29 - chromatography and spectroscopy Flashcards
overall function of chromatography
Used to separate individual components from a mixture of substances
practical uses of chromatography
analysis of drugs, plastics, flavourings, air samples and forensic science
stationary phase in TLC
solid silica (SiO2) or alumina (Al2O3) held on an inert support material (glass/plastic)
mobile phase of TLC
Liquid solvent
advantages of TLC
Quick
inexpensive
how does separation occur in TLC
via relative adsorption
Components in the mixture adsorb differently to the surface of the stationary phase which result in different rf values
outline the method for TLC
Draw a base line approx. 1 cm from the bottom in pencil
Using a capillary tube, spot small sample of solution to be tested onto the baseline
Pour some solvent into the chromatography tank.
Ensure the depth is lower than the pencil line.
Place the TLC plate into the solvent, ensuring the solvent does not cover the spot, and that the edges of the TLC plate do not touch the sides of the tank.
Place a lid on the solvent tank.
Allow the solvent to rise until it is about 1 cm below the top of the plate
Remove the plate, marking the solvent level immediately with pencil
Allow the plate to dry
Circle any visible spots.
may need to hold a UV lamp over the TLC plate or spray the plate with a locating agent to show the position of the spots
compare TLC and paper chromatography
rf formula
distance moved by component / distance moved by solvent front
limitations of TLC (4)
Difficult to measure the exact centre of the component ‘spot’
Similar compounds have similar Rf values – difficult to tell them apart
Difficult to find a solvent which dissolves all components in a sample
may not be a reference chromatogram that exists in the database
overall function of gas chromatography
Separate volatile components in a mixture
what types of chemicals can gas chromatography analyse
components with low boiling points
stationary phase of gas chromatography
high boiling liquid adsorbed onto an inert solid support
mobile phase of gas chromatography
inert carrier gas
e.g. helium / neon
how does separation occur in gas chromatography
Separation via relative solubility
what is retention time
time taken for each component to travel through the column
what are the conditions for adsorption and solubility
Adsorption = solid stationary phase = TLC
Solubility = liquid stationary phase = gas chromatography
explain separation by adsorption
component molecules bind to surface of stationary phase
stronger the adsorption, more the component molecules are slowed down
explain separation by relative solubility
components dissolve in liquid stationary phase
the greater the solubility in the stationary phase, the more the component molecules are slowed down
outline the method for gas chromatography
A small amount of volatile mixture is injected into the gas chromatograph
inert carrier gas carries the components in the sample through the capillary column -contains the liquid stationary phase adsorbed onto the solid support.
components slow down as they interact with the liquid stationary phase
4 reach the detector at different times depending on their interactions with the stationary phase in the column
Component that is in the column for the shortest time has the lowest retention time + detected first
what information does a gas chromatogram give us
each component = detected as a peak
Retention time – used to identify the components present in the sample by comparing these to known components.
Peak integration – can be used to determine the concentrations of components in the sample
(area under the peak)
formula for percentage of component in a mixture
how to plot a calibration curve for concentration of substance against peak area
Prepare standard solutions with known concentrations of the compound being investigated.
Run gas chromatograms for each standard solution.
Plot a calibration curve of peak area against concentration
Run a gas chromatogram of sample.
Use the calibration curve to measure the concentration of the compound.
limitations of gas chromatography
Compounds may have the same retention times
Small amount of one component may hide behind a component with a higher concentration
Unknown components will not have any retention time data to compare it to
test + positive observation for alkenes
add bromine water drop-wise
should decolourise from orange to yellow
test + positive observation for haloalkane
add silver nitrate + ethanol and warm to 50°C in water bath
chloroalkane - white precipitate
bromoalkane - cream precipitate
iodoalkane - yellow precipitate
test + positive observation for carbonyls
add 2,4 DNP
orange precipitate
test + positive observation for aldehyde
add tollens reagent + warm
silver mirror
test + positive observation for primary / secondary alcohol and aldehyde
add acidified potassium dichromate + warm in a water bath
colour change from orange to green
test + positive observation for carboxylic acids
add aqueous sodium carbonate
effervescence
property of a nucleus
has nuclear spin which is ONLY significant if there is odd number of nucleons
(particles in the nucleus)
E.g. H1 and C13
how does NMR work
Uses strong magnetic field strength + radio frequency radiation
Causes nuclei of some atoms to absorb radiation + flip between spin states = resonance
Energy absorbed measured + recorded
describe the spectrometer used
strong superconducting electromagnet
Cooled by liquid helium
what is TMS used for
standard reference chemical which all chemical shifts are measured against
what ppm does TMS have
0
why do we use TMS
Chemically unreactive
1 carbon environment – one peak
Nuclei heavily shielded – rarely peaks below it
Volatile + easily removed from sample after
preparation of sample for NMR
dissolve in a solvent
what is the issue with solvents for NMR + how do we overcome this
Most solvents have H1 and C13
would produce a peak
Must use deuterated solvent – all 1H replaced with 2H
Produces no NMR signal in frequency range
what is the solvent used for NMR
Deuterated solvents
CDCl3
D is an isotope of H with 2 neutrons
describe the method of NMR
Sample dissolved in deuterated solvents + put into NMR sample tube
Tube inside spectrometer + spun to even out imperfections in magnetic field
Spectrometer zeroed against TMS standard
Given pulse of radiation whilst maintaining constant magnetic field
Absorptions of energy from resonance are detected
what information does carbon NMR provide
Number of different carbon environments = number of peaks
Types of carbon environment present = chemical shift
how to identify carbon environments
If two atoms symmetrical – equivalent + have same chemical environment
Carbon environment with higher chemical shift takes priority
what information does proton NMR decide
Number of different proton environments
Types of proton environments
Relative number of each type of proton
Number of non-equivalent protons adjacent to a given proton
what happens if 2+ protons are equivalent
absorb at same chemical shift + increase size of peak
what are splitting patterns caused by
Caused by protons spin interacting with the spin of nearby protons that are in a different environment
what is the rule of splitting patterns
Number of peaks is 1 more than number of adjacent protons causing the splitting
e.g. 4 peaks (quartet) means 3 protons in adjacent environment
how to identify O-H and N-H specifically
proton exchange
describe the process of proton exchange and the end result
Add small volume of deuterium oxide - D2O
Shake mixture
Run NMR as normal
Replaces the OH and NH protons with deuterium atoms
The peak disappears
