Analytical chemistry

Chromatography

Solvent (mobile phase)moves up a piece of absorbent paper(water trapped between the cellulose fibres as stationary phase)
Substances have different affinities for solvent and for water, they move at different rates over paper
Rf value (retardation factor) are calculated-same T & solvent
Coloured substances can be seen directly
Sometimes sprayed with chemicals that forms coloured compound on the chromatogram,ie amino acids: bluish spots in ninhydrin spray

Rf value

Two way chromatography

Two or more components in mixture have similar Rf in a solvent
The spots will overlap & poor separation(identification of amino acid)
Paper chromatography followed by is rotated by 90 O and re-run in a different solvent, separation takes place
Calculate Rf value from the 2nd solvent front

Partition coefficient

Solute between two solvents
In paper chromatography, the different partition coefficient corresponds to their relative solubility in two solvents
the mobile phase is the solvent chosen
The other solvent is the solvent trapped in the paper’s structure (stationary phase)
Greater the relative solubility in the mobile phase, the faster the rate of movement

Thin layer chromatography(TLC)

The stationary phase is a solid that absorbs solute molecules onto its surface
Stationary phase: Usually alumina (Al2O3) or silica [solid stationary phase]on solid support
Mobile phase: polar or non polar solvent
Made into a slurry with water and spread onto a microscope slide
Then put into an oven, dries out into a solid white coating on the glass
Chromatogram is made similar way as paper chromatography
Polar molecules have a greater attraction for a polar solid used as the stationary phase and adsorbed more strongly
They travel more slowly up the thin layer of alumina/silica and separation occurs
So, it depends on the interaction with stationary phase and relative solubility to the mobile phase

TLC

TLC is normally described as adsorption chromatography
Some partioning happens if water is present
Both dried alumina and silica can become rehydrated
When this happens, water also acts as a partitioning stationary phase together with the adsorbing stationary solid phase
TLC is quicker than paper chromatography
Can be used for smaller samples(forensic science)
Cannabis-stationary phase is silica sprayed with AgNO3 solution,which is dried. Mobile phase is methylbenzene(non-polar)

Gas liquid Chromatography(GLC)

Gases sample(gas/liquids/solids) enters the column(contains stationary phase and the sample is moved through by inert carrier gas)
Stationary phase: high boiling non polar liquid on solid support
Mobile phase: inert gas/unreactive gas
All conditions must be controlled to make comparison with the database(same carrier gas, flow rate, stationary phase and temperature)
The detector record the retention times (how long to pass through the column) based on the interaction between the stationary phase

Limitations of GLC

Similar compounds will have similar retention times
Newly discovered compounds will not have a match in the database

Determination of the % composition of mixture by GLC

Component peaks are first identified
Then area of each is measured
The peaks are triangular in shape, so the area is approximately the area of a triangle
GLC measures the area of the peak automatically and can print the results with the chromatogram
If the peaks are very narrow/similar base widths, then the peak height may be used instead of peak area to estimate the proportion of components in a mixture

Use of GLC

Testing for steroids in competing athletes
Testing for fuels used in formula One motor racing
Medical diagnosis in analysis blood samples to determine the percentages of dissolved oxygen, nitrogen, carbon dioxide and carbon monoxide in blood samples
Combined with mass spectrometry to separate and rapidly identify components in mixture

Proton nuclear magnetic resonance

Nucleus of each hydrogen(single proton) atom behaves like a tiny magnet
The proton can spin and create magnetic field
The sample is put in magnetic field
The protons either line with the field or spin in opposite direction
Tiny difference in energy by opposite spinning proton
The difference corresponds to the energy carried by waves in the radiowave range of the electromagnetic radiation spectrum
In NMR, the nuclei ‘flip’ between two energy levels or gets promoted due to the chemical environment of the proton or the strength of the magnetic field
Only atoms with mass number is an odd number (1H or 13C) absorb energy in the range of frequencies that are analysed

Reference compound

Reference compound= tetramethylsilane (TMS)
TSM is a inert, volatile liquid that mixes well with organic compounds
CH3OH, 1H atoms are in two different molecular environments
H atom is –CH3 and H atom in –OH
The energy absorbed by the H in –CH3 is different from the energy absorbed by H in –OH
Magnetic field is changed because easier than changing wavelength of radiowaves
As magnetic field is changed, the H nuclei in different environments flip at different field strengths
The different field strengths are measured using reference compound, value 0

Chemical shift

TMS- all H atoms are in the same environment
TMS gives only one sharp absorption called a peak and this peak is higher than most of other protons
All other absorptions are measured by their shift away from TMS line on the NMR spectrum
This is called by chemical shift and measured in ppm units

Low resolution NMR

Single peak for each non-equivalent hydrogen atom
Ethanol(CH3CH2OH) has three peaks H atoms in –OH, -CH2 and –CH3
Heights of peak vary
The area under each peak tells us the relative number of equivalent H atoms responsible for a particular chemical shift
Largest peak from –CH3 hydrogen atoms
Middle peak from –CH2 hydrogen atoms
Smallest peak from –OH hydrogen atoms

High resolution NMR

we can see that the peak at about 1.2 ppm is caused by the -CH3 hydrogen atoms (range 0.7–1.6 ppm)
the peak at about 3.7 ppm corresponds to -CH2 -hydrogen atoms (range 3.3–4.3 ppm)
and the peak at about 5.4 ppm is due to the -OH hydrogen atom

High resolution NMR

Chemical shifts are given in ranges
Ranges overlap
In some molecules, heavy shielding of hydrogen nuclei by lots of electrons in surrounding atoms, peaks are shifted beyond their usual range
High res NMR is useful for those cases and gives more info to interpret
Peaks that appear as one signal on low NMR are often made up of cluster of grouped peaks
This is because the magnetic fields generated by spinning nuclei interfere slightly with the neighboring nuclei (spin-spin coupling)
The exact splitting depends on the number of hydrogen atoms on the adjacent carbon atom/atoms

The CH3 peak is split into three because there are two 1H ions on the adjacent CH2 group, the formula is (n+1) as n=2 so the peak is a triplet(3).
The CH2 peak is split into four because there are 3 1H ions on the adjacent CH3 group
The OH peak is a singlet because the 1H ions is constantly being exchanged with 1H ions of other ethanol molecules or water. This results in one average peak being produced.
The number of H above the peak represents the number of hydrogen in the same environment and the number of split peaks represent the number of hydrogen from different environments close to it.

Identifying OH/NH signal in NMR

-OH appears as single peak because it exchanges very rapidly with protons in any traces of water/ acids
The hydrogen atoms are coloured above
The exchange takes place so rapidly until the signal becomes single peak
Also happens in amine and amides –NH- group
If heavy water is used, -OH / -NH- disappear, therefore the H in –OH and –NH- are refered as labile proton
Heavy is also known as D20 with 2H ions will not be detected as only atoms with mass of odd numbers can be detected

Carbon 13 NMR

Another tool to analyse
C-12 isotope not used because even number
No signal in NMR
Proton NMR- reference TMS
Appears to be discrete vertical lines without splitting
The heights are not proportional to carbon atoms present • Solvent: CDCl3 small signal near 80ppm (ignored

Mass spectroscopy

mass to charge ratio, the charge is always one

M+1 peak

Always a small peak after the molecular ion peak
One of the carbon atom is carbon-13
1.10%

M+2 and M+4

If the M+2 peak is one third of the height of the M peak, this suggests the presence of one chlorine atom per molecule
If the M+2 is peak is the same as the height of the M peak, this suggest the presence of one bromine atom per molecule
Ratio of M:M+2:M+4 is 1:2:1

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