Edexcel A Level Chemistry:复习笔记7.7.5 Other Chromatography

Types of Column Chromatography

 

Column Chromatography

  • In column chromatography, the column is often a long vertical glass tube or in the laboratory a burette is suitable
  • An inert solid (usually powdered silica gel or alumina) is the stationary phase which is placed in the column
  • A liquid solvent phase, mobile phase, is added into the column until it is saturated with solvent
    • Care should be taken when setting up the column because cracks in the stationary phase can lead to issues during separation

     

  • 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
    • The aim here is to 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
    • 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

     

  • The component with the greatest attraction / affinity to the stationary phase takes the longest time to flow through the column
  • If the components are coloured, then they can be identified using the Rf value
  • If the components are colourless, then other techniques such as fluorescence under UV light can be used to show their position in the column

7.11.3-Column-chromatography

Column chromatography showing the separation of coloured compounds in a glass burette

 

High performance liquid chromatography, HPLC

  • This is essentially the same as column chromatography
  • The main differences are that:
    • 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
      • Retention time is the time taken from the sample being injected to the sample being detected
    • 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 databasehplc

Gas-Liquid Chromatography, GLC

  • Gas-Liquid Chromatography (GLC) is used for analysing:
    • Gases
    • Volatile liquids
    • Solids in their vapour form

     

  • The stationary phase:
    • This method uses a long coiled column for the stationary phase
    • Normally a non-volatile liquid is the stationary phase in GLC

     

  • The Mobile phase
    • An inert carrier gas (e.g. helium, nitrogen) moves the sample molecules through the stationary phase

     

  • The sample is injected into the column through a self-sealing disc and the vapour formed is carried through the stationary phase using the inert-gas mobile phase

Retention times

  • Once sample molecules reach the detector, their retention times are recorded
    • This is the time taken for a component to travel through the column
    • It depends upon the attraction between the solute and the stationary and mobile phases as well as the volatility and nature of the solute

     

  • 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

     

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

8-1-analytical-techniques-glc-chromatogram

 A gas chromatogram of a volatile sample compound has six peaks. Depending on each molecule’s interaction with the stationary phase, each peak has its own retention time

Worked Example

Analysis of a compound by GLC shows the presence of four components, A, B, C and D.7.11.4-GLC-Worked-example-chromatogram

i) Which compound is present in the greatest quantity?

ii) Which compounds were present in equal amounts?

iii) Which compound had the strongest interaction with the stationary phase?

Answers:

i) D (the larger the relative size of the peak, the greater the quantity of that substance present)

ii) B and C (the peak sizes are equal)

iii) D (the larger the retention time, the greater the interaction of that component with the stationary phase)

Uses and limitations of HPLC and GLC

  • HPLC and GLC are used to separate small amounts of components from a mixture
  • They are often used to:
    • Provide forensic evidence
    • Drug testing, particularly in sports
    • Analysis of environmental pollution
    • Detecting explosives in baggage
  • However, they are not very useful at identifying specific compounds, in legal terms - positive identification or beyond reasonable doubt
  • This is because:
    • Different compounds may have the same retention time
    • It can be difficult to manage the conditions, e.g. temperature and pressure
    • Some unknown compounds may not have a reference for comparison in the databases
  • For this reason, HPLC and GLC are often coupled with other analytical techniques, most commonly mass spectrometry
    • This results in HPLC-MS and GC-MS (GLC is sometimes abbreviated to GC)
    • This means that components can be separated from mixtures and then analysed all within one machine

Problems with drug testing 

  • GC-MS is the most common method of drug detection in sports due to the accepted reliability of the results
  • Even then, there can be problems
  • One publicised problem is around the use of anabolic steroids
    • 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 supplementssteroids-nandrolone-and-19-norandrosterone

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