Chromatography and functional group analysis

• Chromatography is used to separate individual components from a mixture of substances.
• all forms of chromatography have a stationary phase and a mobile phase:

  • the stationary phase does not move and is normally a soli or a liquid supported on a solid
  • the mobile phase does move, and is normally a liquid or a gas
• chromatography can be used in the analysis of drugs, plastics, flavourings, air samples and has applications in forensic science
• Thin layer chromatography:

  • a quick and inexpensive analytical technique that indicated how many components are in a mixture
  • the technique uses a TLC plate which is usually a plastic sheet or glass, coated with a thin layer or a solid adsorbent substance - usually silica
• in TLC:

  • the adsorbent is the stationary phase. the different components in the mixture have different affinities for the adsorbent and bind with differing strengths to its surface
• Adsorption is the process by which the solid silica holds the different substances in the mixture to its surface. separation is achieved by the relative adsorptions of substances within the stationary phase
• carrying out TLC:

  • take a TLC plate. using a pencil, draw a line across the plate about 1cm from one end of the plate - base line
  • using a capillary tube, spot a small amount of solution of the sample onto the baseline on the plate
  • prepare a chromatography tank for the TLC plate - can be made from a small beaker with a watch glass placed on top. pour some solvent into the beaker to a depth of about 0.5cm
• • place the prepared TLC plate in the beaker, making sure the solvent does not cover the spot. cover the beaker with the watch glass and leave it undisturbed on the bench - solvent will rise up the TLC plate
  • allow the solvent to rise up the plate until it is about 1cm below the top of the plate. remove the plate from the beaker and immediately mark the solvent front with a pencil. allow the plate to dry
• • if there are any visible spots, circle them with a pencil
  • alternatively hold a UV lamp over the plate and circle any spots you can see
  • sometimes the plate is sprayed with a chemical or locating agent, such as iodine, to show the position of the spots that may be invisible to the naked eye
• Interpretation of a TLC plate:

  • thin layer chromatograms are analysed by calculating the value for the retention factor - Rf for each component
  • each component can be identified by comparing its Rf value with known values recorded using the same solvent system and adsorbent
  • formula: Rf = distance moved by component
  • distance moved by the solvent front
• it is common to run a TLC of a sample alongside pure samples of compounds that may be present. it is then easy to identify the amino acids in the unknown sample visually without needing to calculate any Rf values
• Gas chromatography:

  • useful for separating and identifying volatile organic compounds present in a mixture
• • the stationary phase is a high boiling liquid adsorbed onto an inert solid support
  • the mobile phase is an inert carrier gas such as helium or neon
• • a small amount of the volatile mixture is injected into the apparatus called a gas chromatogram
  • the mobile carrier gas carries the components in the sample through the capillary column which contains the liquid stationary phase adsorbed 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 the liquid stationary phase, the slower it moves through the capillary column
• • the components of 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 the shortest time has the lowest retention time and is detected first
• the retention time is the time taken for each component to travel through the column
• Interpretation of a gas chromatogram:

  • each component is detected as a peak on the gas chromatogram: two pieces of info can be obtained from it
  • retention times can be used to identify the components present in the sample by comparing these to retention times for known components
  • peak integrations (the areas under each peak) can be used to determine the concentrations of components in the sample
• retention times:

  • number of peaks = number of components
  • relative sizes of peaks show different proportions of each drug
• concentration components:

  • the concentration of a component in a sample is determined by comparing its peak integration (peak area) with values obtained from standard solutions of the component
• procedure of working out conc. of components:

  1. prepare standard solutions of known concentrations of the compound being nvestigated
  2. obtain gas chromatograms for each standard solution
  3. plot a calibration curve of peak area against conc. - this is called external calibration and offers a method for converting a peak area into a concentration
  4. obtain a gas chromatogram of the compound being investigated under the same conditions
  5. use the calibration curve to measure the concentration of the compound