6.5 CHROMATOGRAPHY AND SPECTROSCOPY

Question 1

What are the uses of TLC?

Answer 1

The uses of TLC:
- urine samples = test for drugs
- drugs = establish purity or identify components
- food = determine presence of pesticides/contaminants

Question 2

How does TLC work?

Answer 2

TLC works because:
1. components have different solubility’s in mobile phase - more attraction between molecules of compound and molecules of solvent = distance component will travel
2. components have different relative absorptions - surface of stationary phase is very polar so the more polar a compound is the more attracted to the stationary phase it is = will be absorbed more strongly, move slowly, have a greater affinity for stationary phase

Question 3

What are the advantages of TLC?

Answer 3

Advantages of TLC:
- quick = very little time required for separation
- inexpensive = uses very little equipment
- in comparison to paper chromatography it is very sensitive, gives better separation, allows for choice of stationary phase and solvent front travels more evenly through stationary phase

Question 4

What is the distance moved by the substance?

Answer 4

Distance moved by substance is from the pencil line to the centre of the dot.

Question 5

What is the distance moved by the solvent?

Answer 5

Distance moved by the solvent is from the pencil to the solvent front.

Question 6

What are the limitations of TLC?

Answer 6

Limitations of TLC:
- similar compounds often have similar Rf values
- unknown compounds have no reference Rf for comparison
- can be difficult to find a solvent that separates all the components in a mixture
- components of sample can be colourless = spots can be shown by using a location such as iodine of ninhydrin, chemical can be added to stationary phase to make it fluoresce in UV light

Question 7

What is the method of TLC?

Answer 7

TLC method:
1. crush leaf sample with mortar and pestle, pipette 2cm acetone and grind until leaf is broken
2. transfer mixture to TLC plate with capillary tube after drawing pencil line 1cm from bottom
3. add propanone and petroleum spirit [solvent] to glass tank using pipette
4. place TLC paper into tank, make sure solvent is below pencil line
5. add lid to glass tank
6. leave in fume hood to separate
7. take out after separation, mark solvent front, leave to dry
8. calculate Rf

Question 8

What is the mobile phase in gas chromatography?

Answer 8

The mobile phase in GC is an inert carrier gas, e.g helium, nitrogen, neon

Question 9

What is the stationary phase in gas chromatography?

Answer 9

The stationary phase in GC is a high boiling point liquid adsorbed onto an inert solid support, e.g silica

Question 10

What does a gas chromatogram show?

Answer 10

Gas chromatogram:
- number of peaks = number of different compounds
- retention time = reaction time
- area under peak = amount of different compounds

Question 11

What is the retention time?

Answer 11

Retention time:
- for a particular compound the retention time depends of boiling point and solubility in the liquid phase
- high boiling point = long retention time
- greater solubility = long retention time

Question 12

What are the limitations of gas chromatography?

Answer 12

Limitations of GC:
- large, not portable
- expensive
- can take a while to prepare
- similar compounds will have the same retention times
- identifies only substances that have reliable retention times
- the flow rate of the carrier gas/oven temperature may need to be altered in order to improve separation (trial and error)

Question 13

What is nuclear spin?

Answer 13

Nuclear spin:
- nuclei are electrically charged (+ve)
- atoms which contain an odd number of nucleons (protons and neutrons), have significant spin, e.g H-1 C-13
- a spinning nucleus behaves like a spinning charge so it creates a magnetic field comparable to that of a bar magnet

Question 14

Magnets in magnetic field.

Answer 14

Magnets in magnetic field:
- line up in parallel to the field
- to go to anti-parallel requires a higher energy level = magnets would have to be forced into such a position against the repulsion of the magnetic field

Question 15

How could an anti-parallel position be forced in the magnetic field?

Answer 15

The anti-parallel position could be forced by energy (just equal to difference between two positions) supplied by a beam of radio waves flipping the nuclei into a higher energy position

Question 16

What is nuclear resonance?

Answer 16

Nuclear resonance:
- by absorbing energy into the nuclei in a magnetic field rapidly flip between the more stable, parallel, alignment to the less stable, opposed alignment
- the energy required to achieve this depends on the strength of the external magnetic field used
- flipping of the nucleus from one magnetic alignment to the other (between spin states) is known as a resonance

Question 17

When is the energy absorbed by the sample released??

Answer 17

The energy absorbed by the sample, from the beam of radio waves, is emitted when nuclei relax back to the lower energy state. This is measured and plotted against magnetic field (chemical shift).

Question 18

How is NMR used in medical imaging?

Answer 18

NMR is used in medical imaging and is known as an MRI (magnetic resonance imaging). It scans for water levels in body tissues.

Question 19

What are the advantages of MRI?

Answer 19

MRI advantages:
- non-invasive and safe as they do not involve exposure to ionising radiation (unlike CT scans which use x-rays)
- images can be acquired in multiple plans without moving the patient, and over the entire body
- can produce clear, detailed diagnostic images of soft tissue structures that other imaging techniques cannot achieve

Question 20

What the disadvantages of MRI?

Answer 20

MRI disadvantages:
- very expensive, and can take a lot longer to produce imagine (hence why CT scans are primarily used in A&E)
- image clarity is affected by movement = patient must remain completely still
- cannot be used on patients with internal foreign objects, such as pacemakers, certain metal implants, cardiac stents, etc

Question 21

Carbon-13 and proton NMR.

Answer 21

C-13 and proton NMR:
- all organic molecules contain carbon and hydrogen atoms
- hydrogen atoms are mostly H-1
- carbon atoms are mainly C-12
- it is the 1.1% of carbon atoms that are the isotope of C-13 that are relevant to NMR analysis (significant spin due to odd number of nucleons)

Question 22

What is chemical shift and TMS?

Answer 22

Chemical shift and TMS:
- tetramethylsilane (TMS) is the standard reference chemical in which all other chemical shifts are measured against
- chemical shift of TMS is defined as delta = 0ppm
- TMS is chemically unreactive, non-toxic and volatile so it can easily be removed from the sample

Question 23

Solvents used to dissolve samples from NMR.

Answer 23

Solvents used to dissolve samples from NMR:
- can’t use organic solvents such as hexane = they contain C-13 and H-1 atoms which will produce an NMR signal
- deuterated solvents are used instead = deuterium, D or H-2, is an isotope of H with a neutron
- deuterated chloroform (CDCl3) is commonly used for both H-1 and C-13 NMR spectra
- for C-13 spectra the carbon peak from CDCl3 is automatically removed
- CDCl3 can be easily evaporated to restore the sample

Question 24

What can C-13 provide?

Answer 24

C-13 can provide the number of different carbon environments (from number of peaks), and the types of carbon environments present (from the chemical shift)

Question 25

What is proton NMR?

Answer 25

Proton NMR:
- number of peaks = number of different proton environments
- chemical shift of peaks = type of proton environment
- integration/peak area = number of protons in environment
- splitting pattern of peak = number of neighbouring protons on directly adjacent carbon atoms

Question 26

What is low resolution proton NMR?

Answer 26

Low resolution NMR:
- gives one peak for each environmentally different groups of protons
- little (no splitting patterns)

Question 27

What is high resolution proton NMR?

Answer 27

High resolution NMR:
- gives more complex signals = peaks are split into doublets, triplets, quartets, etc
- splitting pattern depends on the number of neighbouring protons on directly adjacent carbon atoms
- the number of neighbouring protons can be readily calculated using the ‘n+1 rule’

Question 28

What the table look like to analyse proton NMR spectra?

Answer 28

Analysing proton NMR:
peak : chemical shift (ppm) : peak area : splitting pattern n+1 rules

Question 29

What are the n+1 rules?

Answer 29

N+1 rules:
singlet (1 peak) = 0 hydrogens on neighbouring carbon
doublet (2 peaks) = 1 hydrogen on neighbouring carbon
triplet (3 peaks) = 2 hydrogens on neighbouring carbon
quartet (4 peaks) = 3 hydrogens on neighbouring carbon
quintet (5 peaks) = 4 hydrogens on neighbouring carbon
sextet (6 peaks) = 5 hydrogens on neighbouring carbon
multiplet (7+ peaks) = 6+ hydrogens on neighbouring carbon