chromatography

what can high resolution mass spectrometry be used for?

• High resolution mass spectrometry can be used to deduce / confirm molecular formulae 

• High resolution mass spectrometers provide data accurate to four, or sometimes more, decimal places

• This means that a compounds molecular formula can sometimes be deduced by using atomic masses accurate to four decimal places

what are the three analytical technique used to separate the components of a mixture

• simple (inc thin layer)

• liquid and HPLC

• Gas

how simple chromatography seperates compounds?

• chromotography works because a mobile phase sweeps over the stationary phase

• the components seperate as the solvent moves over the paper, due to different affinities to the mobile and stationary phase

• a greater affinity for the stationary phase means the component moves up the paper more slowly

how to calculate retardation factor?

distance moved by component/ distance moved by solvent

results compared to book values

problems with chromotography

• similar substances have similar Rf values

• new (novel) structures have nothing to be compared with

• It can be difficult to manage the conditions, e.g. temperature and pressure

use of thin layer chromatography

• Thin-layer chromatography (TLC) is a technique used to analyse small samples via separation

  • For example, we could separate a dye out to determine the mixture of dyes in a forensic sample

how does thin layer chromatography

• a thin layer of an adsorbent material such as silica gel or alumina are supported on a glass/plastic sheet

• The solute molecules adsorb onto the surface

• Depending on the strength of interactions with the stationary phase, the separated components will travel particular distances through the plate

• The more they interact with the stationary phase, the more they will 'stick' to it

what is stationary phase in thin layer chromatography?

• solid silica on a plastic or glass plate

what is mobile phase on TLC

liquid solvent eg water or organic

• Flows over the stationary phase

• It is a polar or non-polar liquid (solvent) that carries components of the compound being investigated

• Polar solvents - water or alcohol

• Non-polar solvents - alkanes

how to locate spots on TLC if sample components are not colored

• To locate the spots we can use:

  • UV light

  • Ninhydrin (carcinogenic)

  • Iodine vapour

conducting a TLC analysis

• Step 1:

  Prepare a beaker with a small quantity of solvent

• Step 2:

  On a TLC plate, draw a horizontal line at the bottom edge (in pencil)

  This is called the baseline

• Step 3:

  Place a spot of pure reference compound on the left of this line, then a spot of the sample to be analysed to the right of the baseline and allow to air dry

  The reference compounds will allow identification of the mixture of compounds in the sample

• Step 4:

  Place the TLC plate inside the beaker with solvent - making sure that the solvent does not cover the spot - and place a lid to cover the beaker

  The solvent will begin to travel up the plate, dissolving the compounds as it does

• Step 5:

  As solvent reaches the top, remove the plate and draw another pencil line where the solvent has reached, indicating the solvent front

  The sample’s components will have separated and travelled up towards this solvent front

how to carry out column chromatography?

• The sample mixture is dissolved in the solvent and introduced at the top of the column

  • A pipette is usually used to carefully add the dissolved sample to the top of the column

  • add the sample without disturbing the surface of the column so that the sample runs from one level through the column

• Once the sample has been added, more solvent (eluent) is added on top of the sample

• As the solvent runs through, fresh solvent is added to the top of the column so that it does not dry out

• The sample flows through the column via gravity

how column chromatography works?

• the four components leave the column separately. The component with the greatest attraction / affinity to the stationary phase takes the longest time to flow through the column. Their retention times are measured

• once through each sample is analysed using a mass spectrometer

how can process of column chromatography be sped up?

• This process can be sped up by pushing the sample and mobile phase through the column

• In school laboratories, this can be achieved by attaching a gas syringe to the top of the chromatography column

• In industrial / research laboratories, this is achieved by attaching an air line to the top of the chromatography column

what is retention time

the time taken from injection to detection

what is stationary phase for column chromatography

solid alumina or silica gel, packed into the HPLC tube

what is mobile phase in column cheomtography

usually an organic solvent or water

what is a high performance liquid chromatography?

• a more elaborate column chromatography

• retention times are recorded for each component in the mixture

main difference between HPLC and column chromatography

• The column doesn't work via gravity, the sample is pumped through by the solvent

• The particles of the stationary phase are much smaller, leading to greater separation of compounds

• There is a detector at the end of the column which measures retention time

• HPLC is automated so the results are obtained quicker 

• The HPLC equipment typically includes a computer which allows for quicker analysis and comparison of results against known compounds in a database 

how gas chromatography works?

• after injection,into the column through a self-sealing disc the sample vaporises and the vaapour formed in arried throught eh statinary phase using the inert-gas mobile phase

• the mobile carrier gas carries the mixture through the column

• Once sample molecules reach the detector, their retention times are recorded

• The retention times are recorded on a chromatogram where each peak represents a volatile compound in the analysed sample

  • The relative sizes (i.e. areas) of the peaks are related to how much of each compound is present in the mixture

• components more strongly attracted to the stationary phase have longer retention times

• Retention times are then compared with data book values to identify unknown molecules

what is G.c- M.S

Mass spectrometry is combined with gas chromatography to roduce a more powerful analytical tool

• G.C- seperates the components

• M.S- identifies each component ( molecular ion for Mr and fragments for detailed structure)

• Concentrations as low as 1×10^-9 mldm^-3 can be detected

uses of gas chromotography and M.S and HPLC

• Provide forensic evidence

• Drug testing, particularly in sports

• Analysis of environmental pollution

• Detecting explosives in baggage

Problems with using GC-MS for drug detection

• Anabolic steroids can be used by athletes to improve muscle growth, increase production of red blood cells and strengthen bones by increasing their density

• They are also used to treat conditions such as osteoporosis, anaemia and some cancers

• One high profile anabolic steroid is nandrolone which is metabolised into a similar chemical called 19-norandrosterone

  • Competitors in the Olympic Games are routinely urine tested for the presence of 19-norandrosterone

  • A urine content above 2 nanograms per cm³ (0.000000002 g per cm³) is a positive test and can result in the athlete being disqualified and risking further sanctions

  • There is debate about nandrolone due to its genuine medical applications and the fact that it may be in some nutritional and dietary supplements

stationary phase in gas chromatography?

• microscopic liquid film on a solid support

mobile phase in gas chromatography?

• inert carrier gas eg nitrogen, Ar, He

what does NMR require?

• low energy radio frequency radiation (radio waves)

• a strong magnetic field (electromagnet)

what happens to nucleons (protons and neutrons) in the nucleus?

• Nucleons pair up and spin in opposite directions, in the nucleus

• protons spin interact with the spin sttes of nearby protons that are in diffrerent environments. his can provide information about the number of protons bonded to adjacent carbon atoms. The splitting of a main peak into sub-peaks is called spin-spin splitting

• even number of nucleons- their spins cancel each other out and so cannot be detected

• odd number of nucleons- the unpaired nucleon produces a small residual spin, that generates a magnetic field

• atoms eg isotopes of atoms with odd mass numbers usually show signals on NMR

how NMR works?

• when placed in a magnetic field, the nucleon can flip and spins in the opposite direction when they absorb energy from the radiowaves

• the energy absorbed is measured and used to plot a graph

• Carbon-12 cannot be detected with 6 P and 6N, it has an even number of nucleons

• Carbon-13, 6P and 7N or hydrogen-1 (1P and 0N) are mainly used, but 15N, 19F, and 31P are also used

what is the solvent in NMR?

the sample is dissolved in a solvent that must not produce a signal

what is the references for NMR

• all NMR spectra are compared to a reference molecule- tetramethysilane, TMS

• TMS shows a single sharp peak on NMR spectra, at a value of zero

• TMS is also used because it is:

  • Non toxic.

  • Does not react with the sample.

  • Easily separated from the sample molecule due to its low boiling point.

  • Produces one strong, sharp absorption peak on the spectrum.

what is chemical shift

describes the displacement of the signal for the sample, to the reference peak

• Identical atoms (carbon or Hydrogen) in the same environment absorb the same frequency of radiation

features of H1 NMR spectrum

• NMR spectra shows the intensity of each peak against their chemical shift

• The area under each peak gives information about the number of protons in a particular environment

• The height of each peak shows the intensity / absorption from protons

• A single sharp peak is seen to the far right of the spectrum

  • This is the reference peak from TMS

  • Usually at chemical shift 0 ppm

what are moelcular environmnts

• ¹H nuclei that have different neighboring atoms (said to have different chemical environments) absorb at slightly different field strengths

• The difference environments are said to cause a chemical shift of the absorption

• Different types of protons are given their own range of chemical shifts

• Each individual peak on a ¹H NMR spectrum relates to protons in the same environment

• Protons in the same chemical environment are chemically equivalent

relative intensities of each splitting pattern

• A doublet has an intensity ratio of 1:1 – each peak is the same intensity as the other

• In a triplet, the intensity ratio is 1:2:1 – the middle of the peak is twice the intensity of the 2 on either side

• In a quartet, the intensity ratio is 1:3:3:1 – the middle peaks are three times the intensity of the 2 outer peaks

how splitting patterns occur

• The number of sub-peaks is one greater than the number of adjacent protons causing the splitting

  • For a proton with n protons attached to an adjacent carbon atom, the number of sub-peaks in a splitting pattern = n+1

• Splitting patterns must occur in pairs, because each protons splits the signal of the other