Chromatography and NMR Flashcards
what is chromatography?
is a technique used for separating the components of a mixture on the basis of difference in their affinities for a stationary and for a moving phase
why is chromatography used?
provides and important method of separating and identifying components in a mixture
in chromatography, there is always a fixed..?
stationary phase through which passes a moving phase containing the mixture to be separated
What does separation in chromatography depends on?
the balance between solubility in the moving phase and retention in the stationary phase
Chromatography can either be..?
Analytical or Preparative
what is analytical chromatography?
Analytical chromatography involves the analysis of small amounts of material
what does analytic chromatography try to identify?
tries to identify and measure the relative amounts of various components present in the mixture
How is the resulting chromatogram compared?
the resulting chromatogram is compared with a known ‘standards; (chromatograms of specific substances of a ,known concentration
What is Analytical chromatography used in?
used routinely in sport drug testing
What is preparative chromatography?
is a larger scale process used to separate a mixture of products at the end of a chemical reaction
Chromatographic separation falls into two categories, what are the two categories?
- Partition chromatography and adsorption chromatography
What is partition chromatography?
- separation depends on the balance between a solutes solubility in the moving phase and retention in the stationary phase
- Stationary phase - Thin, non-volatile liquid film on the surface of an inner solid or fibrous matrix (e.g paper - cellulose fibres)
- moving phase - liquid or gas
- solute molecules equilibrate (partition between 2 phases)
what is Adsorption chromatography?
- strongly adsorbed molecules travel more slowly in the moving phase than those that are only weakly adsorbed
- stationary phase - solid (e.g alumina- aluminium oxide, silica - silicon dioxide)
- moving phase - liquid or gas
- separation occurs when solute molecules become attached to adsorption sites on the solid
What are different chromatographic techniques?
- paper chromatography (Similar to TLC)
- thin-layer chromatography (TLC)
- column chromatography (CG)
- Gas chromatography (GC)
what is Paper Chromatography?
Used in schools to separate the different dyes in an ink solution or the different food additives (colours) in sweets.
What is a chromatogram?
a chromatogram is a pattern of separated substances obtained by chromatography
How can the number of substances be identified?
the number of substances present in a mixture is given by the number of spots on the chromatogram
How can compounds be identified?
Using their Rf values (under standard conditions)
How are mixture patterns compared with?
Known standards on the same chromatogram
How can Rf values be calculated?
Distance moved by substance/ distance moved by solvent front
Why is the Rf value different for the same substances?
The Rf value for a substance is different in different solvents
When is complete separation only possible?
sometimes complete separation is only possible by rotating the paper through 90°, changing the solvent and repeating the process (2D- paper chromatography)
What is Thin-layer Chromatography?
a plate is coated with a thin layer of a solid and a solvent moves up the plate
when is TLC used?
used routinely in laboratories by bench-top scientists -
Explain how TLC differs from Paper chromatography?
similar technique to paper chromatography but the stationary adsorbent phase is usually a thin layer of silica or alumina coated on an aluminium sheet (plate). And an organic solvent e.g ethanol is usually used (moving phase)
In TLC, the mixtures are spotted on…?
a pencil baseline and the resulting chromatogram can be used to identify substances using Rf values
TLC chromatograms are usually viewed under..? which reveals..?
Ultra-violet light and colourless chemicals are revealed using a UV lamp
TLC is used to monitor?
the course of organic reactions
What is Column Chromatography ?
in CC, a column is packed with a solid and a solvent moves down the column
when is this used?
used routinely by top bench scientists
Explain how Column chromatography is carried out?
- the stationary adsorbent phase is usually finely-divided silica gel or alumina house in a vertical glass tube (column)
- the moving phase (usually the organic solvent) is called the eluent
- the mixture solution (compounds dissolved in an organic solvent) is added to the top of the column followed by enough fresh solvent to wash the components down the column
which substances take the longest time to flow through the column?
the most strongly adsorbed substances take the longest time to flow through the column (longer retention time)
what is the retention time?
the retention time is the time each component remains in the column
the more polar a substance, the..?
the more polar the molecule in the mixture, the greater the retention time
How is the solution collected when it exits the column?
- as the solution exits the column it is normally collected as a series of fractions in small beakers
- the fractions that you want (e.g the main product) can then be processes and analysed further
How do you get the compound that you want?
to get the compunds that you want you need to evaporate the solvent and recrystalise
what is column chromatography a good method for?
column chromatography is a great method for separating and purifying a mixture of organic products after a chemical reaction (preparative chromatography)
How can the process be speeded up?
by forcing the solvent through the system under pressure (flash chromatography)
what does separation by column chromatography depend on?
depends on the balance between solubility in the moving phase and retention in the stationary phase
What is Gas Chromatography?
in GC, a column is packed with a solid (or a solid coated by a liquid), and a gas is passed through a column under pressure at high temperatures
What is this technique used by?
This is a powerful technique used by skilled analytical chemists and chemistry technicians
What can GC be used to separate?
mixtures of volatile liquids
Explain how Gas chromatography is carried out?
- uses carrier gas (helium) as the moving phase
- the stationary phase is often an inert powder coated with a film of non-volatile liquid
- the powder is then packed into a long narrow column
- a vapourised mixture sample is injected at the entrance to the column and carried down the column by the carrier gas (the moving phase)
Each component has a..?
characteristics retention time
what is a major importance in the separation process?
the solubility of the different mixture components in the non-volatile liquid film (stationary phase) is of major importance in the separation process
what components present in the mixture will have a longer retention time?
a component that is highly soluble will have in the non-volatile liquid film (stationary phase) takes longer to leave the column (longer retention time)
why does Gas chromatography produce a peak on a graph?
a detector monitors as they exit the column - so that each substance appears as a peak on a cart
what does the number of peaks give?
the number of peaks gives the number of substances present in the mixture
the chart provides the peak retention time to enable..?
Identification
under standard conditions, each substance will have..? And we can use this to..?
a unique retention time - so you can use the retention time to identify the components of the mixture
what are retention times compared with..?
standards to identify substances
the peak area is proportional to..?
the amount of substance present
What can Gas chromatography measure?
can measure and separate tiny quantities of closely related compounds
what is GC usually connected to?
a mass spectrometer
why is it usually connected to a mass spectrometer?
mass spec is used to analyse each component separated by gas chromatography
what is this combined technique called?
GCMS
Explain what this enables?
this enables positive identification of substances through their Mr. substances with similar GC retention times can be distinguished by GCMS (they will have different Mr. values
NMR Spectroscopy
What does NMR stand for?
Nuclear magnetic Resonance (NMR) spectroscopy
what is NMR used for?
is used to determine the structure of a molecule. It provides information on the different environments of atoms in molecules.
what are the two main types of NMR?
- Carbon 13 NMR (13C NMR)
- Proton NMR (1H NMR)
what does carbon 13 NMR tell you?
tells us how carbon atoms in a molecule are arranged
what does proton NMR tell you?
tell us us how hydrogen atoms in a molecule are arranged
How is a weak magnetic field made?
- if an atomic nucleus has an odd number of nucleons (protons and neutrons), then it has a nuclear spin
- the nuclear spin creates a weak magnetic field
Explain how NMR uses this magnetic field?
NMR detects how these weak magnetic fields are affected by a larger external magnetic field
why does both Hydrogen and Carbon have a nuclear spin?
- hydrogen has one proton
- Carbon 13 has
what percentage of Carbons are 13C?
1% of carbons are 13C
In which direction does the nuclei spin in?
What is it affected by
spins in random directions, However, when an external magnetic field is applied. They Align in 2 directions
what are the two directions they spin in?
- nuclei either spin in the direction of the external magnetic field or Against it.
Those that spin in the direction of the magnetic field spin, Have..?
lower energy
Why can NMR be used to identify molecules with a nuclear spin?
- NMR fires out radio waves at a specific frequency
-the nuclei that are aligned with the magnetic field absorb the energy and flip to a higher energy level - those with a higher energy can also drop to a lower energy level and emit radio waves
- initially, there is more nuclei aligned with the magnetic field, so overall more energy is absorbed than emitted
- NMR measures the amount of energy absorbed
why is there more energy absorbed than emitted?
initially, there is more nuclei aligned with the magnetic field, so overall more energy is absorbed than emitted
Rule: The energy absorbed by the nuclei is dependent on…?
the environment it is in.!!!
How can a nucleus be shielded?
What affects the shielding of amount of electron shielding?
- a nucleus can be shielded from the effects of an external magnetic field by it’s surrounding electrons
- any atoms or groups of atoms that are around/adjacent to the nucleus will affect the amount of electron shielding
Give an example of a type of atom that will affect the electron shielding?
- e.g An electronegative atom such as oxygen near the carbon atom will reduce the electron shielding on the carbon atom.
- this is because electrons are pulled further away from the carbon atom. the carbon atom thus is shielded less.
Overall, why is the effect of the external magnetic field different on each nucleus?
depending on the environment within the molecule.
- The magnetic field will be felt by the nuclei differently depending on the environment it is in.
Therefore, NMR picks up the..?
difference in absorption of energy between environments, as they absorb different amounts of energy and various frequencies.
for an atom to be in the same environment, it must be bonded to…?
an atom or group of atoms that are identical
What chemical is used as a standard, when looking at chemical shift in NMR spectra?
(TMS) - Tetramethylsilane
Draw the structure of TMS
Why do we use a standard?
As nuclei absorb different amounts of energy at different frequencies, it is difficult to measure the magnitude of those without a reference or standard chemical to measure it against.
Why is TMS specifically used as a standard?
- TMS has 12 hydrogens all in identical environments, so produces a large single peak well away from sample peaks.
- it is also inert, Non-toxic and volatile so easy to remove from your sample.
What is the chemical shift?
the difference between the TMS peak and the peaks produced by the substance under test is called the chemical shift (δ)
what is chemical shift measure in?
chemical shift is measure in Parts per million (ppm) relative to a standard (TMS)
what do we assign the peak TMS has?
δ = 0
Why will we often see a peak produced as δ=0 on a spectra?
because TMS is added to the test compund for calibration purposes
13C NMR spectroscopy
what does 13C NMR spectroscopy tell us?
Tells us how many different carbon environments there are in a sample being tested.
what does the number of peaks on a Carbon 13 NMR spectrum tell you?
the number of carbon environments there are present in a particular molecule being tested.
describe the chemical shift and electron shielding for a normal C-H bond?
electron shielding is more, lower chemical shift (will be closer to δ=0
Describe the chemical shift for A carbon with an electronegative atom like O, N adjacently attached?
- electron shielding is lower and chemical shift is higher (will be further away from δ = 0)
How can we identify chemical shifts in cyclical compounds?
look for symmetry, use line of symmetry
Describe how many chemical environments there are in Cyclohexane-1,3-diol ?
What would this look like on a graph?
- in cyclohexane-1,3-diol there are 4 different carbon environments
How else could we identify peaks on 13C NMR?
we can use data sheet
what are certain issues with using the data sheet/table?
- the chemical shifts can overlap
- δ = 190 could be carbonyl group in a ketone or aldehyde
- δ= 30 may be caused by a C-C, C-Cl, C-Br
- δ = 60 could be ester, alcohol, amine, or ether
what are the steps we can follow to interpret a 13C spectra?
- count number of peaks - this tells us n of carbon environments
- use table of chemical shifts
What steps would need to be taken in order to interpret 13C NMR spectra of Cyclical compounds? Use example in Chemistry text book with a formula of C6H4CL2
- the spectrum has 4 peaks
- all 4 peaks are between the δ = 125ppm and δ=140 ppm. Looking at the chemical shifts table, this can only be down to alkene groups or carbons in a benzene ring.
- the question tells you that the molecule is cyclic with 6 carbons, so these carbons must be in a benzene ring, as a 6 carbon cyclic molecule with 3 double bonds does not exist
- there are 3 aromatic molecules with the structure
- decipher using the peaks.
1H (Proton) NMR Spectroscopy
What is 1H NMR about?
1H NMR is all about how hydrogen nuclei react to a magnetic field.
what is each peak on the Hydrogen NMR due to?
is due to one or more hydrogen nuclei (protons) in a particular environment
what does 1H NMR Spectroscopy tell us?
tells us how many different hydrogen environments there are and how many hydrogens in each environment in the sample.
when there is a larger peak, this means there are..?
more hydrogens
when there is a smaller peak. this means there are…?
less hydrogens
what does the relative area under each peak tell us?
also tells us the relative number of H atoms in each environment
what does the numbers above each peak tell us?
area ration between hydrogens in each environment
How many peaks will there be present on the 1H NMR spectrum of 1-Chloropropanone? Predict the ratio of the areas of these peaks
- 2 peaks
- ratio = 2:3
what is a splitting pattern or spin-spin splitting?
peaks split into smaller peaks
what are the split peaks called?
multiplets
what does the number of splitting peaks correspond to?
corresponds to the number of hydrogen atoms on the adjacent carbon plus one. Called the n+ 1 rule
what does a singlet peak tell us?
0 hydrogens on the adjacent carbon
what does a doublet peak tell us?
1 hydrogen on the adjacent carbon
what does a triplet peak tell us?
2 hydrogens on the neighbouring carbon
what does a quartet ell us?
3 hydrogens on the adjacent carbon
Integration traces
what do integration traces tell us?
integration traces show area under the peak more clearly. this helps to work out the hydrogen ratio
on an integration trace, the height ratio of the trace corresponds to the..?
area ratio
Proton-free-solvents
is a sample has to be dissolved, then a solvent is needed that doesn’t contain..?
protons or any 1H atoms (so proton-free)
Why can we not use solvents that contain hydrogen?
because these peaks would show up on the spectrum and confuse things
If we can not use hydrogen based solvents what can we use?
Deuterated solvents are often used
what are deuterated solvents often used?
- Their 1H atoms have been replaced by D or 2H.
- Dueterium is an isotope of a hydrogen atom with 2 nucleons ( a proton and a neutron)
- has an even number of nucleons, so not detected by NMR.
other than deuterated solvents what can be used?
Alternative solvents like CCL4 (tetrachloromethane) can be used as a solvent as it doesn’t contain any 1H atoms either
Give examples of proton free solvents that can be used?
- CCL4
- CGCL3,
- D2O
- C6D6
DONE
