29) Chromatography and spectroscopy Flashcards
What is the purpose of chromatography?
to separate individual components from a mixture of substances
Define stationary phase
phase that does not move in chromatography
Define mobile phase
phase that moves in chromatography
Define thin layer chromatography (TLC)
quick and inexpensive analytical technique that indicated how many components are in a mixture
What is the stationary phase in TLC?
TLC plate (plastic or glass) coated with a thin layer of solid adsorbent substance (e.g. silica)
Define adsorption
the process that occurs when a gas or liquid or solute is held to the surface of a solid
How is separation achieved in TLC?
by relative adsorptions of substances with the stationary phase (different components in the mixture have different affinities and bind with different strengths to the surface)
Describe a 5 step method for TLC
1) base line drawn across the plate 1cm from the edge in pencil
2) small amount of sample solution spotted using a capillary tube onto the base line
3) prepare a chromatography tank with a small beaker and watch glass, solvent depth 5mm, then place the TLC plate inside ensuring the solvent doesn’t cover the spot
4) leave undisturbed until the solvent has risen to about 1cm below the top of the plate, remove + mark solvent front with pencil. allow to dry
5) circle any visible spots with pencil. You may need to use a UV lamp or spray with a chemical/locating agent e.g. iodine
How can each component of TLC be identified?
by comparing its Rf value with known values recorded using the same solvent system and stationary phase
OR TLC of a sample could be run against pure/known values
retention factor, Rf = ?
distance moved by component / distance moved by solvent front
What is the purpose of gas chromatography?
separating and identifying volatile organic compounds in a mixture
What is the stationary phase in gas chromatography?
high boiling liquid adsorbed onto an inert solid support
What is the mobile phase in gas chromatography?
inert carrier gas e.g. helium or neon
Describe a 3 step method for gas chromatography
1) small amount of volatile mixture is injected into the gas chromatograph + is carried by the mobile phase through the capillary column
2) components slow down as they interact with the stationary phase. the more soluble the component is in the stationary phase, the slower it moves through the column. therefore, compounds are separated depending on their solubility in the liquid stationary phase
3) components reach the detector at different times. the compound retained in the column for the shortest time has the lowest retention time and is detected first
Define retention time
the time taken for each component to travel through the column (from the column inlet to the detector)
Each component is detected as a _ on the gas chromatogram
peak
How can compounds be identified in GC?
retention times can be compared to known components
Define peak integrations
the areas under each peak
How can you determine the concentrations of components in the sample, in GC? Give a 4 step method
compare peak integrations to values obtained from the standard solutions of each component:
1) prepare standard solutions of known concentrations of the compound being investigated
2) obtain gas chromatograms for each
3) plot a calibration curve of peak area against concentration = external calibration
4) obtain a gas chromatogram of the compound being investigated under the same conditions + use the calibration curve to measure the concentration of the compound
Give the chemical test and observation for an alkene
+ bromine water dropwise
orange -> colourless
Give the chemical test and observation for a haloalkane
\+ silver nitrate, ethanol water bath at 50 degrees C Cl -> white ppt Br -> cream ppt I -> yellow ppt
Give the chemical test and observation for a carbonyl
+ 2,4-DNP
orange ppt
Give the chemical test and observation for an aldehyde
+ Tollens’ reagent and warm
silver mirror
Give the chemical test and observation for an alcohol (primary/secondary) or aldehyde
+ acidified potassium dichromate (VI) and warm in a water bath
orange -> green
Give the chemical test and observation for a carboxylic acid
+ aqueous sodium carbonate
effervescence
Define nuclear magnetic resonance (NMR) spectroscopy
technique using a very strong magnetic field and radio frequency radiation so that the nuclei of some atoms absorb radiation. the energy for the absorption can be measured and recorded as a NMR spectrum
Name two types of NMR
proton NMR and carbon-13 NMR
When is nuclear spin significant?
when there is an odd number of nucleons (protons and neutrons)
Describe resonance and how it can occur
when the nucleus rapidly flips between two spin states
the nucleus has 2 different spin states which have different energies. with the right combination of a strong magnetic field and radio frequency radiation the nucleus can absorb energy
What uses the same technology as an NMR
a MRI
What do the e- surrounding the nucleus do for NMR to take place?
shift the energy and radio frequency
What can frequency shift be measured on?
chemical shift δ, in ppm
a scale that compares the frequency of an NMR absorption with the frequency of the reference TMS at δ = 0ppm
What is tetramethylsilane (TMS), (CH3)4Si used as?
the standard reference chemical against which all chemical shifts are measured
What is the amount of chemical shift determined by and why?
the chemical environment, especially the presence of nearby electronegative atoms.
depending on the chemical environment, NMR requires a different energy and frequency, producing absorption peaks at chemical shifts
Give a 5 step method for running a NMR spectrum
1) sample dissolved in solvent, placed inside a narrow NMR tube with a small amount of TMS and placed inside the NMR spectrometer
2) spun to even out any imperfections inside the NMR spectrometer
3) spectrometer zeroed against TMS standard + sample given a pulse of radiation containing a range of radio frequencies whilst maintaining a constant magnetic field
4) absorptions of energy resulting from resonance are detected and displayed on a computer screen
5) after analysis the sample can be recovered by evaporation of solvent
Why is a deuterated solvent often used in NMR?
common solvents contain C and H atoms which will produce a signal in the spectra
in a deuterated solvent the 1H atoms are replaced by 2H atoms (deuterium) which produce no NMR signal in the frequencies used
What is done in response to deuterated trichloromethane (a common solvent) still producing a peak in carbon-13 spectra?
a computer will usually filter out the peak
In carbon-13 NMR spectroscopy, the number of peaks indicate…?
number of different carbon environments
In carbon-13 NMR spectroscopy, the chemical shift indicates…?
types of carbon environment present
What is the chemical environment of a carbon atom determined by?
the position of the atom within the molecule
Carbon atoms bonded to different atoms/groups have _ environments and will absorb at _ chemical shifts
different
different
If two carbon atoms are positioned symmetrically within a molecule, then they are _ and have _ chemical environment. What implication does this have?
equivalent
same
They will then absorb radiation at the same chemical shift and contribute to the same peak.
In proton NMR spectroscopy, the number of peaks indicate…?
number of different proton environments
In proton NMR spectroscopy, the chemical shift indicates…?
types of proton environments present
In proton NMR spectroscopy, the integration traces / ratio numbers of relative peak areas indicate…?
relative number of each type of proton
In proton NMR spectroscopy, the spin-spin splitting pattern indicates…?
number of non-equivalent protons adjacent to a given proton
What 3 factors may move a peak outside of the normal ranges?
solvent
concentration
substituents
If two or more protons are equivalent, they will absorb at _ chemical shift, _ the size of the peak
the same
increasing
(look for any plane of symmetry)
Protons of different types have _ chemical environments and are _ - they absorb at _ chemical shifts
different
non-equivalent
different
(look for any plane of symmetry)
What does the NMR spectrometer measure?
the area under each peak as an integration trace
What is an integration trace shown as?
an extra line on the spectrum or as a printed number of the relative peak areas
A proton NMR peak can be split into sub-peaks or splitting patterns. How is this caused?
by the proton’s spin interacting with the spin states of nearby protons that are in different environments
Define spin-spin coupling / spin-spin splitting
the splitting of a main peak into sub-peaks
the interaction between spin states of non-equivalent nuclei that results in the splitting of a signal
The number of sub-peaks is _ than the number of adjacent protons causing the splitting.
one greater
State the n+1 rule
for a proton with n protons attached to an adjacent carbon atom, the number of sub-peaks in a splitting pattern is n+1
If a proton has 0 protons attached to an adjacent carbon atom, state the following? n+1 splitting pattern relative peak areas within splitting structural feature
1
singlet
1
no H on adjacent atoms
If a proton has 1 proton attached to an adjacent carbon atom, state the following? n+1 splitting pattern relative peak areas within splitting structural feature
2
doublet
1:1
adjacent CH
If a proton has 2 protons attached to an adjacent carbon atom, state the following? n+1 splitting pattern relative peak areas within splitting structural feature
3
triplet
1:2:1
adjacent Ch2
If a proton has 3 protons attached to an adjacent carbon atom, state the following? n+1 splitting pattern relative peak areas within splitting structural feature
4
quarter
1:3:3:1
adjacent CH3
When you are analysing spin-spin splitting, what are you seeing?
the number of hydrogen atoms on the adjacent carbon atom
Describe a common splitting that results in a heptet (7) splitting pattern
CH(CH3)2
where a CH proton has 6 protons on the adjacent carbon atoms
Sometimes, adjacent protons have different environments so resulting splitting would show as a…?
multiplet
Why does spin-spin coupling occur in pairs?
because each proton splits the signal of the other
Why is proton exchange used?
to identify OH and NH protons which are otherwise difficult to assign
How is proton exchange carried out?
by running a second NMR spectrum with a small amount of deuterium oxide, D2O, added to the mixture (shaken) before
deuterium exchanges and replaces OH and NH protons in the sample and does not absorb in the chemical shift range so OH/NH peaks disappear
What can you find out with elemental analysis?
percentage composition by mass -> empirical formula
What can you find out with mass spectra?
M+ peak -> molecular mass
fragment ions -> determine parts of the molecule
What can you calculate with an empirical formula and molecular mass?
molecular formula
What can you find out with infrared spectra?
absorption peaks -> bonds present and functional groups
What can you find out with NMR spectra?
chemical shifts of peaks -> number and types of carbon and hydrogen atoms
splitting patterns -> order of atoms within molecules
Explain why deuterated dimethylsulfoxide, (CD3)2SO, is used as a solvent in NMR rather than (CH3)2SO
no peaks from interfering protons
What information can be deduced about a compound from an integration trace in proton NMR?
area under graph measured
number of protons present
