Analytical techniques

Resonance

• all covalent bonds are able to vibrate in a number of different ways

• The frequency of vibration occurs in the infra-red region of the electromagnetic spectrum

• if an organic molecule is irradiated with infra-red energy that matches the natural vibration frequency of its bonds, it absorbs some of that energy and the amplitude of vibration increases

Stretching

When a bond absorbs infrared radiation and therefore changes its length

Transmittance

A value in the IR spectrum that shows the amount of radiation absorbed at a particular frequency

IR spectroscopy

• a technique used to identify compounds based on changes in vibrations of atoms when they absorb IR of certain frequencies

• The machine irradiates the sample with the IR radiation and then detects the intensity of IR radiation absorbed by the molecule

• IR energy is absorbed only if a molecule has a permanent dipole that changes as it vibrates - so symmetrical molecules are IR inactive

• The resonance frequency is the specific frequency at which the bonds will vibrate

• Rather than displaying frequency, an IR spectrum shows a unit called a wave number → this is the reciprocal of the wavelength And characteristic absorptions can be matched to specific bonds in molecules so functional group can be determined

Fingerprint region

• the region below 1500cm-1 is the fingerprint region and is unique to every molecule

• The peaks in this region represent the complex vibrational interactions that occur between different bonds within a molecule

• The value of this region is being able to compar IR spectrum to a known compound from a database and coming up with an exact match

• All members of the same series will show the same type of bonds present but no two molecules will have the same fingerprint region

Infrared radiation

Has frequencies below that of red light

Stages of mass spectrometry

• vaporization

• Ionization

• Acceleration

• Deflection

• Detection

Mass spectrometry

A technique that gives you information about the mass of a molecule and its fragments

The formation of molecular ions

When the vaporized organic sample passes into the ionization chamber of a mass spectrometer, it is bombarded by a stream of high energy electrons. These electrons have high enough energy to knock an electron off an organic molecule to form a positive ion. This ion is called the molecular ion or sometimes the parent ion

Analysis of mass spectrometry

these current values are then used in the combination with the flight times to produce a spectra print-out with the relative abundance of each isotope displayed

during the ionisation process, a 2+ charged ion may be produced, these ions are affected more by the magnetic field producing a curved path of smaller radius, therefore the m/z ratio is halved and this can be seen on the spectra as a trace at half the expected value

Fragmentation of the molecular ion

• The molecular ions are energetically unstable and some will break into smaller fragments, in the simplest case the molecular ion breaks into two parts - one of which is another positive ion and the other is an uncharged free radical

• The uncharged radical wont produce a line on the mass spectrum and they will get lost in the machine and then removed by vacuum pump

• The ion will travel through the mass spectrometer just like any other positive, being accelerated by the electric field and deflected by the magnetic field

• The lighter ions are deflected more than the heavier ions and so a spectrum of ions of different mass/charge ratios is produced

The molecular ion peak and the base peak

In the mass spectrum of pentane, the line produced by the heaviest ion passing through the machine is due to the molecular ion

The tallest line in the stick diagram is called the base peak. This is usually given an arbitrary height of 100 and the height of everything else is measured relative to this, it is the tallest peak because it represents the most common fragment ion to be formed - either because there are several ways in which it could be produced during fragmentation of the parent ion, or because it is a more stable ion

Using mass spectroscopy to find the Mr of compounds

One of the peaks shows a unique molecular formula, so there is only one combination of atoms that has that Mr

The low resolution of mass spectrometry does not allow us to determine the Mr of compounds but high resolution spectrometry tells us the specific molecular formula of a compound

Using mass spectroscope to find the Mr of compounds

The first species formed is called the molecular ion

The signals for the molecular ion gives the Mr of the compound - this is the peak with the greatest m/z

Using mass spectroscopy to find the molecular formula of compounds

• high resolution mass spectrometers measure the m/z values to enough accuracy to find the molecular formula

• If more than one compound has the same Mr, high resolution spectrometer can show us what molecular formula they have exactly

General information about NMR

• Nuclei have spin

• If the nucleus has an odd mass number, its spin will produce a magnetic field - for example, H² will not produce a magnetic field but H^1 will

• If/when an external magnetic field is applied, the m.f. Of the nuclei can align with or against it

CNMR

• the spectrum gives signals (peaks) for each different chemical environment that can identified by their chemical shift

• Does not observe splitting and the peak height is not related to the relative concentration of each carbon environment

• Has a much larger chemical shift range

HNMR

• solvent = TMS (non-toxic, inert, low b.p., 1 signal at 0ppm)

• Environments

High resolution mass spectrometry

• high resolution mass spectrometry is a much more sensitive form of mass spectrometry which allows the Mr of a substance to be determined to several decimal places

• precise atomic masses can then be used to calculate the molecular formula of the compound being tested

• after determining molecular formula, you can predict possible structures of the compound

Introduction to NMR

• NMR is an analytical technique that allows the structure of a molecule to be determined by analysing the energy of each bond environment

• each bond environments within a molecule absorb different amounts of energy so they are displayed as different peaks on a spectra

CNMR

• analyses different carbon environments in a molecule

• carbon environments that are near to oxygen atoms have sigma values that are shifted to the right, this is because oxygen is very electronegative and changes the bond environment and affects how it absorbs energy

Molecule symmetry

• molecules that have symmetry may display fewer peaks than the number of carbon atoms in the molecule

• therefore it is important to look at the given molecular formula of the compound in order to decipher its displayed structure

HNMR

• the different hydrogen environments in a molecule are analysed and displayed as peaks on a spectrum, these peaks are also measured against the TMS standard

• the sample being analysed must be dissolved in a non-hydrogen containing solvent so that the solvent doesn’t produce any peaks on the spectrum

• CCl4 is a common solvent used along with deuterated solvents containing deuterium

• the height of the peaks show the relative intensity of each value, and they correspond to the number of hydrogens in that certain environment within a molecule

splitting patterns

• the peaks of a HNMR spectra also inform where each environment is positioned within the molecule

• peaks are split into a small cluster, with smaller peaks indicating how many hydrogens are on the adjacent carbon atom within the molecule

• the smaller peaks are a splitting pattern and follow an ‘n+1’ rule, where n is the number of hydrogens on the adjacent carbon

• singlet - 0H on adjacent C, doublet = 1H on adjacent C, triplet = 2H on adjacent C etc.

Chromatography

• Chromatography is when a sample is dissolved in a solvent and washed through a stationary phase by a mobile phase called the eluent

• substances that are more soluble in the eluent and less adsorbed by the stationary phase move faster through the apparatus

Thin layer chromatography

Stationary phase - silica gel or aluminium oxide

Mobile phase - suitable eluent (liquid)

Method:

• draw the pencil line from the bottom of the stationary phase

• sample is dissolved in solvent and spot it onto the plate

• plate is dipped into suitable eluent

• plate is left in a sealed container until eluent has reached near the top of the plate

• take plate out and draw solvent line in pencil

• if the unknown is amino acid, then spray plate with ninhydrin and heat

High-performance liquid chromatography

• it is used in the pharmaceutical industry to

• separate a mixture of drugs and check their purity

Stationary phase - column packed with solid uniform particle size

Mobile phase - liquid eluent → force through the column under high pressure

Method:

• sample is added to a suitable solvent and added to the top of the column

• liquid eluent is forced through the column under high pressure

• different substances have different strengths of interactions between the stationary and mobile phases therefore pass through the detector one after another

Conditions - room temp → to not decompose biological samples

Retention time - time taken for a component in the sample mixture to the column

Gas chromatography

• used for the analysis of complex molecules that do not decompose on heating

Stationary phase - liquid adsorbed onto the surface of an inert solid

Mobile phase - inert gas

Method:

• gas is injected into chromatography column in a thermostatically controlled oven

• sample evaporates and forced through the column by the flow of inert gas

• the rate at which samples travel through the column is dependent on the interactions with both phases

• detection measures the thermal conductivity of the gas exiting the column