C29 Chromatography And Spectroscopy Flashcards
What are the basic principles of all kinds of chromatography
A family of separation techniques that depend on the principle that a mixture is separated if it’s dissolved in a solvent and this mobile phase is passed over a solid (the stationary phase)
What’s the mobile phase
Carries the soluble components of the mixture
What relationship between a sample and the mobile phase makes the move faster
More soluble components/compounds with more affinity to the solvent move faster
What does stationary phase do
Holds back components of the mixture that are attracted to it
What relationship between a sample and the stationary phase that makes the sample move slower? What kind of bonding does this often involve
More affinity for the stationary phase means that a component moves slower; often attracted by hydrogen bonding
How are substances separated by chromatography
If suitable stationary/mobile phases are chosen, the balance between affinity for the mobile phase and affinity for the stationary phase is different for each component of mixture. Thus, they move at different rates and are separated over time
Why will different substances show different Rf values
They’re bonded differently and have different polarities - more polar bonds mean longer retention time or smaller Rf value, since hydrogen bonding/dipoles are attracted more strongly to the stationary phase
What does TLC stand for
Thin layer chromatography
What’s the stationary phase in TLC
Plastic
Glass
Metal sheet or ‘plate’ coated in silica (SiO2) or alumina (Al2O3)
What are the advantages of TLC over paper chromatography
Runs faster
Smaller amounts of a mixture can be separated
TLC plates are more robust that paper
How can you observe colourless spots
Shine UV light on them
Or
Spray with developing agent (e.g. ninhydrin turns amino acid spots from colourless to purple, so they can be seen)
(Heating needed with ninhydrin)
How do you calculate Rf value.
Measure the distance from the initial line (that the mixture was spotted onto) to the solvent front, and the distance from the initial line to the spot.
Calculate Rf using:
distance moved by spot / distance moved by solvent front
What does Rf value stand for?
Retention factor
A measure of the rate of movement of a component through the chromatography apparatus; a ratio between the rate of movement of the solvent and that component
How could you confirm the identity of a substance from its Rf value
Compare your Rf value to accepted values Rf for that substance run in the same solvent and set-up; if they match, then identity is confirmed.
What’s the stationary phase in gas-liquid chromatography
Powder, coated with oil. Packed into a long, thin, capillary tube (100m long, 0.5mm diameter). Coiled and placed in an oven, the temperature of which can be varied.
What is the mobile phase in gas-liquid chromatography
Carrier gas, inert e.g. N2 or He
What do you measure in gas-liquid chromatography
Retention time; different components of the mixture take different amounts of time to move through
What are the advantages of GLC
Very sensitive; GC can detect minute traces of substances in foodstuffs, and link pollution on beaches to the specific tanker the oil came from.
What are the GLC’s uses
Test athletes’ and horses’ blood and urine for drugs
How can you use GC or GCMS to identify substances
Match gas chromatography to that of a known substance under the same conditions; retention time should exactly match. Substance’s identity can be confirmed by mass spectrometry, NMR or infrared spectroscopy.
How does GCMS work
Gas chromatography is run, retention time is recorded, then mixture is run through a mass spectrometer. Fragmentation pattern/molecular ion peak confirms identity.
How do you test for alkenes
Shake with bromine water
Result is bromine water is decolourised (orange to colourless)
How do you test for haloalkanes
Add NaOH(aq) and warm, acidify with HNO3, add AgNO3(aq)
Result = precipitate of AgX
Cl - white
Br - cream
I - yellow
How do you test for alcohols
Add acidified K2Cr2O7 (potassium dichromate (vi)) and heat
Result =
Colour change from orange to green for 1* and 2*
No change for 3* alcohols
How do you test for aldehydes
Warm with Fehling’s solution
Result = Brick red ppt forms from blue solution
Warm with tollens reagent
Result = silver mirror (Ag solid ppt forms)
How do you test for carboxylic acids
Add Na2CO3(aq)
CO2(g) given off - effervescence
How do you test for phenols
By weak acidity - there is a neutralisation reaction reacted with NaOH but no reaction with CO3^2-
How do you test for carbonyl compounds
React with 2,4- DNP and an orange precipitate should form
What does NMR stand for
Nuclear magnetic resonance
What are the basic principles of NMR
You can find the structures of complex molecules by placing them in a magnetic field and applying EM waves of radio frequency to them. If radio waves of the right frequency are absorbed, the nuclei flips from parallel to applied magnetic to field to anti-parallel. This energy change can be monitored and recorded. Uses the resonance of nuclei with spin.
Give one use of NMR
MRI scans
What kind of nuclei does NMR work with (and examples)
Those with an uneven number of nucleons, meaning they will spin e.g. 1H, 13C
Summarise what number of signals mean for 13C NMR
One signal for each C environment (each set of in equivalent 13C atoms)
Summarise what chemical shift means for 13C NMR
Greater from atoms closer to electronegative atoms or C=C
Summarise what area under peak means for 13C NMR
No meaning
Summarise what splitting means for 13C NMR
There’s no splitting for 13C NMR
Why does the peak from O-H bonds disappear if D2O is used as a solvent?
The O-D bond is formed in preference to O-H due to labile protons that move/swap from one molecule to another.
Why is it easier to get a spectrum of 1H NMR than 13C NMR
Most H atoms are 1H- its much more abundant than 13C
This means almost all H atoms have spin so show up
On a low resolution spectrum, what peaks would you expect to see for H NMR
One peak for each set of inequivalent H atoms (each chemical environment shows 1 peak)
What does the area under the peak represent (for H NMR)
The area under the peak is proportional to the number of 1H atoms represented by the peak
What is the integration trace
A stepped line that makes it easier to measure the area under the curve (height of line = area under that peak)
What is TMS
Tetramethylsilane
Why is TMS used
Can be added to sample to calibrate the NMR equipment. It provides a peak at exactly 0ppm. Is is the reference point against which all ppm are measured.
What are other advantages of using TMS
Inert, non-toxic, easy to remove from the sample (as relatively volatile)
When does splitting/spin-spin coupling occur
Neighbouring hydrogen atoms (3 or fewer bonds away, or on the adjacent carbon) affect the magnetic field of 1H atoms and causes their peaks to split
What is the n+1 rule
If there are n inequivalent 1H atoms on the neighbouring carbon then the peak will split into (n+1) smaller peaks
Why must solvents used for 1H NMR not contain any hydrogen atoms
Signals from the solvent would swamp signals from the sample, as there is much more solvent than sample.
Which solvents are used
Deuterated solvents: CDCl3, D2O, C6D6
CCl4 - tetrachloromethane
Summarise what number of signals mean for 1H NMR
One main signal for each set of inequivalent 1H atoms (for each Hydrogen environment)
Summarise what chemical shift means for 1H NMR
Larger. For 1H atoms closer to electronegative atoms or C=C
Summarise what splitting means for 1H NMR
Number of smaller peaks = 1 + number of inequivalent hydrogen atoms 3 bonds away
Summarise what area under peak means for 1H NMR
Proportional to the number of atoms represented by that peak
