Chromatography- TLC

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Chromatography- TLC HPLC

• By Chloe Holmberg

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TLC- Thin Layer Chromatography
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TLC

• The stationary phase of TLC is a thin layer of
  fine powder (eg. Alumina) spread on a glass or
  plastic plate.
• The mobile phase is the solvent.
• TLC is similar to paper chromatography.
• Is suitable for qualitative analysis

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TLC- Steps involved in TLC

• Place a small spot of the sample being analysed
  at one end of the plate (this is the origin)
• Put the end of the plate in solvent so that the
  origin mark is above the solvent.
• Remove the plate from the solvent before the
  solvent front reaches the top of the plate.

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TLC- The chemistry behind TLC

• The components being analysed in the sample
  undergo continuous adsorption (where the
  substance bonds with the surface of the plate)
  and desorption (where these bonds break) back
  into the liquid mobile phase.
• The rate of movement of each individual component
  in the sample causes different Rf values of each
  of these components.
• This rate of movement is caused by how strongly
  it adsorbs onto the plate and how readily it
  dissolves back into the mobile phase.

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TLC-Interpreting TLC chromatograms

• Rf value can be used to identify the compounds
  present in the mixture by comparing the Rf value
  of each component with the Rf value of a known
  substance under identical conditions.
• Rf value will always be less than 1.
• Rf value can alter if temperature, type of
  stationary phase used, amount of water vapor
  surrounding the plate, or solvent type are
  changed during the chromatography process.
• Also substances can be identified by running
  standards of known chemicals on the same
  chromatogram as the unknown sample.

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TLC- Limitations and advantages

• TLC is only useful for qualitative analysis as it
  will only determine if a substance is present in
  a chemical, not how much is present (quantitative
  analysis).
• However this method is fast, cheaper than column
  chromatography, is able to detect the smallest
  amount of a substance present in the chemical,
  will work with corrosive materials and provides
  better separation of less polar compounds.

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TLC- Elutropic series

• This is a list of popular solvents in order of
  their polarity. Whether a solvent used in the
  mobile phase has high polarity or low polarity
  will depend on the samples polarity
• Where more polar compounds will normally require
  a more polar solvent.
• If unsure of the samples polarity, a solvent such
  as acetone which has medium polarity will be most
  appropriate to use for the experiment.
• The component which adsorbs onto plate the most
  will move the least distance, having the lowest
  Rf value and this will be the more polar
  component of the sample.

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TLC
Elutropic series
Nb.- if the substance you are trying to test is
colourless, by putting the plate under UV light,
the components will come up as dark spots which
are only visible under this light.
http//www.chem-ilp.net/labTechniques/TLCAnimation
.htm
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Bibliography- TLC

• http//www.chem-ilp.net/labTechniques/ElutropicSer
  ies.htm
• http//www.chemguide.co.uk/analysis/chromatography
  /thinlayer.html

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HPLC- High Performance/Pressure Liquid
Chromatography

• Detector sends a signal to the recorder which
  shows the amount of each component in the sample
  in a peak.

HPLC Column
Pump solvent reservoir
Sample injection point
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HPLC

• Stationary phase of HPLC is the solids of very
  small particle size packed into a column.
• Liquid mobile phase is the solvent which is
  dripped into the column from the reservoir above.
  A tap at the bottom of the column allows the
  solvent (now called the eluent) to leave the
  column at the same rate it entered at.
• HPLC is suitable for qualitative and quantitative
  analysis.

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HPLC- Apparatus

• The column
• Has a typical internal diameter of 4.6mm and can
  be 25cm long.
• It is packed with tiny polar silica particles
  (which are the solid stationary phase).
• These tiny particles create resistance of flow
  for the solvent travelling through the column
  causing the need for a pump to force the mobile
  phase through under high pressure of about
  14000kPa.
• Small size of solid silica particles allows more
  frequent adsorption and desorption of components
  (as with the solvent and paper in TLC) between
  the silica particles and the solvent.
• The most strongly adsorbed component of the
  sample will take the longest to pass through the
  column, while the least adsorbent component will
  go through more quickly, this is how HPLC
  separates the components of a compound.

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HPLC- Apparatus

• The solvent (liquid mobile phase)
• Non-polar solvent is used (eg. Hexane) as this
  will not adsorb with the polar silica particles
  for very long, therefore, will pass through the
  column quickly.
• The solvent enters the column from the reservoir
  above and is pumped into the column at around
  14000kPa. It is slowly dripped into the column
  and a tap at the bottom of the column allows the
  solvent to leave the column at the same rate with
  which it entered.
• The solvent which has left the column is called
  the eluent.

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HPLC- Apparatus

• The detector
• Components in the sample are detected by passing
  the solvent, which has been separated in the
  column, through a thin beam of UV light. This
  light is picked up by the detector which measures
  how much light it is receiving.
• As the components pass through this beam they
  absorb some of the UV light and the detector
  picks up this change and sends a signal to the
  recorder screen.
• Different components will absorb different
  amounts of light depending on how much of a
  particular compound is passing through the beam
  at that time.
• This method is called UV absorption.

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HPLC- Interpreting HPLC chromatograms

• Once the detector has picked up how much UV light
  it is receiving, it sends a signal to a recorder
  which moves at a constant pace and as components
  pass through the UV beam, draws a peak on the
  chromatogram, while the solvent will draw up as a
  flat baseline.
• The time taken for the sample to pass through the
  column is called the retention time (Rt).
• Under identical conditions, the same component
  will give the same Rt and by comparing a sample
  with other chromatograms of known solutions, we
  are able to determine the components in the
  unknown sample.
• The amount of a component in the solution is
  proportional to the area under the peak.
• Quantitative analysis of HPLC is done by running
  a series of standards of known concentration and
  comparing their peak size with that of the
  sample.
• Quantitative analysis can also be determined by
  plotting the peak height against the
  concentration on a calibration curve.

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HPLC- Interpreting HPLC chromatograms
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HPLC- Limitations advantages

• Allow chemists to determine very small quantities
  of components present in a sample.
• Able to identify a wide range of materials
• Is able to be linked to mass spectroscopy to
  enhance analysis of components.
• However the equipment is expensive.

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HPLC

• HPLC machines do not require calibration. The
  chromatograms are all done on a computer which
  will scale it automatically and even the
  injection of the sample is automated.
• HPLC is able to identify and separate very
  complex mixtures in similar compounds, such as
  drugs or sleeping tablets in the blood making it
  an ideal procedure for hospitals in testing
  patients for suspected overdose.

http//www.studyhplc.com/animatedsimulation.php
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bibliography- HPLC

• http//www.chemguide.co.uk/analysis/chromatography
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• http//www.google.com.au/imgres?qHPLCchromatogra
  msandcalibrationcurvebenzenehlengbv2biw1
  117bih675tbmischtbnidmR8JtAFDCKmYQMimgrefu
  rlhttp//www.sciencedirect.com/science/article/pi
  i/S0021967307004803docidY5q6zEgG0RFBQMimgurlht
  tp//origin-ars.sciencedirect.com/content/image/1-
  s2.0-S0021967307004803-gr2.gifw301h489eivr-C
  T7W-B6ekiAe_v-HSBAzoom1iacthcvpx792vpy167
  dur15hovh286hovw176tx80ty139sig11039824
  6544391041133page1tbnh147tbnw90start0ndsp
  18ved1t429,r4,s0,i76
• http//www.google.com.au/imgres?qHPLCchromatogra
  msandcalibrationcurvebasichlengbv2biw111
  7bih675tbmischtbnidKYLm71dDWQbAKMimgrefurl
  http//www.boomer.org/c/p3/c03/c0305.htmldocid9
  RfWgWINLl5uxMimgurlhttp//www.boomer.org/c/p3/c0
  3/Fig19.gifw606h393eis76CT8TUN7C0iQe8_8nVBA
  zoom1iacthcvpx334vpy360dur2281hovh181h
  ovw279tx128ty74sig110398246544391041133pag
  e1tbnh131tbnw202start0ndsp15ved1t429,r
  6,s0,i81