chapter 29

• What is chromatography?


  Its used to separate individual components from a mixture of substances;

  
  

  all forms of chromatography have a stationary phase and a mobile phase
• What is the stationary phase?

  Does not move;

  
  

  and may be a solid (as in thin layer chromatography, TLC)

  
  

  or

  
  

  either a liquid or solid on a solid support (as in gas

  chromatography, GC)
• What is the mobile phase?

  Moves and is normally a liquid or a gas.
• How fast does the substances move?

  Depends on:

  
  

  How soluble the compound is in the solvent;

  
  

  How much the compound sticks to the stationary phase - e.g. substance that is strongly adsorbed will move slowly;

  
  

  external variables - e.g. temp
• How does polarity affect solubility?

  If the stationary phase was polar and the moving

  phase was non- polar e.g. Hexane;

  
  

  Then non-polar compounds would pass through the column more quickly than polar compounds as they would have a greater solubility in the non polar moving phase.
• What can chromatography be used for?

  Analysis of drugs;

  
  

  It has applications in forensic use
• What are the 2 types of chromatography?

  Thin-layer chromatography (TLC);

  
  

  Gas chromatography (GC)
• What is thin layer chromatography?

  Separates compounds in a mixture;

  
  

  A mixture can be separated by

  chromatography and identified from the

  amount they have moved. (Can be used with

  mixtures of amino acids)

  
  

  A quick and inexpensive analytical technique;

  
  

  indicates how many components are in a mixture;

  
  

  This technique uses a TLC plate which is usually a plastic sheet or glass, coated with a thin layer of a solid absorbent substance - usually silica.
• How does TLC work?

  Adsorption onto stationary phase;

  
  

  Process?

  
  

  The solvent is the mobile phase e.g. ethanol;

  
  

  The adsorbent is the stationary phase. The stationary phase in TLC is the thin layer of solid e.g. silica gel or aluminium powder on a glass or plastic plate.

  
  

  The mobile phase passes over the stationary phase;

  
  

  The different components in the mixture will have different affinities for the absorbent and bind with differeng strengths to its surface.

  
  

  Adsorption is the process by which the solid silica holds the different substances in the mixture to its surface.

  
  

  A stationary phase is separated by adsorption;

  A liquid stationary phase separates by relative solubility

  
  

  Separation is achieved by the relative absorptions of substances with the stationary phase.
• Method for TLC


  Wearing gloves, draw a pencil line 1 cm above the

  bottom of a TLC plate and mark spots for each sample, equally spaced along line

  
  

  Use a capillary tube to add a tiny drop of each solution to a different spot and allow the plate to air dry.

  
  

  Add solvent to a chamber or large beaker with a lid so that is no more than 1cm in depth

  
  

  Place the TLC plate into the chamber, making sure that the level of the solvent is below the pencil line. Replace the lid to get a tight seal.

  
  

  When the level of the solvent reaches about 1 cm from the top of the plate, remove the plate and mark the solvent front with a pencil. Allow the plate to dry in the fume

  cupboard.

  
  

  Place the plate under a UV lamp in order to see the spots.

  
  

  Draw around them lightly in pencil.

  
  

  Calculate the Rf values of the observed spots

  
• Reason why you do certain steps in TLC

  Wear plastic gloves to prevent contamination

  from the hands to the plate

  
  

  pencil line -will not dissolve in the solvent

  tiny drop - too big a drop will cause different

  spots to merge

  
  

  Depth of solvent- if the solvent is too deep it

  will dissolve the sample spots from the plate

  
  

  Will get more accurate results if the solvent is

  allowed to rise to near the top of the plate but

  the Rf value can be calculated if the solvent

  front does not reach the top of the plate

  
  

  Lid- to prevent evaporation of toxic solvent

  
  

  dry in a fume cupboard as the solvent is toxic

  
  

  UV lamp used if the spots are colourless and

  not visible
• How are thin layer chromatograms analysed?

  (Each substance has its own Rf value)

  
  

  calculate the Rf value;

  
  

  Compare Rf values to those for known

  substances.

  
  

  (using the same solvent system and adsorbent)

  
  

  OR

  
  

  can be run alongside pure samples of compounds, usually without needing Rf values
• How do you calculate the Rf value?

  Distance moved by the component divided by the distance moved by the solvent front

  
  

  see page 243 TB
• Name the process by which TLC separates α-amino acids?

  adsorption
• What is gas chromatography used for?

  For separating and identifying volatile organic compounds present in a mixture
• What is gas chromatography?

  The stationary phase is a high boiling liquid absorbed onto an inert solid support.

  
  

  The mobile phase is an inert carrier gas such as helium or neon.
• What happens in gas chromatography?

  A small amount of the volatile mixture is injected into the apparatus, called a gas chromatograph.

  
  

  The mobile carrier gas carries the components in the sample through the capilliary column which contains the liquid stationary phase absorbed onto the solid support.

  
  

  The components slow down as they interact with the liquid stationary phase inside the column. The more soluble the component is in liquid, the slower it moves through the capillary column.

  
  

  The components in the mixture are separated depending on their solubility in the liquid stationary phase.;

  
  

  The compounds in the mixture reach the detector at different times depending on their interactions with the stationary phase in the column.

  
  

  The compound retained in the column for shortest time has the lowest retention time and is detected first

  
  

  see diagram page 244 TB
• What is the retention time?

  time from injection to detection;

  
  

  (The time taken for a particular compound to travel from the injection of the sample to where

  it leaves the column to the detector)
• What is a gas chromatogram?

  shows a series of peaks when detector senses something leaving tube
• Interpretation of a gas chromatogram?

  Retention timescan be used to identify the components present in the sample by comparing these to retention times for known components.

  
  

  Peak integrations(areas under each peak) can be used to determine the concentrations of components in the sample.

  The area under each peak will be proportional to the abundance of that component

  
  

  The tallest peak wont always represent most abundant; its the one with biggest area
• What factors affect retention time?

  Solubility;

  
  

  Boiling point;

  
  

  Temperature of the gas chromatography instrument - higher temp, move quickly, shorter retention time
• Why is difficult to distinguish substances sometimes based on retention times?

  Some compounds have similar retention times so

  will not be distinguished.
• What is the procedure for identifying the concentration of a substance from a gas chromatogram?

  Prepare standard solutions of known concentrations of the compound being investigated.

  
  

  Obtain gas chromatograms of each standard solution.

  
  

  Plot a calibration curve of peak area against concentration. This is called external callibration and offers a method for converting a peak area into a concetration.

  
  

  Obtain a gas chromatogram of the compound being investigated under the same conditions.

  
  

  Use the calibration curve to measure the concentration of the compound.
• What is a calibration curve?

  Can be a line or curve
• Why is necessary to carry out external calibration?

  To calculate the concentration of each component in the curve it is necessary to complete external calibration curves to confirm concentrations of components.

  
  

  Known amounts of a pure component can be passed through the GC machine.

  
  

  see TB page 245
• When is gas chromatography used in real life?

  forensics;

  
  

  environmental analysis

  
  

  airport security
• What is the alkene test (test for double bonds)?

  Add bromine water drop-wise, shake;

  
  

  Alkene presence decolourises bromine water from orange to colourless.
• What is the haloalkane test?

  Add silver nitrate (aq) and ethanol;

  
  

  warm to 50° (degrees celsius) in a water bath.

  
  

  you will get a precipitate;

  Chloroalkane - white precipitate

  Bromoalkane - cream precipitate

  Iodoalkane - yellow precipitate
• What is the carbonyl test?

  Add 2,4-DNP;

  
  

  Orange precipitate if carbonyl present.
• What is the aldehyde test?

  Add Tollens' reagent and warm.

  
  

  Silver mirror if present (a thin coating of silver forms on the walls of the test tube)
• What is the test for primary and secondary alcohols and aldehydes?

  Add acidified potassium dichromate(VI) and warm in a water bath.

  
  

  Colour change from orange to green.
• What is the test for a carboxylic acid?

  Add aqueous sodium carbonate.

  
  

  Effervescence;

  
  

  (bubble CO2 through limewater -> cloudy)
• What is the test for a phenol?

  one way:

  
  

  phenol's are acidic compounds;

  
  

  Tested using pH indicator paper;

  
  

  indicator paper will show it as pH<7

  
  

  second way

  
  

  first test for a reaction with NaOH:

  -Acid (phenol) -> solid will dissolve and colourless sol of sodium salt

  -Not acidic -> nothing happens

  
  

  then test for a reaction with carbonate:

  -Phenol's dont react with carbonates

  -strong acid will effervescence
• What reactions do phenol's undergo?

  Electrophilic substitution reactions;

  
  

  e.g. they react with bromine at room temp -> decolourised + white precipitate formed
• What is NMR spectroscopy?

  An analytical technique that you can use to work out the structure of an organic substance

  
• What are the two types of NMR spectroscopy?

  ¹³C NMR (carbon 13)

  gives info about the no of carbon environments;

  
  

  ¹H NMR (high resolution proton NMR)

  shows you the number of hydrogen atoms and the no of hydrogen environments
• What is a nuclear spin?

  electrons have a property called spin;

  
  

  NMR only works for odd numbered nuclei atoms
• What is resonance?


  An electron has two different spin states;

  
  

  The nucleus also has two different spin states and these have different energies.

  
  

  With the right combination of a strong magnetic field and radio frequency radiation, the nucleus can absorb energy and rapidly flips between the two spin states.

  
  

  This is called resonance.

  
• What is chemical shift δ?

  the difference is the radio frequency absorbed by the nuclei (hydrogen or carbon) in the molecule being analysed and that absorbed by the same nuclei in TMS

  
  

  All atoms have electrons surrounding the nucleus, which shifts the energy and radio frequency needed for NMR to take place

  
  

  The frequency shift is measured on a scale called chemical shift δ;

  
  

  in units of parts per million (ppm).
• What is TMS?


  Tetramethylsilane, (CH₃)₄Si, is used as the standard reference chemical against which all chemical shifts are measured.

  
  

  TMS is given a chemical shift value of 0 ppm.

  
  

  TMS produces a single absorption peak;

  
  

  All the peaks in other substances are measured as chemical shifts relative to this;

  
  

  The amount of chemical shift is determined by chemical environment, especially the presence of nearby electronegative atoms.