interact with and how?

Answer 33

it will interact with other polar compounds via dispersion, dipole, acid-base interactions or hydrogen bonds

Question 34

what is the purpose of a detector in a gas chromatographer?

Answer 34

to monitor the column effluent and produce an electrical signal proportional to the amout of analyte being eluted

Question 35

what are the 5 types of detector?

Answer 35

FID (flame ionisation detector) TCD (thermal couple device) MS (mass spectrometer) NPD (nitrogen phosphorus detector) - niche ECD (electron capture detector) - niche - many of these are usually used in combination with each other

Question 36

why must FID be used last if used in combination with other detectors?

Answer 36

FID destroys the sample - its a destructive detector

Question 37

what does MS/mass spectroscopy measure?

Answer 37

mass : charge ratio

Question 38

what are the 2 ionisation techniques used in GC-MS?

Answer 38

electric ionisation - ionising agent = 50-70 eV electrons, causes extreme fragmentation, allows structure determination, done under ultra low pressure/vacuum chemical ionisation - ionising agent = gaseous ions, allows molecular mass determination

Question 39

column selectivity definition

Answer 39

the selection is based on achieving the maximum selectivity between the phase and analytes of interest - so separation is increased by exploiting analyte-column interactions that change the progress of some analytes relative to others so as to increase their retentions

Question 40

give 4 other conditions that can be altered to improve separation

Answer 40

initial/final temp, how long we're at this temp for, ramp rate, carrier gas flow rate

Question 41

how does column temp change separation?

Answer 41

increased column temp forces a larger fraction of solute molecules into the vapour phase, reducing retention times and decreasing separation (+ vice versa)

Question 42

what is a necessary condition for internal standards to be used?

Answer 42

sample injected must be liquid

Question 43

why is GC-MS preferred to LC-MS?

Answer 43

GC-MS has libraries of spectra/chromatograms to compare with allowing for structural identification, LC-MS doesn't

Question 44

give 3 conditions to know when using FID

Answer 44

- must know response factor, as sample is combusted, you cannot just take area under peak - for hydrocarbons, response is related to amount of C atoms present - oxygenated hydrocarbons burn less effectively

Question 45

how to calculate response factor?

Answer 45

= area under GC peak / volume % can be flipped: area under GC peak / response factor = calibrated volume

Question 46

how to calculate % selectivity?

Answer 46

= amount of required product / sum of all products - as using gas, moles and volume are interchangeable

Question 47

how does internal standard method work?

Answer 47

each sample is 'doped' with exactly the same amount of hydrocarbon (usually dodecane, as it doesn't interact with anything we want to analyse), and so everything is measured relative to internal standard